WO2023215244A1 - Use of n-acetylcysteine to treat lipid disorders and related organ dysfunctions - Google Patents

Use of n-acetylcysteine to treat lipid disorders and related organ dysfunctions Download PDF

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Publication number
WO2023215244A1
WO2023215244A1 PCT/US2023/020609 US2023020609W WO2023215244A1 WO 2023215244 A1 WO2023215244 A1 WO 2023215244A1 US 2023020609 W US2023020609 W US 2023020609W WO 2023215244 A1 WO2023215244 A1 WO 2023215244A1
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nac
hfd
day
mice
pharmaceutical composition
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PCT/US2023/020609
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French (fr)
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Khalid M. KHAN
Alexander H. K. KROEMER
Wanxing Cui
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Georgetown University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]

Definitions

  • Lipid disorders conditions that cause abnormal levels of lipids, or fats, in the blood—continue to be a global health concern.
  • hypercholesterolaemia which is the presence of high levels of cholesterol in the blood, is associated with an increased risk of cardiovascular disease, and has increased from being the 15th leading risk factor for death in 1990, to 11th in 2007 and 8th in 2019 (Pirillo et al., 2021).
  • Fatty liver disease marked by excess fat build-up in the liver, has a worldwide prevalence of 25% (Cotter and Rinella, 2020) and is associated with increased overall mortality, with ranges for the standardized mortality ratio of 1.34-2.6 compared to the general population (Kwak and Kim, 2015))
  • Treatments for lipid disorder include the use of medications, such statins and cholesterol absorption inhibitors, but these medications carry risks.
  • statins have been proven to be effective in lowering cholesterol levels, they are associated with a variety of side effects such as myopathy, increased risk of diabetes, and myalgia (Mesi et al., 2021).
  • N-acetylcysteine NAC
  • the present application is directed to a method of treating a lipid disorder in a subject in need thereof.
  • the method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in treating a lipid disorder in a subject in need thereof.
  • the lipid disorder may be a triglyceride-related lipid disorder.
  • the lipid disorder may be fatty liver disease, hyperlipidemia, obesity, or lipodystrophy.
  • the present invention is directed to a method of treating a skin disorder in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in treating a skin disorder in a subject in need thereof.
  • the skin disorder is selected from sebaceous hyperplasia; sebostatis; age-related skin changes; atopic dermatitis/eczema; dry skin (xerosis); psoriasis; ichthyosis; Sjogren’s syndrome; skin infections; acne inversa; acne vulgaris; seborrheic dermatitis; dry scalp; hair breakage and damage; hair loss disorders; alopecia; dry hair conditions; wound healing; skin complications and conditions in patients with diabetes mellitus; neurodermatitis; chemotherapy-induced skin complications and conditions; and any combination thereof.
  • the present invention is directed to a method of increasing lipid secretion from sebaceous glands, skin, or a combination thereof, in a subject in need thereof.
  • the method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in increasing lipid secretion from sebaceous glands in a subject in need thereof.
  • the subject has a skin disorder; ageing and/or age-related conditions; obesity and/or obesity-related disorders and conditions; metabolic syndrome; fatty liver disease; dyslipidemias; lipodystrophy; polycystic ovary syndrome (PCOS); diabetes; cardiovascular disease; kidney disease; wound healing; or inflammation or autoimmune condition.
  • the present invention is directed to a method of reducing adipose tissue in a subject in need thereof.
  • the method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in reducing adipose tissue in a subject in need thereof.
  • the adipose tissue is white adipose tissue. In certain embodiments, the adipose tissue is epididymal adipose tissue, inguinal adipose tissue, or a combination thereof.
  • the present invention is directed to a method of inhibiting diet- related weight gain in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In a further aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in inhibiting diet-related weight gain in a subject in need thereof.
  • the diet-related weight gain is due to a high-fat diet.
  • the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier.
  • the pharmaceutical composition is administered orally. In certain embodiments, the pharmaceutical composition is administered in an amount of about 1 to 200 mg/kg/day. In preferred embodiments, the pharmaceutical composition is administered in an amount of about 5 to 100 mg/kg/day. [0019] In some embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, the pharmaceutical composition is administered in an amount of about 25 to 400 mg/kg/day. In preferred embodiments, the pharmaceutical composition is administered in an amount of about 50 to 300 mg/kg/day. [0020] In some embodiments, the pharmaceutical composition comprises an aqueous solution.
  • the pharmaceutical composition comprises NAC in a concentration of about 0.5 to 10 g/L. In preferred embodiments, the pharmaceutical composition comprises NAC in a concentration of about 1 to 5 g/L.
  • FIG.1 shows T1-weighted magnetic resonance imaging (MRI) of mice in vivo, as described in Example 1
  • Panels A and B show hepatic steatosis. Regions of interest (ROI) were localized on the liver of each individual animal on at least three slices, and their mean intensity was measured and averaged to quantify hepatic fat accumulation. This demonstrated that mice fed a high-fat diet (HFD) presented with a an ⁇ 27% increase in liver contrast, as compared to mice fed a normal diet (ND).
  • HFD high-fat diet
  • ND normal diet
  • abdominal fat contained in the entire dataset was quantified by thresholding with ImageJ software.
  • FIG.2 shows hematoxylin and eosin (H&E) analysis of liver tissue, as described in Example 1.
  • Panel A shows H&E sections demonstrating the degree of hepatic steatosis.
  • Panel B shows a histogram corresponding to the percentage of hepatic steatosis in ischemia- reperfusion injury (IRI) specimens.
  • IRI ischemia- reperfusion injury
  • Panel A shows representative images of 26-30 week old male mice undergoing dynamic contrast- enhanced MRI (DCE-MRI), showing T1-weighted images performed before Eovist ® injection and at 4, 12 and 20 minutes post-injection (P-I).
  • DCE-MRI dynamic contrast- enhanced MRI
  • the circle denotes a typical region of interest used to measure mean intensity.
  • Panel B shows time course of relative liver enhanced T1 signal intensities after Eovist ® injection, comparing mice on ND, ND + NAC, HFD, and HFD mice supplemented with NAC (HFD + NAC).
  • Panel C shows area under the curve (AUC) calculated from the data in Panel B.
  • Panel D shows the slope of T1 signal intensity increased for each group shown, corresponding to the rates of change within the first 20 min after Eovist ® injection.
  • FIG.4 shows that the effects of IRI and NAC supplementation on HDF mice, as described in Example 1. HFD mice are prone to more severe IRI, which is significantly improved with NAC supplementation.
  • Panel A shows serum alanine aminotransferase (ALT) level following 24 hours of reperfusion.
  • Panel B shows representative images of baseline T2 mapping of mice on (i) ND, (iii), ND + NAC, (v) HFD, and (vii) HFD + NAC. Representative images of T2 mapping twenty-four hours after IRI surgery of (ii) ND, (iv) ND + NAC, (vi) HFD, and (viii) HFD + NAC. Blue represents liver parenchyma while green denotes edema.
  • FIG.5 shows IRI results in a higher degree of glycolytic stress in HFD mice, as described in Example 1.
  • Panel A shows color mapped Phasor Fluorescence Lifetime Imaging (FLIM) images of the ND and HFD liver samples in the absence (left) and presence (right) of NAC supplementation. The top row shows the sham samples, and the bottom row shows the IRI samples. More green, blue, and red color represents more protein-bound NADH, free NADH and long lifetime species (LLS), respectively.
  • FLIM Phasor Fluorescence Lifetime Imaging
  • Panel B shows Phasor plot of autofluorescence FLIM in liver samples—the three circles represent the lifetime position of the three components: free and bound NADH, and LLS.
  • Panel C shows the concentration ratio of free to bound NADH was calculated from each mouse. The higher value represents more glycolytic stress. There was a higher level of glycolytic stress in HFD and HFD IRI samples. This was reduced in the HFD + NAC IRI samples, but not the HFD + NAC samples, respective to their HFD counterparts.
  • Panel D shows the fractional intensity of LLS calculated using three component calculations to show the increased LLS in high fat liver and how that increased with IRI. [0027]
  • FIG.6 shows effects of NAC treatment on hepatic steatosis in HFD mice, as described in Example 1.
  • Panel A shows the fluorescence intensity image, the LLS mapped image, the calculated size distribution maps, and the corresponding phasor plot.
  • the cursor was used to select the LLS lifetime signature originating from the intensity image and the LLS image was colored according to that phasor signature.
  • the size map was created to show the distribution of the droplets.
  • Panels B and C show the size distribution plots for control (Panel B) and NAC treatment (Panel C).
  • the distribution shows HFD and IRI induced the large lipid droplets and (especially above 10000 pixels) were lower in number in HFD + NAC groups.
  • Panel D shows the average size distribution of the animals from each feeding regiment.
  • FIG.7 shows spatial heterogeneity analysis of phasor-FLIM imaging, as described in Example 1.
  • the analysis was based on quantification of components of phasor plot and their fractional intensity, which allowed for comparison between sample to sample and between different sets of samples. However, calculation of an average value from the total image resulted in loss of the dimensional information and the inherent heterogeneity that these maps can show.
  • the actual distribution of the fractional intensity of LLS and free NADH show how there was embedded spatial information if phasor-FLIM autofluorescence images (Panel A).
  • the composite image of LLS, free and bound NADH (Panel B) and the individual species distribution of LLS, bound NADH, free NADH, and free-bound NADH (Panels C-E, respectively) show how different parts of the image had differential amount of these species.
  • Panel F shows the free and bound NADH map with higher blue and green representing more free and more bound NADH, respectively, and how some areas were more glycolytic than others—information that was harder to see in three color images.
  • the cumulative graphs from 3 samples show that along with a shift of LLS and glycolysis in HFD- IRI samples there could be broadening of these distributions.
  • FIG.8 shows the effects of NAC treatment on the lipid profile within the liver of HFD mice.
  • Panel A shows a generated heat map demonstrating the alterations in lipid composition with the respective tissue.
  • Panel B shows volcano plots demonstrating the differences in upregulated lipid species in HFD vs ND and HFD vs HFD + NAC mice, ultimately revealing changes triacylglycerol (TAG) composition within these tissues.
  • Panel C shows a histogram representing quantitative differences in TAG composition between the treatment groups. These results show that NAC treatment in HFD mice significantly altered the lipid profile within the liver.
  • FIG.9 shows the effects of IRI on gene expression in HFD mice, as described in Example 1. IRI induced specific genetic upregulation in HFD mice.
  • Panel A shows a heat map of cytokine and chemokine PCR analysis.
  • FIG.10 shows role of IFN- ⁇ producing hepatic natural killer T (NKT) cells following IRI, as described in Example 1.
  • Panel A shows representative contour plots of the percentage of CD45+ /CD3+ /NK1.1+ NKT1 cells within hepatic mononuclear cells.
  • Panel B shows representative contour plots of the percentage of IFN- ⁇ producing CD45+ /CD3+ /NK1.1+ NKT cells.
  • Panel C shows a histogram demonstrating the percentage of NKT1 cells illustrated in Panel A.
  • FIG.11 shows changes in bodyweight over time of B6 wildtype ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 2. The results show that NAC treatment protected B6 wildtype mice from high HFD-induced obesity when compared to untreated HFD controls, as shown by substantial reduction in weight gain.
  • FIG.12 shows blood glucose levels over time post-HFD start date of B6 wildtype ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 2.
  • FIG.13 shows changes in bodyweight over time of B6 wildtype HFD and HFD + NAC mice, as described in Example 2. Body weight is shown with NAC treatment started at16 weeks post HFD (Panel A), and started 32 weeks post HFD (Panel B). The results show that NAC treatment reversed HFD-induced obesity in B6 wildtype mice when compared to untreated HFD controls, as shown by weight loss induction.
  • FIG.14 shows consumption rate of B6 wildtype ND + NAC, HFD, and HFD + NAC mice, as described in Example 2.
  • FIG.15 shows triacylglycerols (TAG) levels in HFD + NAC and HFD B6 wildtype mice, as described in Example 2.
  • Panel A shows a heat map and Panel B shows a volcano plot comparing 277 TAGs in HFD + NAC versus HFD controls, in which significantly decreased TAGs (149 in total) are marked as blue dots, and others (128) are marked in grey.
  • TAG triacylglycerols
  • FIG.16 shows images of HFD B6 wildtype ND (Panel A), ND + NAC (Panel B), HFD (Panel C), and HFD + NAC (Panel D) mice, as described in Example 2.
  • the images show that HFD B6 wildtype mice treated with NAC develop fatty and “greasy” hair, which suggested that they lost weight through secreting fat (TAGs) via the skin.
  • FIG.17 shows an image of HFD B6 wildtype HFD + NAC mouse, as described in Example 2.
  • the image shows that HFD B6 wildtype mice treated with NAC possessed fatty and “greasy” fur, which suggested that they lost weight through secreting fat (TAGs) via the skin.
  • FIG.18 shows analysis of the fur of HFD + NAC and HFD mice, as described in Example 2.
  • Panel A shows quantity of hair lipids in HFD + NAC and HFD mice.
  • Panel B shows HFD lipid composition and quantification via thin-layer chromatography. The results show that the hair of HFD B6 wildtype mice treated with NAC contained significantly higher lipid mass and TAG levels than HFD controls, confirming that NAC caused adipose loss through enhanced lipid secretion through the skin.
  • FIG.19 shows analysis of body weight and fur of ND + NAC and ND mice, as described in Example 2.
  • Panel C shows ND lipid composition and quantification via thin-layer chromatography, wherein the hair of ND mice treated with NAC contained significantly lower TAG levels than ND mice (p ⁇ 0.05). [Data are expressed as mean ⁇ SEM, and statistical analysis was conducted using the Mann-Whitney test.
  • FIG.20 shows longitudinal (Panel A) and transverse (Panel B) sebaceous gland area of ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 3.
  • NAC treatment significantly reduced both longitudinal and transverse sebaceous gland area as compared to HFD control.
  • FIG.21 show examples images of longitudinal and transverse cross-sections of sebaceous glands (Panel A), representative images of longitudinal and transverse cross- sections of sebaceous glands in HFD and HFD + NAC (1%) mice (Panel B), and representative images of longitudinal and transverse cross-sections of sebaceous glands in ND and ND + NAC (1%) mice (Panel C), as described in Example 3.
  • the sebaceous glands in longitudinal cross-section views resemble a water or tear drop shape and are adjacent and follow the follicle length, while the sebaceous glands in transverse cross-section view resemble a “ bean” shape and often appear as wrapping around the hair bulb.
  • the size difference between the HFD and HFD + NAC group are apparent in the longitudinal cross-section views of the sebaceous glands in Panel B.
  • FIG.22 shows body weight (Panel A) and percent change in body weight from baseline (Panel B) of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%) mice, as described in Example 4.
  • the effect of NAC treatment on body weight was greater at higher NAC doses.
  • FIG.23 shows food consumption of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%), as described in Example 4.
  • FIG.24 shows epididymal fat pad weight (Panel A) and inguinal fat pad weight (Panel B) of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%) mice, as described in Example 4.
  • FIG.25 shows images of ND (Panel A), ND + NAC (0.1%) (Panel B), ND + NAC (1%) (Panel C), HFD (Panel D), HFD + NAC (0.1%) (Panel E), HFD + NAC (0.2%) (Panel F), and HFD + NAC (1%) (Panel G) mice after one week of treatment, as described in Example 4.
  • FIG.26 shows analysis of fur of HFD and HFD + NAC (0.2%) mice, as described in Example 4.
  • FIG.27 shows effects of NAC treatment on Tbet-/-RAG mice, as described in Example 5.
  • FIG.28 shows effects of NAC treatment on RAG-deficient (or “RAG1”) mice in regard to body weight (Panel A), food consumption (Panel B), and blood glucose levels (Panel C), as described in Example 5.
  • RAG-deficient mice or “RAG1” mice in regard to body weight (Panel A), food consumption (Panel B), and blood glucose levels (Panel C), as described in Example 5.
  • the HFD RAG-deficient mice treated with NAC exhibit reduced body weight and blood glucose, but similar food consumption rates.
  • FIG.29 further shows effects of NAC treatment on RAG-deficient (or “RAG1”) mice as described in Example 5.
  • the results show that the HFD + NAC RAG-deficient mice possessed reduced epididymal and inguinal fat pads as compared to the HFD RAG-deficient mice.
  • the present application is directed to the use of N-acetylcysteine to treat lipid disorders and other conditions in a subject.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, and molecular biology, which are within the skill of the art.
  • certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.
  • the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
  • Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form.
  • Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein.
  • a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.
  • a disclosed range is a disclosure of each individual value encompassed by the range.
  • a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.
  • inhibitor refers to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity.
  • inhibition can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence.
  • An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.
  • induce “increase,” and “stimulate” are used interchangeably and refer to any statistically significant increase in occurrence or activity, including full blocking of the occurrence or activity.
  • “stimulate” can refer to an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence.
  • a “stimulator” is a molecule, factor, or substance that produces a statistically significant increase in the occurrence or activity of a process, pathway, or molecule.
  • “quantify” or “quantity” refers to the measurement of the amount of a component in a sample, and is typically calculated as a concentration such as a mass concentration (mass of the antibody divided by volume of the sample). Generally, quantity is determined in a sample that is collected from a subject.
  • the terms “treat,” “treating,” and “treatment” refer to curing, slowing down, lessening symptoms of, and/or halting progression of a diagnosed pathologic disease, condition, or disorder.
  • such terms include, but are not limited to, lessening symptoms of a disease, condition, or disorder; slowing down the worsening of symptoms of a disease, condition, or disorder; slowing down and/or halting the progression of a disease, condition, or disorder; causing regression of a disease, condition, or disorder; and the like.
  • a subject is successfully “treated” for a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.
  • the present invention provides a method of treating a lipid disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of NAC.
  • the lipid disorder may be a triglyceride-related lipid disorder. Examples of lipid disorders that can be treated include, but are not limited to, fatty liver disease, hyperlipidemia, obesity, and lipodystrophy.
  • the present invention provides a method of treating a skin disorder or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • Examples of skin disorders or conditions that can be treated include, but are not limited to, sebaceous hyperplasia; sebostatis; age-related skin changes such as aging skin and wrinkles; atopic dermatitis/eczema, such as dermatitis due to dry skin; dry skin (xerosis); psoriasis; ichthyosis; Sjogren’s syndrome; skin infections; acne inversa; acne vulgaris; dermatitis due to dry skin; dry scalp; hair breakage and damage; hair loss disorders; alopecia, such as traction alopecia; dry hair conditions; wound healing; skin complications and conditions in patients with diabetes mellitus; neurodermatitis; chemotherapy-induced skin complications and conditions; and any combination thereof.
  • the present invention provides a method of increasing lipid secretion from sebaceous glands and/or from skin in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the lipid secretion comprises triacylglycerols.
  • the subject may have a condition or disorder selected from the group consisting of a skin disorder or condition as described herein; ageing and/or age-related condition; obesity and/or obesity-related disorders or conditions, including an obesity-related cancer; metabolic syndrome; fatty liver disease, such as nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH); dyslipidemia; lipodystrophy; PCOS; diabetes mellitus type I or II; cardiovascular disease, such as coronary heart disease or stroke; kidney disease; wound healing; and inflammation and/or autoimmune condition.
  • the subject may have a need for support as a prosthetic device use.
  • the present invention provides a method of reducing fat mass in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • fat mass is an indicator of the quantity of adipose tissue in a body or a portion of a body. Fat mass may be calculated as a weight, e.g., the weight of the adipose tissue in the body or a portion of the body; or may be calculated as a percentage, for example, a percentage of the weight of the body or portion of the body that constitutes adipose tissue.
  • the present invention provides a method of reducing adipose tissue in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • the adipose tissue is white adipose tissue.
  • the adipose tissue is epididymal adipose tissue, inguinal adipose tissue, or a combination thereof.
  • the present provides a method of inhibiting diet-related weight gain in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject.
  • diet-related weight gain refers to an increase weight that is primarily due to diet, as opposed to a disorder or condition such as high insulin levels or insulin resistance, leptin resistance, use of medications that can cause weight gain, hyperthyroidism, Cushing’s syndrome, etc.
  • the diet-related weight gain is due to a high-fat diet.
  • high-fat diet or “HFD” refers to a diet in which the amount of diet in the diet is greater than recommended for a healthy diet, for example, greater than recommended by Food and Nutrition Board of the National Academys of Sciences Engineering, and Medicine, or by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture.
  • a high-fat diet may be a diet in which at least 35% of total calories is consumed from fats, both unsaturated and saturated.
  • the term “subject” encompasses all mammalian species, including, but not limited to, humans, non-human primates, dogs, cats, rodents (such as rats, mice and guinea pigs), cows, pigs, sheep, goats, horses, and the like – including all mammalian animal species used in animal husbandry, as well as animals kept as pets and in zoos, etc. In some embodiments the subjects are human. [0074] The efficacy of a given composition or method in treatment can be demonstrated or assessed using standard methods known in the art, such as methods that compare the efficacy of a given / “test” composition or method to a “control” composition or method.
  • the efficacy of a given composition or method in treating a lipid disorder may be demonstrated or assessed by comparing its ability to improve one or more clinical indicators or symptoms of the lipid disorder as compared to that of a control composition or control method, such as a placebo control. For instance, a comparison can be made between different subjects (e.g., between a test group of subjects or a control group of subjects).
  • the efficacy of a given composition or method in treatment can be demonstrated or assessed in a single subject by comparing one or more clinical indicators or symptoms of the lipid disorder in the subject before and after treatment.
  • any suitable method or route of administration can be used to deliver the active agents or combinations thereof described herein.
  • administration includes any route of introducing or delivering the specified compositions or agents to subjects.
  • the active agents or combinations thereof are administered systemically.
  • the active agents or combinations thereof are administered locally.
  • Systemic administration refers to introducing or delivering to a subject a specified composition or agent via a route which introduces or delivers the composition or agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems.
  • local administration refers to introducing or delivering to a subject a specified composition or agents via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount.
  • administration can be carried out by any suitable route known in the art, including intratumoral, intravenous, subcutaneous, oral, topical, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like.
  • parenteral e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques
  • Administration includes self-administration and administration by another. The suitability of a given route or means of administration can be readily determined by a physician. In preferred embodiments, administration is oral.
  • the term “effective amount” refers to an amount of NAC that is sufficient to achieve, or contribute towards achieving, one or more of the outcomes listed in the “treatment” description herein.
  • An appropriate “effective” amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the desired route of administration (e.g., systemic vs. local), the desired frequency of dosing, etc.
  • an “effective amount” may be determined in the context of any co-administration to be used.
  • the dose of NAC may be calculated based on studies in humans or other mammals carried out to determine efficacy and/or effective amounts of NAC.
  • the dose amount and frequency or timing of administration may be determined by methods known in the art and may depend on factors such as the pharmaceutical form, route of administration, whether it is used in combination with other active agents (for example, the dosage of NAC required may be lower when i is used in combination with another active agent), and patient characteristics including age, body weight, or the presence of any medical conditions affecting drug metabolism.
  • the dosage of NAC required may be lower when i is used in combination with another active agent
  • patient characteristics including age, body weight, or the presence of any medical conditions affecting drug metabolism.
  • suitable doses of NAC described herein can be determined by performing dosing studies of the type that are standard in the art, such as dose escalation studies, for example using the dosages shown to be effective in mice in the Examples section of this patent application as a starting point.
  • Dosing regimens can also be adjusted and optimized by performing studies of the type that are standard in the art, for example using the dosing regimens shown to be effective in mice in the Examples section of this patent application as a starting point.
  • the active agents are administered daily, or twice per week, or weekly, or every two weeks, or monthly.
  • the pharmaceutical composition may be administered orally.
  • the pharmaceutical composition may be administered in an amount of about 1 to 200 mg/kg/day or any sub-range therebetween, for example, about 1 to 190 mg/kg/day, or about 1 to 180 mg/kg/day, or about 1 to 170 mg/kg/day, or about 1 to 160 mg/kg/day, or about 1 to 150 mg/kg/day, or about 1 to 140 mg/kg/day, or about 1 to 130 mg/kg/day, or about 1 to 120 mg/kg/day, or about 1 to 110 mg/kg/day, or about 1 to 100 mg/kg/day, or about 5 to 200 mg/kg/day, or about 5 to 190 mg/kg/day, or about 5 to 180 mg/kg/day, or about 5 to 170 mg/kg/day, or about 5 to 160 mg/kg/day, or about 5 to 150 mg/kg/day, or about 5 to 140 mg/kg/day, or about 5 to 130 mg/kg/day, or about 5 to 120 mg/kg/day, or about 5 to 110 mg/kg
  • the pharmaceutical composition may be administered intravenously.
  • the pharmaceutical composition may be administered in an amount of about 25 to 400 mg/kg/day or any sub-range therebetween, for example, about 25 to 390 mg/kg/day, or about 25 to 380 mg/kg/day, or about 25 to 370 mg/kg/day, or about 25 to 360 mg/kg/day, or about 25 to 350 mg/kg/day, or about 25 to 340 mg/kg/day, or about 25 to 330 mg/kg/day, or about 25 to 320 mg/kg/day, or about 25 to 310 mg/kg/day, or about 25 to 300 mg/kg/day, or about 30 to 400 mg/kg/day, or about 30 to 390 mg/kg/day, or about 30 to 380 mg/kg/day, or about 30 to 370 mg/kg/day, or about 30 to 360 mg/kg/day, or about 30 to 350 mg/kg/day, or about 30 to 340 mg/kg/day, or about 30 to
  • compositions and methods provided herein may be employed together with other compositions and treatment methods known to be useful for treatment of lipid disorders or skin disorders, reducing adipose tissue, inhibiting diet-related weight gain, etc., including, but not limited to, pharmaceuticals (e.g., statins, cholesterol absorption inhibitors), surgical methods (e.g., adjustable gastric banding, gastric bypass surgery, sleeve gastrectomy), and the like, as well as other non-pharmaceutical and non- invasive methods such as dietary changes and exercise/activity.
  • pharmaceuticals e.g., statins, cholesterol absorption inhibitors
  • surgical methods e.g., adjustable gastric banding, gastric bypass surgery, sleeve gastrectomy
  • non-pharmaceutical and non- invasive methods such as dietary changes and exercise/activity.
  • the methods of treatment provided herein may be employed together with procedures used to monitor progression of lipid or skin disorders, adipose tissue reduction, weight gain, etc.
  • NAC NAC Pharmaceutical Compositions
  • NAC may be formulated in a pharmaceutical composition.
  • the pharmaceutical composition may comprise one or more carriers, diluents, excipients, or other additives.
  • Carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile.
  • Sterile water can be a vehicle when the compound of the invention is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
  • the composition can comprise one or more stabilizing agents (e.g., dextran 40, glycine, lactose, mannitol, trehalose, maltose), one or more buffers (e.g., acetate, citrate, histidine, lactate, phosphate, Tris), one or more pH adjusting agents (e.g., hydrochloric acid, nitric acid, potassium hydroxide, sodium hydroxide), one or more surfactants (polysorbate, sodium lauryl sulfate, polyethylene glycol-fatty acid esters, lecithins), and/or one or more diluents (e.g., water, physiological saline).
  • stabilizing agents e.g., dextran 40, glycine, lactose, mannitol, trehalose, maltose
  • buffers e.g., acetate, citrate, histidine, lactate, phosphate, Tris
  • the composition may also include excipients such as starch, glucose, lactose, methyl cellulose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • excipients such as starch, glucose, lactose, methyl cellulose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the present compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, or solubilizers (e.g., sulfobutylether b- cyclodextrin).
  • the pH is between about 4.0 and 7.0, or between about 5.0 and 6.5.
  • the present formulations can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • the compositions of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to subjects.
  • pharmaceutical compositions of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical compositions may also include a solubilizing agent.
  • compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • a local anesthetic such as lignocaine
  • the ingredients of the pharmaceutical compositions of the present invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.
  • NAC is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the composition is an aqueous solution.
  • the composition comprises NAC in a concentration of about 0.05 to 10 g/L or any sub-range therebetween, for example, about 0.05 to 9 g/L, or about 0.05 to 8 g/L, or about 0.05 to 7 g/L, or about 0.05 to 6 g/L, or about 0.05 to 5 g/L, or about 0.05 to 4 g/L, or about 0.05 to 3 g/L, of about 0.05 to 2 g/L, or about 0.1 to 10 g/L, or about 0.1 to 9 g/L, or about 0.1 to 8 g/L, or about 0.1 to 7 g/L, or about 0.1 to 6 g/L, or about 0.1 to 5 g/L, or about 0.1 to 4 g/L, or about 0.1 to 3 g/L, of about 0.1 to 2 g/L, or about 0.25 to 10 g/L, or about 0.25
  • the pharmaceutical compositions of the invention can be administered orally.
  • Formulations for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • the pharmaceutical compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention.
  • fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • a time delay material such as glycerol monostearate or glycerol stearate may also be used.
  • Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical composition of the invention may be prepared by methods known in the art. For example, the methods may comprise admixing a NAC and a pharmaceutically acceptable carrier to prepare the composition.
  • An aspect of the present invention relates to the NAC pharmaceutical composition thereof, as described herein, for use in any of the methods of the present invention described herein.
  • the invention further provides pharmaceutical packs or kits comprising one or more containers filled with NAC or pharmaceutical composition thereof.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • *** [0095] The invention is further described in the following non-limiting Examples, as well as the Figures referred to therein.
  • Example 1 Oral NAC ameliorates IRI and inflammation in steatotic livers Introduction
  • IRI Expression Hepatic ischemia-reperfusion injury
  • IRI is a complex and incompletely understood process that leads to the disruption of cellular integrity with the release of damages-associated molecular patterns and subsequent innate immune system activation, though identification of key mediators remains the point of ongoing research.
  • the degree of IRI is more profound in the setting of hepatic steatosis and has historically been shown to increase the risk of primary non-function, dysfunction, and reduce graft and patient survival.
  • “marginal” allografts including those with hepatic steatosis, are being considered despite an elevated risk of IRI.
  • NAC N-acetylcysteine
  • Invariant natural killer T (NKT) cells are a unique group of cells that can recognize both self and lipid antigens that are presented to them by MHC class I-like CD1d molecules.
  • Type I invariant Natural Killer T-cells (NKT1 cells) are not only CD1d bound, but also express markers of both T cells (CD3+) and conventional NK cells (NK1.1+) and can potently secrete IFN- ⁇ and TNF ⁇ .
  • NKT1 cells have emerged as key mediators in hepatic IRI and have been shown to worsen this process in a NAFLD murine model, despite lower baseline populations.
  • NKT1 cells have a wide variety of activating antigens to include endogenous lipid antigens such as phosphorylcholine, which may lead to an enhanced response to IRI.
  • endogenous lipid antigens such as phosphorylcholine
  • NAC has also been demonstrated to alter metabolism and have benefit in the treatment of metabolic syndrome, in addition to known benefit in IRI.
  • the immunomodulatory capacity of NAC in liver injury remains understudied.
  • NKT1 cells were activated by lipid antigens and potential roles in hepatic IRI.
  • HFD high-fat diet
  • advanced phasor-FLIM is used to interrogate tissue following IRI and further evaluate the innate immune response to IRI.
  • This study successfully demonstrates NKT1 cells as key mediators in IRI in the setting of hepatic steatosis and shows an improved response following NAC supplementation at least in part secondary to reduced NKT1 cell activation.
  • the normal diet (ND) #5001, was purchased from Lab Diet, St.
  • HFD high-fat diet
  • NAC was purchased from Sigma, St. Louis, MO, and was added to the treatment group drinking water in a concentration of 10 gram/liter.
  • mice All procedures performed on C57BL/6 mice were fully approved by the Georgetown University Institutional Animal Care and Use Committee under protocol number 2016-1341. Male mice were used for all experiments. Food and water were provided ab libitum in the four experimental groups: ND, ND + NAC, HFD, and HFD + NAC.
  • the ND diets were administered at the beginning of the study and maintained for 19-23 weeks.
  • the HFD was started at week seven of age and continued for 19-23 weeks.
  • NAC drinking water was administered to the ND + NAC and HFD + NAC groups following three weeks of ND or HFD and maintained for 16-20 weeks. Animal weights were measured weekly.
  • C57BL/6 mice were purchased at 25 weeks of age from The Jackson Laboratory, Bar Harbor, ME on a ND, #5K52 from Lab Diet (3.50 kcal/gram, 16.6% fat) for use in select groups.ps. [00103] All procedures performed on C57BL/6 mice were fully approved by the Georgetown University Institutional Animal Care and Use Committee under protocol number 2016-1341. Mice were anesthetized with 2% isoflurane and oxygen inhalation. A midline laparotomy was performed, and an atraumatic micro clip was placed across the hepatic hilus, which interrupted the blood supply to the left and median lobes of the liver. The abdomen was temporarily closed with skin staples, and the mice remained anesthetized.
  • a 45-minute ischemic time was utilized to represent a clinically relevant model, more closely corresponding to the average warm ischemia time in human liver transplantation. This also prevented excess animal death under anesthesia, given the metabolic changes accompanied by HFD murine model.
  • the clip was removed to initiate hepatic reperfusion. The abdominal wall was closed with sutures, the skin was reapproximated with staples, and the animals were returned to their cage. All mice undergoing Magnetic Resonance Imaging following IRI, had their abdominal wall and skin reapproximated with sutures. After 24 hours of reperfusion, mice were anesthetized and underwent a second laparotomy. Whole blood was obtained via direct cardiac puncture as a terminal procedure.
  • Magnetic resonance imaging was performed in the Georgetown-Lombardi Preclinical Imaging Research Laboratory on either a 7T/20 Avance III/ParaVision 5 or a 7T/30 USR Avance NEO/ParaVision 360 (S10 OD025153) scanner.
  • mice were anesthetized (1.5% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide) and placed on a custom-manufactured (ASI Instruments, Warren, MI) stereotaxic device, with built-in temperature and cardio-respiratory monitoring as described (B, C), and compatible with a 40 mm Bruker mouse body volume coil.
  • ASI Instruments, Warren, MI custom-manufactured stereotaxic device
  • MRI of abdominal and liver fat was performed with a three-dimensional rapid acquisition with rapid enhancement (3D-RARE) sequence in the coronal orientation with the following parameters: TR: 2855 ms, TE: 12 ms, RARE Factor: 4, Matrix: 220 x 220, FOV: 50 mm x 40 mm, Averages: 4, Slice thickness: 0.75 mm, Slices: 50 and respiratory gating
  • TR 2855 ms
  • TE 12 ms
  • RARE Factor 4
  • Matrix 220 x 220
  • FOV 50 mm x 40 mm
  • Averages 4
  • Slice thickness 0.75 mm
  • Slices 50 and respiratory gating
  • Quantification of visceral fat depots in the imaging datasets was performed by thresholding and voxel-counting with ImageJ software (NIH).
  • NIR ImageJ software
  • the abdominal region analyzed was defined by superior and inferior anatomical landmarks, that is, the proximal border of the left kidney and the convergence of the left and right common iliac veins, respectively.
  • the lateral landmark was the abdominal wall, avoiding subcutaneous fat.
  • the percentage area corresponding to fat depots within the abdomen was calculated via the sum of the visceral fat voxels versus total abdominal voxels.
  • Mouse liver fat was measured by quantifying the mean intensity of the region of interest (ROI) localized on the mouse liver placed in homogenous areas, avoiding structures such as large vessels and ducts. ROIs on three separate slices were selected and averaged for each mouse.
  • ROI region of interest
  • a 3D T1-weighted RARE sequence in the sagittal orientation was run with TR: 2437 ms, TE: 15 ms, FA: 74.1, Matrix: 256 x 256, FOV: 40 mm x 40 mm, Slice thickness: 0.5 mm, Averages: 4, Slices: 35.
  • the mean intensity of nuchal brown adipose tissue (BAT) and white adipose tissue (WAT) was measured by localizing ROIs on the BAT and the WAT. ROIs on three separate slices were selected and averaged for each mouse.
  • DCE-MRI Dynamic Contrast-Enhanced MRI
  • Eovist® Gadoxetate Disodium
  • TE 8.2 ms
  • TR 400 ms
  • slice thickness 1 mm
  • rare factor 2
  • fat suppression and a duration of 25 s.
  • Ten baseline (pre-contrast) scans were run, followed by subcutaneous parenteral administration of 0.025 mmol/kg Eovist. Immediately after injection, 60 MRI scans were acquired repetitively over approximately 30 minutes, and the uptake and excretion of contrast was measured over time.
  • ROI regions of interest
  • a two-dimensional multi-echo multi-slice sequence in the axial orientation was used for T2 mapping with TR: 2000 ms, Flip Angle: 180 deg, Averages: 2, and the following echo times, TE: 10, 20, 30, 40, 50, 60, 70 and 80 ms, with respiratory gating.
  • TE 10, 20, 30, 40, 50, 60, 70 and 80 ms
  • TE 10, 20, 30, 40, 50, 60, 70 and 80 ms
  • ALT levels were measured using a multichannel analyzer, Alfa Wassermann Vet Axcel, from the clinical diagnostics laboratory of VRL Maryland, LLC.
  • the autofluorescence lifetime images of the liver sections (5 ⁇ m thick) were measured using a modified Olympus FVMPERS (Waltham, MA) microscope equipped with a Spectra-Physics Insight X3 (Milpitas, CA) laser and FastFLIM (ISS, Champaign, IL) acquisition card.
  • the samples were excited with the 740 nm laser line using a 20X air objective (LUCPLFLN 0.45NA, Olympus) applying a two-photon excitation scheme.
  • the fluorescence was collected using the DIVER (Deep Imaging Via Enhanced Recovery) detector (16-18) assembly and recorded using a FastFLIM card (ISS, Champaign, IL).
  • To obtain a high signal-to- noise ratio 16 frames were collected for each area. The data from each pixel were recorded and analyzed using the SimFCS software (Laboratory for Fluorescence Dynamics, University of California, Irvine, CA).
  • the FLIM data were collected using the passive mode, where the raster scanning was done using the Olympus software, and the images were collected using the FLIMBox/FastFLIM system, and the scanning parameters were matched to ensure proper image acquisition. SHG and FLIM images were obtained using two separate filter assemblies in DIVER and further separated based on their lifetime and phasor position.
  • the Phasor approach to FLIM is a fit free method of analysis where fluorescence decay information collected at each pixel of an image is transformed to the phasor plot and designated a coordinate based on the lifetime. Being a fit free method, phasor is a much faster analysis method and is used increasingly in biological fluorescence imaging with a large data set.
  • the phasor transformation results in the formation of a heat map in the phasor plot where each pixel of an image is represented by a single phasor point and segmentation and filtering methods and of image analysis can be applied without loss of image definition of the original intensity images. Recent advances in analysis techniques allow us to quantify the contribution of multiple components and this is applied in this work.
  • the distribution of phasor points originating from FLIM measurements appears on (for mono-exponential decays) or inside (for multi-exponential decays) the universal circle (please refer to SMI in the phasor supplemental methods).
  • the linear combinations are shown by the blue. If each component has a multi-exponential decay, its location will be represented by phasors not in the universal circle, but the principle of linear combination remains valid. More details can be found in the phasor section of the supplemental methods.
  • the long lifetime species has a lifetime of ⁇ 8ns, and the corresponding phasor position is selected by the red circle.
  • LLS long lifetime species
  • the autofluorescence FLIM phasor in the NADH channel shifts away from the metabolic trajectory and inside the triangle whose vertices are occupied by the central positions of red, green, and blue circles.
  • Phase section we have used multiple component analysis to obtain the quantitative information about the concentration ratio of free and protein-bound NADH and fractional intensity of NADH.
  • SHG is generated from the non-centrosymmetric collagen fiber bundles and has a different lifetime signature than rest of the tissue autofluorescence.
  • Snap-frozen liver tissue was dissolved in 300 ⁇ L of extraction buffer (IPA) and subjected to two cycles of freezing and rapid thawing into a 37 °C water bath or 90 seconds. Then, tissue samples were sonicated at 30 kHz for 30 seconds and mixed with 100 ⁇ l of ice- chilled isopropyl alcohol (IPA) with internal standards (IS).
  • IPA extraction buffer
  • IS internal standards
  • MultiQuant 3.0.3 (Sciex) software was used to obtain QC normalized data and imputed MRM data. To determine relative quantification values of analytes, the ratio of peak areas of sample transitions normalized to the peak area of the IS specific for every class was computed. Data postprocessing statistical analysis including heatmap, volcano plot, and ANOVA was conducted with the software MetaboAnalyst.
  • the left lobe of the liver was collected into RPMI-1640 culture medium (Gibco). Liver tissues were harvested, passed through a 70- ⁇ m cell strainer (Fisher Scientific), and leukocyte fractions were isolated via Percoll (Cyntiva) density gradient.
  • Recombinant mouse cytokine concentrations used were 10 ng/ml IL-12, 50 ng/ml IL-18, and 50 ng/ml IL-15 (R&D Systems).
  • Cells were stimulated for 4 hours with Cell Activation Cocktail containing phorbol-12myristate-13-acetate (PMA, 50 ng/ml) and ionomycin, (BioLegend) in the presence of 5 ⁇ g/ml brefeldin A (BioLegend).
  • PMA phorbol-12myristate-13-acetate
  • BioLegend ionomycin
  • the cells were stained with the following antibodies to detect cytokines: Brilliant Violet 650TM -conjugated anti-IFN- ⁇ (BD Biosciences) and Brilliant Violet 605TM - conjugated anti-TNF- ⁇ (BioLegend).
  • mice liver specimens collected 24 hours-post sham and IRI surgeries and stored in the Allprotect Tissue Reagent (Qiagen) were used for the total RNA extraction.
  • RIN RNA integrity number
  • Standard procedures were performed to eliminate mouse genomic DNA, synthesize first-strand and set up RT2 profiler PCR array for cytokines and chemokines.
  • Raw Ct values from the array were analyzed using the GeneGlobe web tool (https://geneglobe.qiagen.com/us/) to check the quality of the PCR arrays, normalize threshold cycle (Ct) values using Beta-2-Microglobulin (B2m) as a housekeeping gene and estimate fold regulation with ND (Sham) as a control group.
  • Unsupervised hierarchical clustering for the 2 - ⁇ Ct was performed using ClustVis (35) to identify gene clusters specific to sham and IRI groups under two different diet regimens.
  • Venn diagrams for genes with fold upregulation ⁇ 2 were created using Venny to identify common genes among all the IRI groups and exclusively diet-specific gene signatures. [00120] All data are expressed as mean ⁇ standard error of the mean (SEM). Comparison between groups was performed by a Mann-Whitney U test. Statistical significance was established at p ⁇ 0.05 with all p-values being two-sided and stratified in the following order: “*” 0.05 to 0.01, “**”0.01 to 0.001, “***” 0.001 to 0.0001, and “****” ⁇ 0.0001. Prism Software (GraphPad, Inc. San Diego) was used for all statistical analyses.
  • mice were presented with a significant accumulation of hepatic steatosis and visceral abdominal fat and accumulation of white adipose tissue (WAT) in the nuchal region.
  • WAT white adipose tissue
  • NAC treatment prevented the development of hepatic steatosis and supported the transformation of WAT into brown adipose tissue (BAT) (FIG.1).
  • hepatic steatosis varied between both ND and ND + NAC when compared to HFD mice, with HFD mice demonstrating 80% hepatic steatosis on average (p ⁇ 0.0001). This difference was significantly reduced in mice on NAC supplementation, which resulted in a 50% reduction in hepatic steatosis (FIG.2).
  • the baseline liver function of the HFD murine model was assessed by performing DCE-MRI with Eovist®. Mice on HFD showed a significant decrease in relative liver enhancement as compared to ND mice (FIG.3).
  • mice from each group were subjected to T2-weighted MRI prior to the terminal procedure to assess the magnitude, distribution, and bilaterality of hepatic edema and associated inflammation, pre-and post-IRI T2 maps were acquired. Specifically, baseline T2 mapping of the liver was performed on ND and HFD, with and without NAC.
  • mice Twenty- four hours after partial hepatic IRI surgery, the mice again underwent T2 mapping. The T2 maps were generated, and the T2 values quantified. Pre-IRI T2 values were similar between ND mice and ND + NAC mice, while the T2 values trended towards a slight increase in the livers of mice on HFD (FIG.4, Panel B). Ischemia-reperfusion injury exposure did not affect the T2 values of either the ND or ND + NAC livers. Conversely, the hepatic T2 values of mice on HFD were noticeably elevated after IRI, while the T2 values of HFD + NAC were comparable to those observed with ND, both before and after IRI.
  • LLS was then evaluated for differences in lipid oxidation between the samples. There was a noticeable increase in LLS in the HFD, as seen by the increasing red color of images, as compared to the ND mice. This fractional intensity of LLS was then quantified (FIG.5, Panel D), as described in the supplemental methods. HFD and HFD IRI mice both demonstrate statistically significant higher quantifications of LLS compared to ND and ND IRI mice, respectively. [00128] Upon establishing that a higher degree of IRI is present on a background of hepatic steatosis, as demonstrated by biochemical, MRI, and glycolytic analysis, hepatic steatosis was quantified more accurately within the samples.
  • Phasor FLIM imaging allowed to quantifiably calculate steatosis based on the accumulation of the LLS.
  • Selection of the phasor signature of LLS (long lifetime species, red circle in FIG.6, Panel A), enabled the identification of the lipid droplets in autofluorescence FLIM images, where they are mapped according to the color of the selection (red here).
  • Steatosis is then quantified by calculating the size of the droplets occupying the image. The data show that droplets are much smaller or are nonexistent in ND diet mice and are much larger in HFD diet mice, both in the IRI and sham samples, which correlates to pathological evaluation of H&E staining.
  • the Y axis is in log scale and shows that exceptionally large droplets are seen in HFD livers in both sham and IRI mice as compared to ND mice.
  • the average size of the lipid droplets increases in both HFD and HFD IRI mice, this is visibly reduced in HFD + NAC and HFD + NAC (IRI) mice (FIG.6, Panels B and C).
  • HFD + NAC and HFD + NAC (IRI) mice FIG.6, Panels B and C.
  • HFD + NAC mice This was significantly decreased in the HFD + NAC mice, as demonstrated in the volcano plots showing differences in upregulated lipid profile in HFD vs ND and HFD + NAC vs HFD mice (FIG.8, Panel B). Interestingly, the HFD + NAC mice had a statistically reduced composition of TAG, as compared to HFD mice (6.281 normalized intensity vs.2.681 normalized intensity, p ⁇ 0.0001; FIG.8, Panel C). There were no statistically significant reductions in the HFD + NAC mice for the remaining 20 classes of lipids.
  • HFD IRI mice exhibited an exclusive upregulation of five genes: TGF ⁇ -2, Pf4 (Platelet Factor 4), IL-1 ⁇ , IL-10, and IFN- ⁇ .
  • HFD + NAC mice that underwent IRI exhibited significantly lower expression levels of the genes that were exclusively upregulated in HFD IRI and shared between ND IRI and HFD IRI mice.
  • Hepatic IRI was then elicited and evaluated for NKT1 cell changes.
  • NKT cells frequencies amongst ND + NAC IRI mice or the ND + NAC sham mice, despite the increased frequencies in ND + NAC mice, as previously demonstrated.
  • the HFD + NAC IRI mice showed no significant differences in NKT cell populations as compared to the HFD + SHAM counterparts; however, the frequency of NKT cells was drastically reduced when compared to both HFD IRI and ND IRI groups.
  • Ex vivo cell stimulation with PMA from samples obtained from all IRI groups was employed to identify the level of IFN- ⁇ produced from CD45+CD3+NK1.1+NKT cells.
  • TAG synthesis is known to be regulated via a PPAR- ⁇ dependent pathway, and previous studies have demonstrated that PPAR- ⁇ activity is downregulated as a result of NAC supplementation.
  • PPAR- ⁇ has also been identified as a key mediator in the synthesis of CD1d, which serves a crucial function in priming of NKT cells. This expands on work that previously demonstrated NKT cell activation occurs secondary to lipid excess, which has been linked to CD1d expression on adipocytes. It has further been demonstrated that NKT cell activation is secondarily decreased in ApoE -/- mice. Further work demonstrated that NAC supplementation can abolish V ⁇ 14iNKT cells and IFN- ⁇ signaling in fulminant liver failure.
  • NAC causes adipose loss in HFD mice, but not ND mice, through enhanced lipid secretion via skin and/or sebaceous glands
  • mice were obtained at six weeks of age from Charles River Laboratories and The Jackson Laboratory, Bar Harbor, ME. Food and water were provided ab libitum in four experimental groups: normal chow diet (ND), ND + NAC, 60% high fat diet (HFD), and HFD + NAC.
  • NAC drinking water (10 gram/liter) was administered to the ND + NAC and HFD + NAC groups following three weeks of ND or HFD and maintained for up to 20 weeks. Animal weights, random blood glucose levels, and food consumption rates as defined by food intake in grams per day and cage were measured weekly.
  • the results demonstrate that NAC treatment caused a substantial and persistent reduction in weight gain in HFD mice when compared to untreated HFD controls (FIG.11).
  • NAC treatment also markedly improved metabolic parameters under HFD conditions as indicated by normalization of random blood glucose levels in HFD NAC animals when compared to HFD and ND controls (FIG.12).
  • NAC treatment did not only prevent but also reverse obesity as demonstrated by weight loss induction in obese HFD B6 mice, which received delayed onset NAC treatment after 16 and 32 weeks on HFD, respectively (FIG.13).
  • NAC treatment was not associated with reduced caloric intake, as the average food consumption rates were comparable between NAC-treated HFD and ND mice and untreated HFD controls (FIG.14).
  • HFD mice treated with NAC developed fatty and greasy hair as early as 1 week after treatment initiation (FIGS.16 and 17), suggesting that NAC treatment causes negative energy balance and adipose loss under HFD conditions through enhanced secretion of fat including TAGs through the skin.
  • hair samples were collected from HFD and HFD + NAC mice for hair lipid isolation and subsequent thin layer chromatography analysis. HFD mice treated with NAC demonstrated significantly increased hair lipid mass in comparison to untreated HFD controls (FIG.18, Panel A).
  • NAC affects sebaceous gland morphology in HFD mice [00145] The impact of NAC on sebaceous gland morphology was investigated in B6 wildtype mice.
  • mice were euthanized and skin samples were collected from their backs. The samples were fixed in formalin, paraffin- embedded, and sectioned into five-micron slices, which were then subjected to hematoxylin and eosin (H&E) staining.
  • H&E hematoxylin and eosin
  • the H&E-stained slides were scanned at 40x magnification using an Aperio GT450 slide scanner, and image analysis was conducted using QuPath software. Sebaceous glands in each skin sample were identified and navigated at magnifications ranging from 10x to 40x, manually annotated, and classified as either transverse (T) or longitudinal (L) based on histological features determined by the sample's orientation and cut. QuPath software measured the area of each annotation, corresponding to sebaceous glands, in square micrometers. [00146] Histological examination of the cutaneous tissue in HFD mice subjected to the NAC treatment demonstrated a reduction in sebaceous gland size in comparison to untreated HFD mice (see FIGS.20 and 21).
  • NAC causes adipose loss in HFD mice through enhanced lipid secretion in the skin across a range of NAC dosing
  • NAC treatment resulted in a significant decrease in white adipose tissue mass in HFD mice, even at lower concentrations of 0.1% and 0.2%, as determined by analysis of inguinal and epididymal fat pad weights (see FIG.24).
  • HFD mice treated with NAC at concentrations of 0.1%, 0.2%, and 1% exhibited fatty and greasy hair as early as 1 week after treatment initiation (see FIG.25), suggesting that NAC treatment causes a negative energy balance and adipose loss under HFD conditions through enhanced lipid secretion through the skin and/or sebaceous glands, even at lower NAC concentrations of 0.1% and 0.2%.
  • NAC treatment causes a negative energy balance and adipose loss under HFD conditions through enhanced lipid secretion through the skin and/or sebaceous glands, even at lower NAC concentrations of 0.1% and 0.2%.
  • hair samples from HFD and HFD + NAC 0.2% mice were collected for hair lipid isolation and subsequent thin layer chromatography (TLC) analysis.
  • HFD mice treated with 0.2% NAC exhibited a significantly increased hair lipid mass in comparison to untreated HFD controls (see FIG.26).
  • Tbet-/-RAG and RAG-deficient mice both of which lack T and B cells, were used to understand the role that adaptive T and B cells and thymic stromal lymphopoietin (TSLP) may play in the effects of NAC in two separate studies.
  • TSLP thymic stromal lymphopoietin
  • HFD Tbet-/-RAG mice treated with NAC displayed similar adipose loss as compared to wildtype mice treated with NAC, as indicated by comparable reduction in weight gain (compare HFD Tbet-/-RAG mice results shown in FIG.27, Panel A with wildtype results shown in FIG.11) and presence of greasy fur changes (compare HFD Tbet- /-RAG mice results shown in FIG.27, Panel B with wildtype results shown in FIGS.16 and 17).

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Abstract

Methods of treating a lipid disorder or skin disorder, of increasing lipid secretion from sebaceous glands, or of reducing adipose tissue in a subject in need thereof, the methods comprising administering to the subject a pharmaceutical composition comprising an effective amount of N-acetylcysteine. The lipid disorder may be fatty liver disease, hyperlipidemia, obesity, or lipodystrophy.

Description

TITLE USE OF N-ACETYLCYSTEINE TO TREAT LIPID DISORDERS AND RELATED ORGAN DYSFUNCTIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/337,603, filed on May 2, 2022, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under AI130800 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION [0003] Lipid disorders—conditions that cause abnormal levels of lipids, or fats, in the blood—continue to be a global health concern. For instance, hypercholesterolaemia, which is the presence of high levels of cholesterol in the blood, is associated with an increased risk of cardiovascular disease, and has increased from being the 15th leading risk factor for death in 1990, to 11th in 2007 and 8th in 2019 (Pirillo et al., 2021). Fatty liver disease, marked by excess fat build-up in the liver, has a worldwide prevalence of 25% (Cotter and Rinella, 2020) and is associated with increased overall mortality, with ranges for the standardized mortality ratio of 1.34-2.6 compared to the general population (Kwak and Kim, 2015)) [0004] Treatments for lipid disorder include the use of medications, such statins and cholesterol absorption inhibitors, but these medications carry risks. For instance, while statins have been proven to be effective in lowering cholesterol levels, they are associated with a variety of side effects such as myopathy, increased risk of diabetes, and myalgia (Mesi et al., 2021). Other methods for treating lipid disorder include the use of supplements (e.g., omega-3 fatty acids, niacin), which vary in efficacy, and lifestyle changes (e.g., healthy diet, exercise), which for many is difficult to maintain. [0005] There is thus a need in the art for novel compositions and/or methods for treating lipid disorders. SUMMARY OF THE INVENTION [0006] The present application is directed to the use of N-acetylcysteine (NAC) to treat lipid disorders and other conditions in a subject. The present invention is based, in part, on a series of important discoveries that are described in more detail in the Examples section of this patent specification. For example, administration of NAC was surprisingly discovered to enhance secretion of lipids through the sebaceous glands and/or skin, as well as reverse weight gain and induce adipose tissue loss in HFD mice. In addition, NAC supported the transformation of white adipose tissue into brown adipose tissue, and significantly reduced the size of sebaceous glands. These results demonstrate that NAC can be used to treat disorders and conditions that can be treated or alleviated by increased lipid secretion and and/or lowering of lipid levels. [0007] Accordingly, in one aspect, the present application is directed to a method of treating a lipid disorder in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In another aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in treating a lipid disorder in a subject in need thereof. [0008] In some embodiments, the lipid disorder may be a triglyceride-related lipid disorder. In certain embodiments, the lipid disorder may be fatty liver disease, hyperlipidemia, obesity, or lipodystrophy. [0009] In another aspect, the present invention is directed to a method of treating a skin disorder in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In yet another aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in treating a skin disorder in a subject in need thereof. [0010] In some embodiments, the skin disorder is selected from sebaceous hyperplasia; sebostatis; age-related skin changes; atopic dermatitis/eczema; dry skin (xerosis); psoriasis; ichthyosis; Sjogren’s syndrome; skin infections; acne inversa; acne vulgaris; seborrheic dermatitis; dry scalp; hair breakage and damage; hair loss disorders; alopecia; dry hair conditions; wound healing; skin complications and conditions in patients with diabetes mellitus; neurodermatitis; chemotherapy-induced skin complications and conditions; and any combination thereof.. [0011] In a further aspect, the present invention is directed to a method of increasing lipid secretion from sebaceous glands, skin, or a combination thereof, in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In yet a further aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in increasing lipid secretion from sebaceous glands in a subject in need thereof. [0012] In some embodiments, the subject has a skin disorder; ageing and/or age-related conditions; obesity and/or obesity-related disorders and conditions; metabolic syndrome; fatty liver disease; dyslipidemias; lipodystrophy; polycystic ovary syndrome (PCOS); diabetes; cardiovascular disease; kidney disease; wound healing; or inflammation or autoimmune condition. [0013] In one aspect, the present invention is directed to a method of reducing adipose tissue in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In a further aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in reducing adipose tissue in a subject in need thereof. [0014] In some embodiments, the adipose tissue is white adipose tissue. In certain embodiments, the adipose tissue is epididymal adipose tissue, inguinal adipose tissue, or a combination thereof. [0015] In another aspect, the present invention is directed to a method of inhibiting diet- related weight gain in a subject in need thereof. The method comprises administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In a further aspect, the present invention is directed to a pharmaceutical composition comprising an effective amount of NAC for use in inhibiting diet-related weight gain in a subject in need thereof. [0016] In some embodiments, the diet-related weight gain is due to a high-fat diet. [0017] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier. [0018] In some embodiments, the pharmaceutical composition is administered orally. In certain embodiments, the pharmaceutical composition is administered in an amount of about 1 to 200 mg/kg/day. In preferred embodiments, the pharmaceutical composition is administered in an amount of about 5 to 100 mg/kg/day. [0019] In some embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, the pharmaceutical composition is administered in an amount of about 25 to 400 mg/kg/day. In preferred embodiments, the pharmaceutical composition is administered in an amount of about 50 to 300 mg/kg/day. [0020] In some embodiments, the pharmaceutical composition comprises an aqueous solution. In certain embodiments, the pharmaceutical composition comprises NAC in a concentration of about 0.5 to 10 g/L. In preferred embodiments, the pharmaceutical composition comprises NAC in a concentration of about 1 to 5 g/L. [0021] These and other aspects of the present invention are described further in the below Detailed Description, Drawings, Examples and Claims sections of this patent disclosure. Furthermore, one of skill in the art will recognize that the various embodiments of the present invention described throughout this patent disclosure can be combined in various different ways, and that such combinations are within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS [0022] FIG.1 shows T1-weighted magnetic resonance imaging (MRI) of mice in vivo, as described in Example 1 Panels A and B show hepatic steatosis. Regions of interest (ROI) were localized on the liver of each individual animal on at least three slices, and their mean intensity was measured and averaged to quantify hepatic fat accumulation. This demonstrated that mice fed a high-fat diet (HFD) presented with a an ~27% increase in liver contrast, as compared to mice fed a normal diet (ND). In Panels C and D, abdominal fat contained in the entire dataset was quantified by thresholding with ImageJ software. Exposure to HFD caused a marked increase in abdominal fat to ~47% of the total ROI voxels, as compared with ND demonstrating ~18%. In Panels E and F, nuchal fat content was measured to assess metabolic and thermogenic capacity. HFD exposure resulted in the marked transformation of the brown adipose tissue (BAT) into white adipose tissue (WAT), as represented by the whitening of the nuchal adipose tissue. [0023] FIG.2 shows hematoxylin and eosin (H&E) analysis of liver tissue, as described in Example 1. Panel A shows H&E sections demonstrating the degree of hepatic steatosis. Panel B shows a histogram corresponding to the percentage of hepatic steatosis in ischemia- reperfusion injury (IRI) specimens. Overall, hepatic steatosis varied greatly between both ND and normal-fed mice supplemented with NAC (ND + NAC) when compared to HFD mice, with HFD mice demonstrating 80% hepatic steatosis on average (p<0.0001). This difference was significantly reduced in HFD mice on NAC supplementation, which allowed for a 50%. [0024] FIG.3 shows the effects of HFD on liver function as assessed by DCE-MRI with the contrast agent Gadoxetate Disodium (Eovist® Bayer), as described in Example 1. Panel A shows representative images of 26-30 week old male mice undergoing dynamic contrast- enhanced MRI (DCE-MRI), showing T1-weighted images performed before Eovist® injection and at 4, 12 and 20 minutes post-injection (P-I). The circle denotes a typical region of interest used to measure mean intensity. Panel B shows time course of relative liver enhanced T1 signal intensities after Eovist® injection, comparing mice on ND, ND + NAC, HFD, and HFD mice supplemented with NAC (HFD + NAC). Panel C shows area under the curve (AUC) calculated from the data in Panel B. Panel D shows the slope of T1 signal intensity increased for each group shown, corresponding to the rates of change within the first 20 min after Eovist® injection. [0025] FIG.4 shows that the effects of IRI and NAC supplementation on HDF mice, as described in Example 1. HFD mice are prone to more severe IRI, which is significantly improved with NAC supplementation. Panel A shows serum alanine aminotransferase (ALT) level following 24 hours of reperfusion. Panel B shows representative images of baseline T2 mapping of mice on (i) ND, (iii), ND + NAC, (v) HFD, and (vii) HFD + NAC. Representative images of T2 mapping twenty-four hours after IRI surgery of (ii) ND, (iv) ND + NAC, (vi) HFD, and (viii) HFD + NAC. Blue represents liver parenchyma while green denotes edema. Graphical representation (xi) of the average of the T2 relaxation times of ROIs placed on the right and left areas of the corresponding livers. These results show that HFD mice were prone to more severe IRI, which is significantly improved with NAC supplementation. [0026] FIG.5 shows IRI results in a higher degree of glycolytic stress in HFD mice, as described in Example 1. Panel A shows color mapped Phasor Fluorescence Lifetime Imaging (FLIM) images of the ND and HFD liver samples in the absence (left) and presence (right) of NAC supplementation. The top row shows the sham samples, and the bottom row shows the IRI samples. More green, blue, and red color represents more protein-bound NADH, free NADH and long lifetime species (LLS), respectively. Panel B shows Phasor plot of autofluorescence FLIM in liver samples—the three circles represent the lifetime position of the three components: free and bound NADH, and LLS. Panel C shows the concentration ratio of free to bound NADH was calculated from each mouse. The higher value represents more glycolytic stress. There was a higher level of glycolytic stress in HFD and HFD IRI samples. This was reduced in the HFD + NAC IRI samples, but not the HFD + NAC samples, respective to their HFD counterparts. Panel D shows the fractional intensity of LLS calculated using three component calculations to show the increased LLS in high fat liver and how that increased with IRI. [0027] FIG.6 shows effects of NAC treatment on hepatic steatosis in HFD mice, as described in Example 1. Panel A shows the fluorescence intensity image, the LLS mapped image, the calculated size distribution maps, and the corresponding phasor plot. The cursor was used to select the LLS lifetime signature originating from the intensity image and the LLS image was colored according to that phasor signature. The size map was created to show the distribution of the droplets. Panels B and C show the size distribution plots for control (Panel B) and NAC treatment (Panel C). The distribution shows HFD and IRI induced the large lipid droplets and (especially above 10000 pixels) were lower in number in HFD + NAC groups. Panel D shows the average size distribution of the animals from each feeding regiment. These results show that NAC treatment significantly reduced hepatic steatosis in HFD mice. [0028] FIG.7 shows spatial heterogeneity analysis of phasor-FLIM imaging, as described in Example 1. The analysis was based on quantification of components of phasor plot and their fractional intensity, which allowed for comparison between sample to sample and between different sets of samples. However, calculation of an average value from the total image resulted in loss of the dimensional information and the inherent heterogeneity that these maps can show. The actual distribution of the fractional intensity of LLS and free NADH show how there was embedded spatial information if phasor-FLIM autofluorescence images (Panel A). The composite image of LLS, free and bound NADH (Panel B) and the individual species distribution of LLS, bound NADH, free NADH, and free-bound NADH (Panels C-E, respectively) show how different parts of the image had differential amount of these species. Panel F shows the free and bound NADH map with higher blue and green representing more free and more bound NADH, respectively, and how some areas were more glycolytic than others—information that was harder to see in three color images. The cumulative graphs from 3 samples (Panels F and G) show that along with a shift of LLS and glycolysis in HFD- IRI samples there could be broadening of these distributions. In case of broadening, e.g., HFD-NAC vs ND in Panel G—the central position of the distribution may not shift a lot, there was more metabolic heterogeneity in HFD-NAC samples than ND diet. Observation of the spatial difference was inherent to the imaging methods and multicomponent analysis of phasor-FLIM allowed for a quantitative map of these species. [0029] FIG.8 shows the effects of NAC treatment on the lipid profile within the liver of HFD mice. Panel A shows a generated heat map demonstrating the alterations in lipid composition with the respective tissue. Panel B shows volcano plots demonstrating the differences in upregulated lipid species in HFD vs ND and HFD vs HFD + NAC mice, ultimately revealing changes triacylglycerol (TAG) composition within these tissues. Panel C shows a histogram representing quantitative differences in TAG composition between the treatment groups. These results show that NAC treatment in HFD mice significantly altered the lipid profile within the liver. [0030] FIG.9 shows the effects of IRI on gene expression in HFD mice, as described in Example 1. IRI induced specific genetic upregulation in HFD mice. Panel A shows a heat map of cytokine and chemokine PCR analysis. Panel B shows a Venn Diagram demonstrating genes that were upregulated in HFD IRI and those shared amongst ND IRI and HFD IRI. These results show that IRI induced specific genetic upregulation in HFD mice. [0031] FIG.10 shows role of IFN-γ producing hepatic natural killer T (NKT) cells following IRI, as described in Example 1. Panel A shows representative contour plots of the percentage of CD45+ /CD3+ /NK1.1+ NKT1 cells within hepatic mononuclear cells. Panel B shows representative contour plots of the percentage of IFN-γ producing CD45+ /CD3+ /NK1.1+ NKT cells. Panel C shows a histogram demonstrating the percentage of NKT1 cells illustrated in Panel A. Panel D shows a histogram demonstrating the percentage of NKT1 cells illustrated in Panel B. These results show that IFN-γ producing hepatic NKT cells were key contributors following IRI and successfully ameliorated with NAC supplementation. [0032] FIG.11 shows changes in bodyweight over time of B6 wildtype ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 2. The results show that NAC treatment protected B6 wildtype mice from high HFD-induced obesity when compared to untreated HFD controls, as shown by substantial reduction in weight gain. [0033] FIG.12 shows blood glucose levels over time post-HFD start date of B6 wildtype ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 2. The results show that NAC treatment protected B6 wildtype HFD mice from obesity-related metabolic complications, as shown by normalization of random blood glucose levels. [0034] FIG.13 shows changes in bodyweight over time of B6 wildtype HFD and HFD + NAC mice, as described in Example 2. Body weight is shown with NAC treatment started at16 weeks post HFD (Panel A), and started 32 weeks post HFD (Panel B). The results show that NAC treatment reversed HFD-induced obesity in B6 wildtype mice when compared to untreated HFD controls, as shown by weight loss induction. [0035] FIG.14 shows consumption rate of B6 wildtype ND + NAC, HFD, and HFD + NAC mice, as described in Example 2. The results show that HFD B6 wildtype mice treated with NAC had comparable caloric intake to HFD controls as shown by similar food consumption rates. [0036] FIG.15 shows triacylglycerols (TAG) levels in HFD + NAC and HFD B6 wildtype mice, as described in Example 2. Panel A shows a heat map and Panel B shows a volcano plot comparing 277 TAGs in HFD + NAC versus HFD controls, in which significantly decreased TAGs (149 in total) are marked as blue dots, and others (128) are marked in grey. The results show that HFD B6 wildtype mice treated with NAC show significant reduction in TAG compared to untreated HFD controls. [0037] FIG.16 shows images of HFD B6 wildtype ND (Panel A), ND + NAC (Panel B), HFD (Panel C), and HFD + NAC (Panel D) mice, as described in Example 2. The images show that HFD B6 wildtype mice treated with NAC develop fatty and “greasy” hair, which suggested that they lost weight through secreting fat (TAGs) via the skin. [0038] FIG.17 shows an image of HFD B6 wildtype HFD + NAC mouse, as described in Example 2. The image shows that HFD B6 wildtype mice treated with NAC possessed fatty and “greasy” fur, which suggested that they lost weight through secreting fat (TAGs) via the skin. [0039] FIG.18 shows analysis of the fur of HFD + NAC and HFD mice, as described in Example 2. Panel A shows quantity of hair lipids in HFD + NAC and HFD mice. Panel B shows HFD lipid composition and quantification via thin-layer chromatography. The results show that the hair of HFD B6 wildtype mice treated with NAC contained significantly higher lipid mass and TAG levels than HFD controls, confirming that NAC caused adipose loss through enhanced lipid secretion through the skin. [n = 8; p < 0.05] [0040] FIG.19 shows analysis of body weight and fur of ND + NAC and ND mice, as described in Example 2. Panel A shows body weight (in grams) of ND + NAC and ND mice, in which no statistically significant difference in body weight was observed between the groups (p=0.63). Panel B shows fur lipid weights of ND + NAC and ND mice, wherein no statistically significant difference in fur lipid weight was found between the groups (p=0.53). Panel C shows ND lipid composition and quantification via thin-layer chromatography, wherein the hair of ND mice treated with NAC contained significantly lower TAG levels than ND mice (p<0.05). [Data are expressed as mean ± SEM, and statistical analysis was conducted using the Mann-Whitney test. Error bars represent SEM.] [0041] FIG.20 shows longitudinal (Panel A) and transverse (Panel B) sebaceous gland area of ND, ND + NAC, HFD, and HFD + NAC mice, as described in Example 3. In HFD mice, NAC treatment significantly reduced both longitudinal and transverse sebaceous gland area as compared to HFD control. [Statistical analysis was conducted using the Mann-Whitney test.] [0042] FIG.21 show examples images of longitudinal and transverse cross-sections of sebaceous glands (Panel A), representative images of longitudinal and transverse cross- sections of sebaceous glands in HFD and HFD + NAC (1%) mice (Panel B), and representative images of longitudinal and transverse cross-sections of sebaceous glands in ND and ND + NAC (1%) mice (Panel C), as described in Example 3. As shown in Panel A, the sebaceous glands in longitudinal cross-section views resemble a water or tear drop shape and are adjacent and follow the follicle length, while the sebaceous glands in transverse cross-section view resemble a “ bean” shape and often appear as wrapping around the hair bulb. The size difference between the HFD and HFD + NAC group are apparent in the longitudinal cross-section views of the sebaceous glands in Panel B. [0043] FIG.22 shows body weight (Panel A) and percent change in body weight from baseline (Panel B) of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%) mice, as described in Example 4. In HFD mice, the effect of NAC treatment on body weight was greater at higher NAC doses. [Data are expressed as mean ± SEM, and statistical analysis was conducted using the Mann-Whitney test.] [0044] FIG.23 shows food consumption of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%), as described in Example 4. [0045] FIG.24 shows epididymal fat pad weight (Panel A) and inguinal fat pad weight (Panel B) of ND, ND + NAC (0.1%), ND + NAC (1%), HFD, HFD + NAC (0.1%), HFD + NAC (0.2%), and HFD + NAC (1%) mice, as described in Example 4. In HFD mice, NAC treatment reduced both epididymal and inguinal fat pad weight, with NAC dosing amount appearing to have an impact only on the epididymal fat pad weight. [0046] FIG.25 shows images of ND (Panel A), ND + NAC (0.1%) (Panel B), ND + NAC (1%) (Panel C), HFD (Panel D), HFD + NAC (0.1%) (Panel E), HFD + NAC (0.2%) (Panel F), and HFD + NAC (1%) (Panel G) mice after one week of treatment, as described in Example 4. [0047] FIG.26 shows analysis of fur of HFD and HFD + NAC (0.2%) mice, as described in Example 4. The analysis compares lipid weight (Panel A), free cholesterol (Panel B), free fatty acids (Panel C), TAG (Panel D), wax ester (Panel E), and cholesterol ester (Panel F) between the fur of HFD mice and the further of HFD + NAC (0.2%) mice. These results show that, compared to HFD mice, HFD + NAC 0.2% mice possessed significantly higher fur lipid weight; displayed trends of higher fur lipid densities for free cholesterol, free fatty acid, and wax ester; and showed significantly higher TAG and cholesterol ester levels. [0048] FIG.27 shows effects of NAC treatment on Tbet-/-RAG mice, as described in Example 5. Panel A shows the body weight of Tbet-/-RAG ND, HFD, and HFD + NAC mice over time, and Panel B shows images of the mice. The results show that HFD Tbet-/- RAG mice treated with NAC show adipose loss, comparable to wildtype mice treated with NAC. [0049] FIG.28 shows effects of NAC treatment on RAG-deficient (or “RAG1”) mice in regard to body weight (Panel A), food consumption (Panel B), and blood glucose levels (Panel C), as described in Example 5. Compared to HFD RAG-deficient mice, the HFD RAG-deficient mice treated with NAC exhibit reduced body weight and blood glucose, but similar food consumption rates. [0050] FIG.29 further shows effects of NAC treatment on RAG-deficient (or “RAG1”) mice as described in Example 5. Weights of epididymal (Panel A) and inguinal (Panel B) fat pads of HFD and HFD + NAC RAG-deficient mice, as well as images of the HFD (Panel C) and HFD + NAC (Panel D) RAG-deficient mice, are displayed. The results show that the HFD + NAC RAG-deficient mice possessed reduced epididymal and inguinal fat pads as compared to the HFD RAG-deficient mice. DETAILED DESCRIPTION OF THE INVENTION [0051] The present application is directed to the use of N-acetylcysteine to treat lipid disorders and other conditions in a subject. [0052] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, and molecular biology, which are within the skill of the art. [0053] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. [0054] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0055] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art. Definitions [0056] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [0057] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably. [0058] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). [0059] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included. [0060] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10. [0061] The terms “inhibit,” “reduce,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule. [0062] The terms “induce,” “increase,” and “stimulate” are used interchangeably and refer to any statistically significant increase in occurrence or activity, including full blocking of the occurrence or activity. For example, “stimulate” can refer to an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. A “stimulator” is a molecule, factor, or substance that produces a statistically significant increase in the occurrence or activity of a process, pathway, or molecule. [0063] As used herein, “quantify” or “quantity” refers to the measurement of the amount of a component in a sample, and is typically calculated as a concentration such as a mass concentration (mass of the antibody divided by volume of the sample). Generally, quantity is determined in a sample that is collected from a subject. Methods of Treatment [0064] The present invention provides various methods of treatment. As used herein, the terms “treat,” “treating,” and “treatment” refer to curing, slowing down, lessening symptoms of, and/or halting progression of a diagnosed pathologic disease, condition, or disorder. For example, such terms include, but are not limited to, lessening symptoms of a disease, condition, or disorder; slowing down the worsening of symptoms of a disease, condition, or disorder; slowing down and/or halting the progression of a disease, condition, or disorder; causing regression of a disease, condition, or disorder; and the like. In certain embodiments, a subject is successfully “treated” for a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder. [0065] Thus, in one aspect, the present invention provides a method of treating a lipid disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of NAC. [0066] In some embodiments, the lipid disorder may be a triglyceride-related lipid disorder. Examples of lipid disorders that can be treated include, but are not limited to, fatty liver disease, hyperlipidemia, obesity, and lipodystrophy. [0067] In one aspect, the present invention provides a method of treating a skin disorder or condition in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject. Examples of skin disorders or conditions that can be treated include, but are not limited to, sebaceous hyperplasia; sebostatis; age-related skin changes such as aging skin and wrinkles; atopic dermatitis/eczema, such as dermatitis due to dry skin; dry skin (xerosis); psoriasis; ichthyosis; Sjogren’s syndrome; skin infections; acne inversa; acne vulgaris; dermatitis due to dry skin; dry scalp; hair breakage and damage; hair loss disorders; alopecia, such as traction alopecia; dry hair conditions; wound healing; skin complications and conditions in patients with diabetes mellitus; neurodermatitis; chemotherapy-induced skin complications and conditions; and any combination thereof. [0068] In another aspect, the present invention provides a method of increasing lipid secretion from sebaceous glands and/or from skin in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In some embodiments, the lipid secretion comprises triacylglycerols. In some embodiments, the subject may have a condition or disorder selected from the group consisting of a skin disorder or condition as described herein; ageing and/or age-related condition; obesity and/or obesity-related disorders or conditions, including an obesity-related cancer; metabolic syndrome; fatty liver disease, such as nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH); dyslipidemia; lipodystrophy; PCOS; diabetes mellitus type I or II; cardiovascular disease, such as coronary heart disease or stroke; kidney disease; wound healing; and inflammation and/or autoimmune condition. In some embodiments, the subject may have a need for support as a prosthetic device use. [0069] In another aspect, the present invention provides a method of reducing fat mass in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject. As used herein, “fat mass” is an indicator of the quantity of adipose tissue in a body or a portion of a body. Fat mass may be calculated as a weight, e.g., the weight of the adipose tissue in the body or a portion of the body; or may be calculated as a percentage, for example, a percentage of the weight of the body or portion of the body that constitutes adipose tissue. Methods of measuring fat mass as a percentage are known in the art, for example, by using skin calipers, body circumference measurements, body fat scales, waist circumference, dual-energy X-ray absorptiometry, hydrodensitometry, air displacement plethysmography, three-dimensional body scanner, etc. [0070] In yet another aspect, the present invention provides a method of reducing adipose tissue in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject. In some embodiments, the adipose tissue is white adipose tissue. In some embodiments, the adipose tissue is epididymal adipose tissue, inguinal adipose tissue, or a combination thereof. [0071] In a further aspect, the present provides a method of inhibiting diet-related weight gain in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of NAC to the subject. As used herein, the term “diet-related weight gain” refers to an increase weight that is primarily due to diet, as opposed to a disorder or condition such as high insulin levels or insulin resistance, leptin resistance, use of medications that can cause weight gain, hyperthyroidism, Cushing’s syndrome, etc. [0072] In some embodiments, the diet-related weight gain is due to a high-fat diet. As used herein, “high-fat diet” or “HFD” refers to a diet in which the amount of diet in the diet is greater than recommended for a healthy diet, for example, greater than recommended by Food and Nutrition Board of the National Academies of Sciences Engineering, and Medicine, or by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture. In some embodiments, a high-fat diet may be a diet in which at least 35% of total calories is consumed from fats, both unsaturated and saturated. [0073] As used herein the term “subject” encompasses all mammalian species, including, but not limited to, humans, non-human primates, dogs, cats, rodents (such as rats, mice and guinea pigs), cows, pigs, sheep, goats, horses, and the like – including all mammalian animal species used in animal husbandry, as well as animals kept as pets and in zoos, etc. In some embodiments the subjects are human. [0074] The efficacy of a given composition or method in treatment can be demonstrated or assessed using standard methods known in the art, such as methods that compare the efficacy of a given / “test” composition or method to a “control” composition or method. For example, the efficacy of a given composition or method in treating a lipid disorder may be demonstrated or assessed by comparing its ability to improve one or more clinical indicators or symptoms of the lipid disorder as compared to that of a control composition or control method, such as a placebo control. For instance, a comparison can be made between different subjects (e.g., between a test group of subjects or a control group of subjects). Similarly, the efficacy of a given composition or method in treatment can be demonstrated or assessed in a single subject by comparing one or more clinical indicators or symptoms of the lipid disorder in the subject before and after treatment. [0075] In carrying out the treatment methods described herein, any suitable method or route of administration can be used to deliver the active agents or combinations thereof described herein. The term “administration” includes any route of introducing or delivering the specified compositions or agents to subjects. In some embodiments the active agents or combinations thereof, are administered systemically. In some embodiments the active agents or combinations thereof, are administered locally. “Systemic administration” refers to introducing or delivering to a subject a specified composition or agent via a route which introduces or delivers the composition or agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to introducing or delivering to a subject a specified composition or agents via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration, but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. [0076] In some embodiments, administration can be carried out by any suitable route known in the art, including intratumoral, intravenous, subcutaneous, oral, topical, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. Administration includes self-administration and administration by another. The suitability of a given route or means of administration can be readily determined by a physician. In preferred embodiments, administration is oral. [0077] As used herein the term “effective amount” refers to an amount of NAC that is sufficient to achieve, or contribute towards achieving, one or more of the outcomes listed in the “treatment” description herein. An appropriate “effective” amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the desired route of administration (e.g., systemic vs. local), the desired frequency of dosing, etc. Furthermore, an “effective amount” may be determined in the context of any co-administration to be used. One of skill in the art can readily perform such dosing studies (whether using single agents or combinations of agents) to determine appropriate doses to use, for example using assays such as those described in the Examples section of this patent application—which involve administration of the agents described herein to subjects—such as animal subjects routinely used in the pharmaceutical sciences for performing dosing studies. [0078] For example, in some embodiments the dose of NAC may be calculated based on studies in humans or other mammals carried out to determine efficacy and/or effective amounts of NAC. The dose amount and frequency or timing of administration may be determined by methods known in the art and may depend on factors such as the pharmaceutical form, route of administration, whether it is used in combination with other active agents (for example, the dosage of NAC required may be lower when i is used in combination with another active agent), and patient characteristics including age, body weight, or the presence of any medical conditions affecting drug metabolism. [0079] In those embodiments described herein that refer to specific doses of NAC to be administered based on mouse studies, one of skill in the art can readily determine comparable doses for human studies based on the mouse doses, for example using the types of dosing studies and calculations described herein. [0080] In some embodiments suitable doses of NAC described herein can be determined by performing dosing studies of the type that are standard in the art, such as dose escalation studies, for example using the dosages shown to be effective in mice in the Examples section of this patent application as a starting point. [0081] Dosing regimens can also be adjusted and optimized by performing studies of the type that are standard in the art, for example using the dosing regimens shown to be effective in mice in the Examples section of this patent application as a starting point. In some embodiments the active agents are administered daily, or twice per week, or weekly, or every two weeks, or monthly. [0082] In some embodiments, the pharmaceutical composition may be administered orally. In certain embodiments, the pharmaceutical composition may be administered in an amount of about 1 to 200 mg/kg/day or any sub-range therebetween, for example, about 1 to 190 mg/kg/day, or about 1 to 180 mg/kg/day, or about 1 to 170 mg/kg/day, or about 1 to 160 mg/kg/day, or about 1 to 150 mg/kg/day, or about 1 to 140 mg/kg/day, or about 1 to 130 mg/kg/day, or about 1 to 120 mg/kg/day, or about 1 to 110 mg/kg/day, or about 1 to 100 mg/kg/day, or about 5 to 200 mg/kg/day, or about 5 to 190 mg/kg/day, or about 5 to 180 mg/kg/day, or about 5 to 170 mg/kg/day, or about 5 to 160 mg/kg/day, or about 5 to 150 mg/kg/day, or about 5 to 140 mg/kg/day, or about 5 to 130 mg/kg/day, or about 5 to 120 mg/kg/day, or about 5 to 110 mg/kg/day, or about 5 to 100 mg/kg/day, or about 10 to 200 mg/kg/day, or about 10 to 190 mg/kg/day, or about 10 to 180 mg/kg/day, or about 10 to 170 mg/kg/day, or about 10 to 160 mg/kg/day, or about 10 to 150 mg/kg/day, or about 10 to 140 mg/kg/day, or about 10 to 130 mg/kg/day, or about 10 to 120 mg/kg/day, or about 10 to 110 mg/kg/day, or about 10 to 100 mg/kg/day, or about 15 to 200 mg/kg/day, or about 15 to 190 mg/kg/day, or about 15 to 180 mg/kg/day, or about 15 to 170 mg/kg/day, or about 15 to 160 mg/kg/day, or about 15 to 150 mg/kg/day, or about 15 to 140 mg/kg/day, or about 15 to 130 mg/kg/day, or about 15 to 120 mg/kg/day, or about 15 to 110 mg/kg/day, or about 15 to 100 mg/kg/day, or about 20 to 200 mg/kg/day, or about 20 to 190 mg/kg/day, or about 20 to 180 mg/kg/day, or about 20 to 170 mg/kg/day, or about 20 to 160 mg/kg/day, or about 20 to 150 mg/kg/day, or about 20 to 140 mg/kg/day, or about 20 to 130 mg/kg/day, or about 20 to 120 mg/kg/day, or about 20 to 110 mg/kg/day, or about 20 to 100 mg/kg/day, or about 25 to 200 mg/kg/day, or about 25 to 190 mg/kg/day, or about 25 to 180 mg/kg/day, or about 25 to 170 mg/kg/day, or about 25 to 160 mg/kg/day, or about 25 to 150 mg/kg/day, or about 25 to 140 mg/kg/day, or about 25 to 130 mg/kg/day, or about 25 to 120 mg/kg/day, or about 25 to 110 mg/kg/day, or about 25 to 100 mg/kg/day, or about 30 to 200 mg/kg/day, or about 30 to 190 mg/kg/day, or about 30 to 180 mg/kg/day, or about 30 to 170 mg/kg/day, or about 30 to 160 mg/kg/day, or about 30 to 150 mg/kg/day, or about 30 to 140 mg/kg/day, or about 30 to 130 mg/kg/day, or about 30 to 120 mg/kg/day, or about 30 to 110 mg/kg/day, or about 30 to 100 mg/kg/day, or about 40 to 200 mg/kg/day, or about 40 to 190 mg/kg/day, or about 40 to 180 mg/kg/day, or about 40 to 170 mg/kg/day, or about 40 to 160 mg/kg/day, or about 40 to 150 mg/kg/day, or about 40 to 140 mg/kg/day, or about 40 to 130 mg/kg/day, or about 40 to 120 mg/kg/day, or about 40 to 110 mg/kg/day, or about 40 to 100 mg/kg/day, or about 50 to 200 mg/kg/day, or about 50 to 190 mg/kg/day, or about 50 to 180 mg/kg/day, or about 50 to 170 mg/kg/day, or about 50 to 160 mg/kg/day, or about 50 to 150 mg/kg/day, or about 50 to 140 mg/kg/day, or about 50 to 130 mg/kg/day, or about 50 to 120 mg/kg/day, or about 50 to 110 mg/kg/day, or about 50 to 100 mg/kg/day; or any dosage therebetween, such as 1 mg/kg/day, or about 5 mg/kg/day, or about 10 mg/kg/day, or about 15 mg/kg/day, or about 20 mg/kg/day, or about 25 mg/kg/day, or about 30 mg/kg/day, or about 35 mg/kg/day, or about 40 mg/kg/day, or about 45 mg/kg/day, or about 50 mg/kg/day, or about 60 mg/kg/day, or about 70 mg/kg/day, or about 80 mg/kg/day, or about 90 mg/kg/day, or about 100 mg/kg/day, or about 110 mg/kg/day, or about 120 mg/kg/day, or about 130 mg/kg/day, or about 140 mg/kg/day, or about 150 mg/kg/day, or about 160 mg/kg/day, or about 170 mg/kg/day, or about 180 mg/kg/day, or about 190 mg/kg/day, or about 200 mg/kg/day. [0083] In some embodiments, the pharmaceutical composition may be administered intravenously. In certain embodiments, the pharmaceutical composition may be administered in an amount of about 25 to 400 mg/kg/day or any sub-range therebetween, for example, about 25 to 390 mg/kg/day, or about 25 to 380 mg/kg/day, or about 25 to 370 mg/kg/day, or about 25 to 360 mg/kg/day, or about 25 to 350 mg/kg/day, or about 25 to 340 mg/kg/day, or about 25 to 330 mg/kg/day, or about 25 to 320 mg/kg/day, or about 25 to 310 mg/kg/day, or about 25 to 300 mg/kg/day, or about 30 to 400 mg/kg/day, or about 30 to 390 mg/kg/day, or about 30 to 380 mg/kg/day, or about 30 to 370 mg/kg/day, or about 30 to 360 mg/kg/day, or about 30 to 350 mg/kg/day, or about 30 to 340 mg/kg/day, or about 30 to 330 mg/kg/day, or about 30 to 320 mg/kg/day, or about 30 to 310 mg/kg/day, or about 30 to 300 mg/kg/day, or about 40 to 400 mg/kg/day, or about 40 to 390 mg/kg/day, or about 40 to 380 mg/kg/day, or about 40 to 370 mg/kg/day, or about 40 to 360 mg/kg/day, or about 40 to 350 mg/kg/day, or about 40 to 340 mg/kg/day, or about 40 to 330 mg/kg/day, or about 40 to 320 mg/kg/day, or about 40 to 310 mg/kg/day, or about 40 to 300 mg/kg/day, or about 50 to 400 mg/kg/day, or about 50 to 390 mg/kg/day, or about 50 to 380 mg/kg/day, or about 50 to 370 mg/kg/day, or about 50 to 360 mg/kg/day, or about 50 to 350 mg/kg/day, or about 50 to 340 mg/kg/day, or about 50 to 330 mg/kg/day, or about 50 to 320 mg/kg/day, or about 50 to 310 mg/kg/day, or about 50 to 300 mg/kg/day, or about 60 to 400 mg/kg/day, or about 60 to 390 mg/kg/day, or about 60 to 380 mg/kg/day, or about 60 to 370 mg/kg/day, or about 60 to 360 mg/kg/day, or about 60 to 350 mg/kg/day, or about 60 to 340 mg/kg/day, or about 60 to 330 mg/kg/day, or about 60 to 320 mg/kg/day, or about 60 to 310 mg/kg/day, or about 60 to 300 mg/kg/day, or about 80 to 400 mg/kg/day, or about 80 to 390 mg/kg/day, or about 80 to 380 mg/kg/day, or about 80 to 370 mg/kg/day, or about 80 to 360 mg/kg/day, or about 80 to 350 mg/kg/day, or about 80 to 340 mg/kg/day, or about 80 to 330 mg/kg/day, or about 80 to 320 mg/kg/day, or about 80 to 310 mg/kg/day, or about 80 to 300 mg/kg/day, or about 100 to 400 mg/kg/day, or about 100 to 390 mg/kg/day, or about 100 to 380 mg/kg/day, or about 100 to 370 mg/kg/day, or about 100 to 360 mg/kg/day, or about 100 to 350 mg/kg/day, or about 100 to 340 mg/kg/day, or about 100 to 330 mg/kg/day, or about 100 to 320 mg/kg/day, or about 100 to 310 mg/kg/day, or about 100 to 300 mg/kg/day; or any dosage therebetween, such as 25 mg/kg/day, or about 30 mg/kg/day, or about 35 mg/kg/day, or about 40 mg/kg/day, or about 45 mg/kg/day, or about 50 mg/kg/day, or about 60 mg/kg/day, or about 70 mg/kg/day, or about 80 mg/kg/day, or about 90 mg/kg/day, or about 100 mg/kg/day, or about 110 mg/kg/day, or about 120 mg/kg/day, or about 130 mg/kg/day, or about 140 mg/kg/day, or about 150 mg/kg/day, or about 160 mg/kg/day, or about 170 mg/kg/day, or about 180 mg/kg/day, or about 190 mg/kg/day, or about 200 mg/kg/day, or about 210 mg/kg/day, or about 220 mg/kg/day, or about 230 mg/kg/day, or about 240 mg/kg/day, or about 250 mg/kg/day, or about 260 mg/kg/day, or about 270 mg/kg/day, or about 280 mg/kg/day, or about 290 mg/kg/day, or about 300 mg/kg/day, or about 310 mg/kg/day, or about 320 mg/kg/day, or about 330 mg/kg/day, or about 340 mg/kg/day, or about 350 mg/kg/day, or about 360 mg/kg/day, or about 370 mg/kg/day, or about 380 mg/kg/day, or about 390 mg/kg/day, or about 400 mg/kg/day. [0084] In certain embodiments the compositions and methods provided herein may be employed together with other compositions and treatment methods known to be useful for treatment of lipid disorders or skin disorders, reducing adipose tissue, inhibiting diet-related weight gain, etc., including, but not limited to, pharmaceuticals (e.g., statins, cholesterol absorption inhibitors), surgical methods (e.g., adjustable gastric banding, gastric bypass surgery, sleeve gastrectomy), and the like, as well as other non-pharmaceutical and non- invasive methods such as dietary changes and exercise/activity. Similarly, in certain embodiments the methods of treatment provided herein may be employed together with procedures used to monitor progression of lipid or skin disorders, adipose tissue reduction, weight gain, etc. [0085] For example, in some embodiments, the methods described herein and/or the compositions described herein may be employed or administered to a subject prior to surgery, for instance, in order to reduce adipose tissue or prevent weight gain if either is too high for gastric bypass surgery. In other embodiments the methods described herein and/or the compositions described herein may be employed or administered to a subject both before and after performing surgery. NAC Pharmaceutical Compositions [0086] In embodiments of the invention, NAC may be formulated in a pharmaceutical composition. The pharmaceutical composition may comprise one or more carriers, diluents, excipients, or other additives. Carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Sterile water can be a vehicle when the compound of the invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. [0087] As further examples, the composition can comprise one or more stabilizing agents (e.g., dextran 40, glycine, lactose, mannitol, trehalose, maltose), one or more buffers (e.g., acetate, citrate, histidine, lactate, phosphate, Tris), one or more pH adjusting agents (e.g., hydrochloric acid, nitric acid, potassium hydroxide, sodium hydroxide), one or more surfactants (polysorbate, sodium lauryl sulfate, polyethylene glycol-fatty acid esters, lecithins), and/or one or more diluents (e.g., water, physiological saline). The composition may also include excipients such as starch, glucose, lactose, methyl cellulose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, or solubilizers (e.g., sulfobutylether b- cyclodextrin). The pH of the composition is preferably between about 3.0 and 8.0. In certain embodiments, the pH is between about 4.0 and 7.0, or between about 5.0 and 6.5. [0088] The present formulations can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. [0089] In some embodiments, the compositions of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to subjects. Typically, pharmaceutical compositions of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical compositions may also include a solubilizing agent. Pharmaceutical compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients of the pharmaceutical compositions of the present invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where NAC is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where NAC is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [0090] In preferred embodiments, the composition is an aqueous solution. In some embodiments, the composition comprises NAC in a concentration of about 0.05 to 10 g/L or any sub-range therebetween, for example, about 0.05 to 9 g/L, or about 0.05 to 8 g/L, or about 0.05 to 7 g/L, or about 0.05 to 6 g/L, or about 0.05 to 5 g/L, or about 0.05 to 4 g/L, or about 0.05 to 3 g/L, of about 0.05 to 2 g/L, or about 0.1 to 10 g/L, or about 0.1 to 9 g/L, or about 0.1 to 8 g/L, or about 0.1 to 7 g/L, or about 0.1 to 6 g/L, or about 0.1 to 5 g/L, or about 0.1 to 4 g/L, or about 0.1 to 3 g/L, of about 0.1 to 2 g/L, or about 0.25 to 10 g/L, or about 0.25 to 9 g/L, or about 0.25 to 8 g/L, or about 0.25 to 7 g/L, or about 0.25 to 6 g/L, or about 0.25 to 5 g/L, or about 0.25 to 4 g/L, or about 0.25 to 3 g/L, of about 0.25 to 2 g/L, or about 0.5 to 10 g/L, or about 0.5 to 9 g/L, or about 0.5 to 8 g/L, or about 0.5 to 7 g/L, or about 0.5 to 6 g/L, or about 0.5 to 5 g/L, or about 0.5 to 4 g/L, or about 0.5 to 3 g/L, of about 0.5 to 2 g/L, or about 1 to 10 g/L, or about 1 to 9 g/L, or about 1 to 8 g/L, or about 1 to 7 g/L, or about 1 to 6 g/L, or about 1 to 5 g/L, or about 1 to 4 g/L, or about 1 to 3 g/L, of about 1 to 2 g/L; or any concentration therebetween, such as about 0.05 g/L, or about 0.1 g/L, or about 0.2 g/L, or about 0.25 g/L, or about 0.3 g/L, or about 0.4 g/L, or about 0.5 g/L, or about 0.6 g/L, or about 0.7 g/L, or about 0.75 g/L, or about 0.8 g/L, or about 0.9 g/L, or about 1 g/L, or about 1.5 g/L, or about 2 g/L, or about 2.5 g/L, or about 3 g/L, or about 3.5 g/L, or about 4 g/L, or about 4.5 g/L, or about 5 g/L, or about 5.5 g/L, or about 6 g/L, or about 6.5 g/L, or about 7 g/L, or about 7.5 g/L, or about 8 g/L, or about 8.5 g/L, or about 9 g/L, or about 9.5 g/L, or about 10 g/L. [0091] In some embodiments, the pharmaceutical compositions of the invention can be administered orally. Formulations for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the pharmaceutical compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention. In some platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. [0092] The pharmaceutical composition of the invention may be prepared by methods known in the art. For example, the methods may comprise admixing a NAC and a pharmaceutically acceptable carrier to prepare the composition. [0093] An aspect of the present invention relates to the NAC pharmaceutical composition thereof, as described herein, for use in any of the methods of the present invention described herein. [0094] The invention further provides pharmaceutical packs or kits comprising one or more containers filled with NAC or pharmaceutical composition thereof. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. *** [0095] The invention is further described in the following non-limiting Examples, as well as the Figures referred to therein. EXAMPLES Example 1: Oral NAC ameliorates IRI and inflammation in steatotic livers Introduction [0096] Expression Hepatic ischemia-reperfusion injury (IRI) remains a significant complication of both surgical liver resection and liver transplantation. IRI is a complex and incompletely understood process that leads to the disruption of cellular integrity with the release of damages-associated molecular patterns and subsequent innate immune system activation, though identification of key mediators remains the point of ongoing research. In liver transplantation, the degree of IRI is more profound in the setting of hepatic steatosis and has historically been shown to increase the risk of primary non-function, dysfunction, and reduce graft and patient survival. Unfortunately, due to the increasing demand for liver transplantation and a lack of transplantable livers, “marginal” allografts, including those with hepatic steatosis, are being considered despite an elevated risk of IRI. [0097] Currently, there are few available therapeutic options in the prevention and treatment of IRI, and N-acetylcysteine (NAC), a thiol-containing synthetic compound, remains the mainstay in the treatment. While the efficacy of NAC has been highly established in the treatment of acetaminophen toxicity and has shown some efficacy against IRI and fulminant liver failure, its mechanisms, optimization of treatment, and impact on long-term survival remain understudied. Given the limited treatment options for IRI and the increasing demand for liver transplantation, significant research efforts are required to not only identify key mediators and therapeutic options for IRI, but also accurately evaluate donor allografts for high-risk stigmata in order to safely utilize these “marginal” organs while decreasing the associated risk. [0098] While no standard exists, donor allograft biopsy is frequently performed to aid in allograft selection, and greater than 30% hepatic macro steatosis is often a deterrent for allograft use. However, a recent study by Patel et al. has shown that steatotic allografts can be successfully utilized with appropriate recipient selection. To aid in the selection process, advanced methods to investigate the integrity and function of the allograft may be essential for the use of marginal allografts. Both Magnetic Resonance Imaging (MRI) and Phasor Fluorescence Lifetime Imaging (FLIM) are promising modalities to evaluate liver function and injury. T2 mapping by MRI has previously been shown as a feasible, non-invasive technique in assessing acute liver injury, while FLIM has been demonstrated to assess for liver injury more accurately via the evaluation of steatosis, fibrosis, and glycolytic activity in experimental models. [0099] Invariant natural killer T (NKT) cells are a unique group of cells that can recognize both self and lipid antigens that are presented to them by MHC class I-like CD1d molecules. Type I invariant Natural Killer T-cells (NKT1 cells) are not only CD1d bound, but also express markers of both T cells (CD3+) and conventional NK cells (NK1.1+) and can potently secrete IFN-γ and TNFα. NKT1 cells have emerged as key mediators in hepatic IRI and have been shown to worsen this process in a NAFLD murine model, despite lower baseline populations. Interestingly, it is also known that NKT1 cells have a wide variety of activating antigens to include endogenous lipid antigens such as phosphorylcholine, which may lead to an enhanced response to IRI. Thus, preventing activation of NKT1 cells following IRI in NAFLD could help develop potential therapeutic approaches that merit further investigation. NAC has also been demonstrated to alter metabolism and have benefit in the treatment of metabolic syndrome, in addition to known benefit in IRI. However, the immunomodulatory capacity of NAC in liver injury remains understudied. [00100] Given the activation of NKT1 cells by lipid antigens and potential roles in hepatic IRI, it was hypothesized that NAC supplementation would ameliorate IRI in a high-fat diet (HFD) murine model via decreased activation of NKT1 cells. In the present work, advanced phasor-FLIM is used to interrogate tissue following IRI and further evaluate the innate immune response to IRI. This study successfully demonstrates NKT1 cells as key mediators in IRI in the setting of hepatic steatosis and shows an improved response following NAC supplementation at least in part secondary to reduced NKT1 cell activation. Methods [00101] The normal diet (ND) , #5001, was purchased from Lab Diet, St. Louis MO, (4.09 kcal/gram,13.4% kJ/fat). The high-fat diet (HFD), 58Y1 blue, was purchased from TestDiet, St. Louis, MO (5.10 kcal/gram, 60% kJ/fat). HFD was stored in dry refrigerated conditions at 4 ^C. NAC was purchased from Sigma, St. Louis, MO, and was added to the treatment group drinking water in a concentration of 10 gram/liter. [00102] C57BL/6 mice were obtained at six weeks of age from Charles River Laboratories and The Jackson Laboratory, Bar Harbor, ME. The mice were maintained in the Division of Comparative Medicine at Georgetown University Medical Center, with a standard 12-hour light-dark cycle. All procedures performed on C57BL/6 mice were fully approved by the Georgetown University Institutional Animal Care and Use Committee under protocol number 2016-1341. Male mice were used for all experiments. Food and water were provided ab libitum in the four experimental groups: ND, ND + NAC, HFD, and HFD + NAC. The ND diets were administered at the beginning of the study and maintained for 19-23 weeks. The HFD was started at week seven of age and continued for 19-23 weeks. NAC drinking water was administered to the ND + NAC and HFD + NAC groups following three weeks of ND or HFD and maintained for 16-20 weeks. Animal weights were measured weekly. Additional C57BL/6 mice were purchased at 25 weeks of age from The Jackson Laboratory, Bar Harbor, ME on a ND, #5K52 from Lab Diet (3.50 kcal/gram, 16.6% fat) for use in select groups.ps. [00103] All procedures performed on C57BL/6 mice were fully approved by the Georgetown University Institutional Animal Care and Use Committee under protocol number 2016-1341. Mice were anesthetized with 2% isoflurane and oxygen inhalation. A midline laparotomy was performed, and an atraumatic micro clip was placed across the hepatic hilus, which interrupted the blood supply to the left and median lobes of the liver. The abdomen was temporarily closed with skin staples, and the mice remained anesthetized. A 45-minute ischemic time was utilized to represent a clinically relevant model, more closely corresponding to the average warm ischemia time in human liver transplantation. This also prevented excess animal death under anesthesia, given the metabolic changes accompanied by HFD murine model. Following 45 minutes of partial hepatic ischemia, the clip was removed to initiate hepatic reperfusion. The abdominal wall was closed with sutures, the skin was reapproximated with staples, and the animals were returned to their cage. All mice undergoing Magnetic Resonance Imaging following IRI, had their abdominal wall and skin reapproximated with sutures. After 24 hours of reperfusion, mice were anesthetized and underwent a second laparotomy. Whole blood was obtained via direct cardiac puncture as a terminal procedure. The left lobe of the liver and the whole spleen were collected. Sham controls underwent the same procedure but without vascular occlusion. [00104] Magnetic resonance imaging (MRI) was performed in the Georgetown-Lombardi Preclinical Imaging Research Laboratory on either a 7T/20 Avance III/ParaVision 5 or a 7T/30 USR Avance NEO/ParaVision 360 (S10 OD025153) scanner. The mice were anesthetized (1.5% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide) and placed on a custom-manufactured (ASI Instruments, Warren, MI) stereotaxic device, with built-in temperature and cardio-respiratory monitoring as described (B, C), and compatible with a 40 mm Bruker mouse body volume coil. [00105] MRI of abdominal and liver fat was performed with a three-dimensional rapid acquisition with rapid enhancement (3D-RARE) sequence in the coronal orientation with the following parameters: TR: 2855 ms, TE: 12 ms, RARE Factor: 4, Matrix: 220 x 220, FOV: 50 mm x 40 mm, Averages: 4, Slice thickness: 0.75 mm, Slices: 50 and respiratory gating Quantification of visceral fat depots in the imaging datasets was performed by thresholding and voxel-counting with ImageJ software (NIH). A maximum intensity projection algorithm of the 3D-reconstructed image with an intensity threshold intended to segment fat only was used. The abdominal region analyzed was defined by superior and inferior anatomical landmarks, that is, the proximal border of the left kidney and the convergence of the left and right common iliac veins, respectively. The lateral landmark was the abdominal wall, avoiding subcutaneous fat. The percentage area corresponding to fat depots within the abdomen was calculated via the sum of the visceral fat voxels versus total abdominal voxels. Mouse liver fat was measured by quantifying the mean intensity of the region of interest (ROI) localized on the mouse liver placed in homogenous areas, avoiding structures such as large vessels and ducts. ROIs on three separate slices were selected and averaged for each mouse. For imaging of the nuchal fat, a 3D T1-weighted RARE sequence in the sagittal orientation was run with TR: 2437 ms, TE: 15 ms, FA: 74.1, Matrix: 256 x 256, FOV: 40 mm x 40 mm, Slice thickness: 0.5 mm, Averages: 4, Slices: 35. The mean intensity of nuchal brown adipose tissue (BAT) and white adipose tissue (WAT) was measured by localizing ROIs on the BAT and the WAT. ROIs on three separate slices were selected and averaged for each mouse. [00106] Dynamic Contrast-Enhanced MRI (DCE-MRI) using Gadoxetate Disodium (Eovist®, Bayer) was performed with a T1-weighted RARE protocol in the coronal orientation, without respiratory gating, with TE: 8.2 ms, TR: 400 ms, slice thickness: 1 mm, matrix: 128 x128, FOV: 40 mm x 40 mm, rare factor: 2, fat suppression and a duration of 25 s. Ten baseline (pre-contrast) scans were run, followed by subcutaneous parenteral administration of 0.025 mmol/kg Eovist. Immediately after injection, 60 MRI scans were acquired repetitively over approximately 30 minutes, and the uptake and excretion of contrast was measured over time. Relative liver enhancement (RLE) was used to quantify hepatic function. Briefly, the mean intensity of regions of interest (ROI) localized in homogenous areas of the liver were quantified in baseline and post-injection images. Liver function was calculated using the formula RLE = (SILiver enh – SILiver unenh)/SILiver unenh x 100. The area-under- the-curve (AUC) and the slope were derived from the corresponding RLE data. The slopes were measured for the first 20 minutes post-injection, where after this time point, the curves reached their plateau. Both the AUC and the slopes were calculated with a 95% confidence interval. [00107] A two-dimensional multi-echo multi-slice sequence in the axial orientation was used for T2 mapping with TR: 2000 ms, Flip Angle: 180 deg, Averages: 2, and the following echo times, TE: 10, 20, 30, 40, 50, 60, 70 and 80 ms, with respiratory gating. In order to measure T2 relaxation times, we used the Paravision360 Image Sequence Analysis tool to generate T2-fit and T2-maps. ROI were localized on the left and right liver areas in four slices per mouse, excluding non-homogenous structures such as large hepatic vessels or ducts, after which T2 values were quantified and averaged. [00108] Whole blood was obtained by direct cardiac puncture as a terminal procedure. ALT levels were measured using a multichannel analyzer, Alfa Wassermann Vet Axcel, from the clinical diagnostics laboratory of VRL Maryland, LLC. [00109] The autofluorescence lifetime images of the liver sections (5 µm thick) were measured using a modified Olympus FVMPERS (Waltham, MA) microscope equipped with a Spectra-Physics Insight X3 (Milpitas, CA) laser and FastFLIM (ISS, Champaign, IL) acquisition card. The samples were excited with the 740 nm laser line using a 20X air objective (LUCPLFLN 0.45NA, Olympus) applying a two-photon excitation scheme. The fluorescence was collected using the DIVER (Deep Imaging Via Enhanced Recovery) detector (16-18) assembly and recorded using a FastFLIM card (ISS, Champaign, IL). The pixel dwell time for the acquisitions was 20 µs, and the images were taken with sizes of 256x256 pixels with a field of view of 318.8 µm (Zoom =2X). To obtain a high signal-to- noise ratio, 16 frames were collected for each area. The data from each pixel were recorded and analyzed using the SimFCS software (Laboratory for Fluorescence Dynamics, University of California, Irvine, CA). The FLIM data were collected using the passive mode, where the raster scanning was done using the Olympus software, and the images were collected using the FLIMBox/FastFLIM system, and the scanning parameters were matched to ensure proper image acquisition. SHG and FLIM images were obtained using two separate filter assemblies in DIVER and further separated based on their lifetime and phasor position. [00110] The Phasor approach to FLIM is a fit free method of analysis where fluorescence decay information collected at each pixel of an image is transformed to the phasor plot and designated a coordinate based on the lifetime. Being a fit free method, phasor is a much faster analysis method and is used increasingly in biological fluorescence imaging with a large data set. The phasor transformation results in the formation of a heat map in the phasor plot where each pixel of an image is represented by a single phasor point and segmentation and filtering methods and of image analysis can be applied without loss of image definition of the original intensity images. Recent advances in analysis techniques allow us to quantify the contribution of multiple components and this is applied in this work.
[00111] A brief description of the method follows below. A much more detailed description of the method, along with individual analysis schemes, flowcharts, and assumptions for both phasor-FLIM quantification and SHG analysis is explained in the supplemental information. FastFLIM (ISS, Champaigne, IL) is a frequency domain FLIM imaging instrument. In frequency-domain fluorescence lifetime measurements, the transformation to the phasor plot uses the following relations, gi,j(ω) = mi,j ·cos (Φi,j) 3 si,j(ω) = mi,j.·sin (Фi,j) 4 where, gi,j(ω) and si,j(ω) re the X and Y coordinates of the phasor plot, respectively, and a mri,j e and Ф i,j = are= the modulation and phase at the image pixel i, j. The longer lifetime is represented by the larger phase angle in the phasor plot - movement towards S=0, G=0 around universal semi-circle. The distribution of phasor points originating from FLIM measurements appears on (for mono-exponential decays) or inside (for multi-exponential decays) the universal circle (please refer to SMI in the phasor supplemental methods). The linear combinations are shown by the blue. If each component has a multi-exponential decay, its location will be represented by phasors not in the universal circle, but the principle of linear combination remains valid. More details can be found in the phasor section of the supplemental methods.
[00112] According to this principle, if a pixel of an image has multiple components originating from multiples species, then the position of the corresponding phasor point is inside the polygon whose vertices are occupied by the phasor points originating from the individual components. The distance of the image phasor point from any of the vertices is reciprocal to the fractional intensity component of that particular component. The higher the fractional intensity contribution of that particular species towards the total intensify of the image pixel, the closer the phasor point of that image pixel is to the corresponding component phasor position. For a three component system, an algebraic solution of this system exists, and this allows the breakdown of a phasor cloud from an image to the individual fractional intensity components. Our recent work created a framework where the phasor clouds originating from multiple components can be quantitatively resolved, and their individual fractional intensities can be calculated. For a system where the quantum yield can be assumed based on their lifetimes or can be quantified—the fractional intensity ratios can be modified to molar fraction ratios. Please see supplemental methods for additional details of the analysis and stepwise processes. [00113] In this work, the positions of the three original phasor positions were selected based on previously published work. Free and protein bound NADH have a lifetime of 0.4 ns and ~3.4 ns, respectively, and their positions are on universal semicircle, and are shown by the blue and green circles, respectively (FIG.5, Panel A). The line joining the two cursors is named metabolic trajectory. The long lifetime species (LLS) has a lifetime of ~8ns, and the corresponding phasor position is selected by the red circle. In the presence of LLS, the autofluorescence FLIM phasor in the NADH channel shifts away from the metabolic trajectory and inside the triangle whose vertices are occupied by the central positions of red, green, and blue circles. As mentioned above and in supplemental methods (Phasor section)—we have used multiple component analysis to obtain the quantitative information about the concentration ratio of free and protein-bound NADH and fractional intensity of NADH. SHG is generated from the non-centrosymmetric collagen fiber bundles and has a different lifetime signature than rest of the tissue autofluorescence. The autofluorescence signal appears inside the universal semicircle and the SHG signal appears at S=0,G=1 in the phasor plot. This allows for separation of the two signals and the calculation of a SHG based fibrosis score. [00114] Snap-frozen liver tissue was dissolved in 300 µL of extraction buffer (IPA) and subjected to two cycles of freezing and rapid thawing into a 37 °C water bath or 90 seconds. Then, tissue samples were sonicated at 30 kHz for 30 seconds and mixed with 100 μl of ice- chilled isopropyl alcohol (IPA) with internal standards (IS). Upon 30 minutes of incubation on ice and additional incubation at −20 °C for 30 minutes, the tissue in suspension was spun down at 14000xg at 4 °C for 20 minutes to collect the supernatant. Targeted LCMS-MS was performed using Xbridge Amide 3.5 μm, 4.6 × 100 mm column (Phenomenex) online with a triple quadrupole mass spectrometer (5500 QTRAP, SCIEX) equipped in the multiple reaction monitoring (MRM) modes. Each metabolite, declustering potential, collision energies, cell exit potential, and entrance potential were optimized to acquire maximum ion intensity using Analyst 1.6.3 software (SCIEX, United States). [00115] To ensure the most intense precursor and fragment ion pair selection, for the MRM- based quantitation, individual analyte signal intensities were ranked for all MRM Q1/Q3 ion pairs of that specific analyte. To obtain metabolite ratio, we used normalized peak area of endogenous metabolites within samples with normalized IS for every class of lipid molecule. Appropriate measures were taken to randomize sample queue and to assess sample carryover. Pooled quality control (pooled QC) samples were injected periodically to check for instrumental variation and National Institute of Standards (NIST) plasma samples were injected for lipidomic data analysis. Data normalized to QC variance. MultiQuant 3.0.3 (Sciex) software was used to obtain QC normalized data and imputed MRM data. To determine relative quantification values of analytes, the ratio of peak areas of sample transitions normalized to the peak area of the IS specific for every class was computed. Data postprocessing statistical analysis including heatmap, volcano plot, and ANOVA was conducted with the software MetaboAnalyst. [00116] The left lobe of the liver was collected into RPMI-1640 culture medium (Gibco). Liver tissues were harvested, passed through a 70-μm cell strainer (Fisher Scientific), and leukocyte fractions were isolated via Percoll (Cyntiva) density gradient. Samples were centrifuged at 1000g (25 °C), without brake for 20 minutes, and the upper layer was carefully discarded. The leukocyte layer was washed and resuspended in 1x PBS (Gibco). Liver leukocytes were stained with the following antibodies (BioLegend, San Diego, CA) for flow cytometry analysis: Alexa Fluor700 conjugated CD 45, Brilliant Violet 510 conjugated CD 4, Brilliant Violet 421 conjugated CD 8, Brilliant Violet 605 conjugated NK-1.1, and Fluorescein isothiocyanate-conjugated CD 3. Data were acquired using a BD FACSAria III Cytometer (BD Biosciences) at our Flow Cytometry Core Facility. Any samples with viability of 60% or lower (as determined by staining with live dead marker Zombie NIR™, BioLegend) were excluded from all analyses. FlowJo v10 (BD, Franklin Lakes, NJ) was used for all subsequent data analyses. [00117] Intracellular staining for the detection of cytokines was carried out from liver leukocytes. Approximately 1x106 cells/ml RPMI supplemented with 10% FBS, 1% Penicillin Streptomycin, and 0.5% Gentamycin were cultured for 20 hours at 37 °C in a cell culture flask. Recombinant mouse cytokine concentrations used were 10 ng/ml IL-12, 50 ng/ml IL-18, and 50 ng/ml IL-15 (R&D Systems). Cells were stimulated for 4 hours with Cell Activation Cocktail containing phorbol-12myristate-13-acetate (PMA, 50 ng/ml) and ionomycin, (BioLegend) in the presence of 5 μg/ml brefeldin A (BioLegend). Following stimulation, the cells were stained with the following antibodies to detect cytokines: Brilliant Violet 650™ -conjugated anti-IFN-γ (BD Biosciences) and Brilliant Violet 605™ - conjugated anti-TNF-α (BioLegend). Cells were fixed (IC Fixation buffer, Invitrogen) and permeabilized (Permeabilzation buffer, Invitrogen) according to the manufacturer’s instructions. [00118] To assess the effect of IRI on mouse-specific cytokines and chemokines, the SYBR- green based RT² Profiler™ Polymerase Chain Reaction (PCR) Array Mouse Cytokines & Chemokines (PAMM-150ZC-12, Qiagen) was used to evaluate the gene expression of 84 different cytokines and chemokines. For this experiment, mice liver specimens collected 24 hours-post sham and IRI surgeries and stored in the Allprotect Tissue Reagent (Qiagen) were used for the total RNA extraction. All tissues were lysed using 1.0 mm Zirconia/Silica beads (BioSpec Products) in FastPrep 24 Tissue Homogenizer (MP Biomedical). We extracted total mouse liver RNA as instructed in the RNeasy Mini kit with RNase-free DNase set (Qiagen). RNA quality and quantity were assessed using 2100 Agilent Bioanalyzer, and RNA samples with RNA integrity number (RIN) ≥ 7 were used for the PCR arrays. [00119] For each RT² Profiler™ PCR Array, 1.25 ug of total RNA was pooled from at least three separate mice livers in an equal proportion. Standard procedures were performed to eliminate mouse genomic DNA, synthesize first-strand and set up RT2 profiler PCR array for cytokines and chemokines. Raw Ct values from the array were analyzed using the GeneGlobe web tool (https://geneglobe.qiagen.com/us/) to check the quality of the PCR arrays, normalize threshold cycle (Ct) values using Beta-2-Microglobulin (B2m) as a housekeeping gene and estimate fold regulation with ND (Sham) as a control group. Unsupervised hierarchical clustering for the 2-ΔCt was performed using ClustVis (35) to identify gene clusters specific to sham and IRI groups under two different diet regimens. Venn diagrams for genes with fold upregulation ≥ 2 were created using Venny to identify common genes among all the IRI groups and exclusively diet-specific gene signatures. [00120] All data are expressed as mean ± standard error of the mean (SEM). Comparison between groups was performed by a Mann-Whitney U test. Statistical significance was established at p<0.05 with all p-values being two-sided and stratified in the following order: “*” 0.05 to 0.01, “**”0.01 to 0.001, “***” 0.001 to 0.0001, and “****” <0.0001. Prism Software (GraphPad, Inc. San Diego) was used for all statistical analyses. Results [00121] To establish the efficacy of our HFD murine model, the adiposity of the liver, the abdomen, and the nuchal areas of mice from each group were assessed in vivo using 3D- RARE magnetic resonance (MRI). HFD mice were presented with a significant accumulation of hepatic steatosis and visceral abdominal fat and accumulation of white adipose tissue (WAT) in the nuchal region. NAC treatment prevented the development of hepatic steatosis and supported the transformation of WAT into brown adipose tissue (BAT) (FIG.1). These findings were correlated with H&E-stained liver sections evaluated by an expert pathologist. Consistently, hepatic steatosis varied between both ND and ND + NAC when compared to HFD mice, with HFD mice demonstrating 80% hepatic steatosis on average (p<0.0001). This difference was significantly reduced in mice on NAC supplementation, which resulted in a 50% reduction in hepatic steatosis (FIG.2). [00122] Given the profound reduction of hepatic steatosis in the HFD + NAC mice, the baseline liver function of the HFD murine model was assessed by performing DCE-MRI with Eovist®. Mice on HFD showed a significant decrease in relative liver enhancement as compared to ND mice (FIG.3). These data, expressed as AUC analyses, correlated strongly with other imaging parameters such as in vivo and ex vivo liver fat content. Corresponding contrast enhancement slopes were calculated during the first 20 minutes post-injection, which represents the transitional phase before the contrast levels begin to plateau during the hepatobiliary phase. These data were strongly correlated with the AUC and overall hepatic relative liver enhancement values, where the rates of T1 enhancement in the HFD group were slower and failed to achieve the signal intensity levels observed in the ND mice. However, when performed on HFD + NAC mice, the contrast uptake is comparable to that obtained by ND mice. Collectively, these data indicate a deficit in Eovist uptake in the HFD mice that is restored in HFD + NAC mice to a level consistent with ND mice. [00123] Partial warm IRI was induced in mice from all groups. Both ND and HFD groups experienced a significant elevation in serum ALT levels as compared to sham-operated mice (p<0.0001 and p=0.0009, respectively). However, HFD mice experienced a significantly sharper rise in serum ALT following IRI than did ND mice (79 u/L vs.408 u/L, p<0.0001), suggesting a higher degree of injury. The role of NAC supplementation in the prevention of IRI was evaluated, and demonstrated a significant reduction in serum ALT following IRI in HFD + NAC mice as compared to HFD mice alone (201.5 u/L vs 408 u/L, p<0.0001), and in fact, there was no significant difference between ND IRI and HFD + NAC IRI groups, suggesting a normalization of injury (FIG.3, Panel A). [00124] Representative mice from each group were subjected to T2-weighted MRI prior to the terminal procedure to assess the magnitude, distribution, and bilaterality of hepatic edema and associated inflammation, pre-and post-IRI T2 maps were acquired. Specifically, baseline T2 mapping of the liver was performed on ND and HFD, with and without NAC. Twenty- four hours after partial hepatic IRI surgery, the mice again underwent T2 mapping. The T2 maps were generated, and the T2 values quantified. Pre-IRI T2 values were similar between ND mice and ND + NAC mice, while the T2 values trended towards a slight increase in the livers of mice on HFD (FIG.4, Panel B). Ischemia-reperfusion injury exposure did not affect the T2 values of either the ND or ND + NAC livers. Conversely, the hepatic T2 values of mice on HFD were noticeably elevated after IRI, while the T2 values of HFD + NAC were comparable to those observed with ND, both before and after IRI. It is important to note that baseline and post-IRI T2 values were not significantly different between the right and left liver lobes in any group. Overall, the T2 maps enabled the quantification and visualization of hepatic edema and inflammation resulting from IRI in HFD mice, which demonstrated an increase in hepatic T2 relaxation times in HFD as compared to ND mice. NAC ameliorated the impact of IRI in HFD mice, making T2 values comparable to those seen in the ND + NAC mice. [00125] Given recent advances in Phasor FLIM and SHG imaging of steatotic human and mouse liver tissue, this methodology was employed to evaluate the differences in liver tissue following IRI as a novel approach to evaluate liver injury. Samples were examined for glycolysis and lipid oxidation based on the phasor FLIM signatures. A higher ratio of free to protein-bound NADH is indicative of increased glycolysis and a larger amount of long lifetime species (LLS) is indicative of higher lipid peroxidation. The FLIM images (FIG.5, Panel A) were colored according to the three cursor positions for bound NADH (green), free NADH (blue), and the LLS (red) selected using the phasor map (FIG.5, Panel B). [00126] The models were first evaluated for increased glycolysis, as demonstrated by free NADH (blue). Visually, there is increasing blue color present in all groups following IRI, while the ND, ND + NAC, and HFD + NAC groups show more green color, thus more bound NADH. These data, when converted to the concentration ratio of free/bound NADH (FIG.5, Panel C), establish that the ratio increases in HFD mice compared to ND mice and this is further exacerbated following IRI, again demonstrating a greater ratio of free/bound NADH ratio in HFD IRI mice compared to ND IRI mice. This increasing ratio correlates to higher glycolytic stress, thus a greater degree of IRI. Importantly, glycolytic stress is significantly reduced in the HFD + NAC IRI mice compared to the HFD IRI mice, suggestive of a protective effect of NAC in the HFD group. [00127] LLS was then evaluated for differences in lipid oxidation between the samples. There was a noticeable increase in LLS in the HFD, as seen by the increasing red color of images, as compared to the ND mice. This fractional intensity of LLS was then quantified (FIG.5, Panel D), as described in the supplemental methods. HFD and HFD IRI mice both demonstrate statistically significant higher quantifications of LLS compared to ND and ND IRI mice, respectively. [00128] Upon establishing that a higher degree of IRI is present on a background of hepatic steatosis, as demonstrated by biochemical, MRI, and glycolytic analysis, hepatic steatosis was quantified more accurately within the samples. Phasor FLIM imaging allowed to quantifiably calculate steatosis based on the accumulation of the LLS. Selection of the phasor signature of LLS (long lifetime species, red circle in FIG.6, Panel A), enabled the identification of the lipid droplets in autofluorescence FLIM images, where they are mapped according to the color of the selection (red here). Steatosis is then quantified by calculating the size of the droplets occupying the image. The data show that droplets are much smaller or are nonexistent in ND diet mice and are much larger in HFD diet mice, both in the IRI and sham samples, which correlates to pathological evaluation of H&E staining. Note that the Y axis is in log scale and shows that exceptionally large droplets are seen in HFD livers in both sham and IRI mice as compared to ND mice. Importantly, while the average size of the lipid droplets increases in both HFD and HFD IRI mice, this is visibly reduced in HFD + NAC and HFD + NAC (IRI) mice (FIG.6, Panels B and C). It is further notable that, while not statistically significant, there is a visible and quantifiable increase in lipid droplet size in ND IRI and HFD IRI mice when compared to the respective ND and HFD counterparts. It is important to note that upon evaluation of the individual distributions of lipid droplet within each sample (FIG.7), it demonstrated that a large degree of variation in lipid droplet sizes is seen in each sample. [00129] Prepared snap-frozen liver tissue from sham mice was subsequently subjected to metabolomic profiling of 21 classes of lipid metabolites. Significant changes in lipid composition between the tissues were identified, as visible in the heat map (FIG.8, Panel A). Of the 21 classes analyzed, triacylglycerols (TAG) were significantly upregulated in HFD mice as compared to normal diet (0.222 normalized intensity vs.6.783 normalized intensity, p<0.0001). This was significantly decreased in the HFD + NAC mice, as demonstrated in the volcano plots showing differences in upregulated lipid profile in HFD vs ND and HFD + NAC vs HFD mice (FIG.8, Panel B). Interestingly, the HFD + NAC mice had a statistically reduced composition of TAG, as compared to HFD mice (6.281 normalized intensity vs.2.681 normalized intensity, p<0.0001; FIG.8, Panel C). There were no statistically significant reductions in the HFD + NAC mice for the remaining 20 classes of lipids. [00130] In order to identify specific transcriptomic changes that occur in the liver following IRI, array-based PCR transcriptional profiling was performed using the RT2 profiler PCR array for 84 distinct Chemokine and Cytokines (Qiagen, Germantown, MD). Utilizing an unsupervised hierarchical clustering analysis, a distinct difference in gene expression patterns between HFD mice and those mice that underwent IRI was demonstrated (FIG.9, Panel A). Both shared and discrete genes with a minimum of two-fold upregulation amongst the mouse groups that underwent IRI were identified (FIG.9, Panel B). [00131] 45 genes of interest that were upregulated within the collective specimens were identified. Among these genes, seven were uniformly upregulated in both ND + IRI and HFD + IRI groups, including CSF3, CXCL1, CCL17, CCL19, TNFSF13b (TNF Superfamily Member 13b), IL-6 and IL17a. Uniquely, HFD IRI mice exhibited an exclusive upregulation of five genes: TGFβ-2, Pf4 (Platelet Factor 4), IL-1β, IL-10, and IFN-γ. In comparison, HFD + NAC mice that underwent IRI exhibited significantly lower expression levels of the genes that were exclusively upregulated in HFD IRI and shared between ND IRI and HFD IRI mice. [00132] Given the significant upregulation of INF-γ and its related genes shown by RT-PCR array analysis and the variations in lipidomic analysis, specifically TAGs, the role of IFN-γ producing NKT cells in hepatic IRI was evaluated by polychromatic flow cytometry. NKT1 cells were defined as CD45+/CD3+/NK1.1+ cells. Utilizing this strategy, the sham mice from each group were first evaluated. ND mice harbored NKT cells at an average frequency of 13.65%. This was significantly higher than the HFD mice (6.97%, p=0.004). Interestingly, while there was no significant difference between the HFD and HFD + NAC groups, the ND + NAC mice were shown to harbor a larger frequency of NKT1 cells as compared to ND mice (27.5%, p=0.017). [00133] Hepatic IRI was then elicited and evaluated for NKT1 cell changes. ND IRI mice had a significant rise in the percentage of CD45+/CD3+/NK1.1+ cells as compared to the ND sham mice (p=0.0095). There were no significant differences in NKT cells frequencies amongst ND + NAC IRI mice or the ND + NAC sham mice, despite the increased frequencies in ND + NAC mice, as previously demonstrated. Interestingly, following reperfusion injury, HFD IRI mice underwent a drastic rise in NKT cells as compared to HFD sham mice (28.9%, p=0.0079), which was statistically similar to that seen in the ND IRI group (FIG.10, Panels A and C). This effect was ameliorated following NAC treatment in HFD mice. The HFD + NAC IRI mice showed no significant differences in NKT cell populations as compared to the HFD + SHAM counterparts; however, the frequency of NKT cells was drastically reduced when compared to both HFD IRI and ND IRI groups. [00134] Ex vivo cell stimulation with PMA from samples obtained from all IRI groups was employed to identify the level of IFN-γ produced from CD45+CD3+NK1.1+NKT cells. While the level of IFN-γ produced was similar between ND IRI and ND + NAC IRI mice, there was a significantly greater frequency of IFN-γ produced by HFD IRI mice (FIG.10, Panels B and D), corresponding to the previously demonstrated cell populations (p=0.0177). This rise in IFN-γ production was ameliorated in the HFD + NAC IRI mice as compared to the HFD IRI mice (p=0.0001). Discussion [00135] The work described here demonstrated that oral NAC treatment decreased hepatic steatosis and increased BAT as detected by T1 weighted MRI. In this sense, BAT is characteristic of high metabolism and thermogenic capacity, which supports that NAC alters thermogenic gene expression. The decreased hepatic steatosis was correlated with improved liver functional capacity using Eovist® DCE MRI, which has emerged as a useful hepatobiliary contrast agent that clinically, and non-invasively, allows for stratification of steatotic liver damage. A stark difference in contrast uptake in HFD mice in comparison to the ND mice was demonstrated, which was normalized with NAC supplementation. While the mechanisms by which NAC improves Eovist® uptake in the HFD mice are presently unknown, previous work in cirrhotic rats suggests alterations in the expression of associated membrane proteins may be a key factor, although alternations in hepatic perfusion cannot be ruled out and additional studies are warranted to establish these relationships. Overall, these findings collectively established this HFD model with NAC treatment group as an effective method to study IRI mitigation. [00136] Despite the known effect of IV NAC in IRI, little was known regarding the effectiveness of oral NAC supplementation in the prevention of IRI prior to the present work. Utilizing the clinically relevant model, an improvement of IRI in HFD mice on NAC supplementation as compared to HFD only using serum ALT and T2 mapping MRI mapping was demonstrated. Interestingly, T2 relaxation times, which correlate with hepatic edema and inflammation, show a similar effect in all lobes of the liver. This demonstrates the scope of full liver sequelae following segmental injury in HFD mice, which is significantly reduced in HFD + NAC mice. Previous work has demonstrated the vasodilatory effects of NAC in models via Nitric Oxide dependent and decreased Vascular Endothelial Growth Factor (VEGF)/VEGFR2 mechanisms, which may offer insight into the reduced edema visualized in our samples. However, this does not provide insight into the key roles of innate immune system cytokine generation and metabolic changes at a cellular level which occur following IRI. [00137] PhasorFLIM and SHG imaging was used in the evaluation of hepatic IRI, which demonstrated increased glycolytic stress and lipid oxidation within the HFD murine model that was worsened following IRI. This is consistent with known requirements of NADPH and FAD metabolism. It was determined that NAC supplementation decreased glycolytic stress in HFD mice following IRI, correlating to a less severe degree of injury and inflammation that was corroborated by previous findings in serum ALT and T2 mapping. Additionally, utilizing LLS imaging of lipid droplets, there was a visual reduction in lipid droplet size in HFD + NAC mice when compared to HFD mice. However, when an average value was calculated for total image, this resulted in loss of dimensional information and the inherent, visually depicted heterogeneity (FIG.7). The actual distribution of the fractional intensity of LLS (red) and free NADH (blue) demonstrate that embedded spatial information is a key component phasor-FLIM auto fluorescent image analysis. This is demonstrated in the composite image of LLS, free and bound NADH and the individual species distribution (FIG.7, Panels C-F), which demonstrates how different parts of the image have a differential amount of each species. The cumulative graphs from 3 samples show that along with a shift of LLS in HFD IRI samples, there may also be broadening of these distributions. In case of broadening, e.g., HFD + NAC vs. ND – the central position of the distribution may not shift appreciably, however more metabolic heterogeneity is seen in the HFD-NAC samples than in the ND diet samples, which is apparent. Further, while previous work had shown a downregulation of genes involved in lipid oxidation in obesity induced mice on NAC supplementation, this was not apparent on LLS imaging for lipid oxidation. Importantly, LLS is a complex and incompletely understood representation of lipid oxidation byproducts that depends greatly on the different pathways of oxidation. Thus, absence of changes in LLS does not confidently correlate to a lack of lipid oxidation throughout the tissue. [00138] To further classify metabolic changes occurring with NAC supplementation, a quantitative lipid profile analysis of whole liver tissue was performed. This may be the first time a full lipidomic profile has been generated from liver tissue of HFD mice on NAC supplementation. This demonstrated significant downregulation of all TAGs species when compared to mice on HFD alone. TAG synthesis is known to be regulated via a PPAR-γ dependent pathway, and previous studies have demonstrated that PPAR-γ activity is downregulated as a result of NAC supplementation. Recently, PPAR- γ has also been identified as a key mediator in the synthesis of CD1d, which serves a crucial function in priming of NKT cells. This expands on work that previously demonstrated NKT cell activation occurs secondary to lipid excess, which has been linked to CD1d expression on adipocytes. It has further been demonstrated that NKT cell activation is secondarily decreased in ApoE -/- mice. Further work demonstrated that NAC supplementation can abolish Vα14iNKT cells and IFN-γ signaling in fulminant liver failure. Taken together, the findings and previous work suggest that NAC treatment can directly prevent NKT cell activation by decreasing lipid excess and CD1d production. [00139] Subsequent experiments were formulated around determination of NKT cell activity. RT-PCR array for cytokine and chemokine expression was performed from liver tissue in the maximally affected left lobe. This ultimately alluded to significant upregulation in HFD IRI mice that was not established elsewhere, specifically IFN-γ and its related genes, including IL-12. Upon further analysis, significantly upregulated in genes that have been linked to the NF-κB pathway—CXCL1 and CXCL10—was shown. This is in line with previous reports that the NAC treatment results in a downregulation of NF-κB, and also furthered our suspicion that NKT cells were in fact mediators in the model, as IFN-γ and, more specifically, TCR-β both result in direct activation of the NF-κB pathway. Further, IFN-γ also results in NF-κB activation by means of JAK/STAT activation pathways. Finally, a complete downregulation of these associated genes in HFD + NAC IRI mice was elicited. Taken together, NKT1 cell activation was suspected as a key effector component of IRI and was directly downregulated following NAC treatment. This was confirmed using flow cytometry. [00140] The work demonstrates the effectiveness of oral NAC supplementation in preventing IRI through an NKT1-cell dependent manner and demonstrates novel methods of both accurately detecting injury within this model. Potential downstream pathways and targets were identified for future research into the prevention of hepatic IRI. Further work will be required to identify specific lipid antigens that result in NKT activation in the HFD murine model, as well as the contribution and interplay of INF-γ producing NKT cells with other NKT cell subsets. Example 2: NAC causes adipose loss in HFD mice, but not ND mice, through enhanced lipid secretion via skin and/or sebaceous glands [00141] The effects of NAC were tested in C57BL/6 (B6) wildtype mice. B6 wildtype mice were obtained at six weeks of age from Charles River Laboratories and The Jackson Laboratory, Bar Harbor, ME. Food and water were provided ab libitum in four experimental groups: normal chow diet (ND), ND + NAC, 60% high fat diet (HFD), and HFD + NAC. NAC drinking water (10 gram/liter) was administered to the ND + NAC and HFD + NAC groups following three weeks of ND or HFD and maintained for up to 20 weeks. Animal weights, random blood glucose levels, and food consumption rates as defined by food intake in grams per day and cage were measured weekly. [00142] The results demonstrate that NAC treatment caused a substantial and persistent reduction in weight gain in HFD mice when compared to untreated HFD controls (FIG.11). Moreover, NAC treatment also markedly improved metabolic parameters under HFD conditions as indicated by normalization of random blood glucose levels in HFD NAC animals when compared to HFD and ND controls (FIG.12). NAC treatment did not only prevent but also reverse obesity as demonstrated by weight loss induction in obese HFD B6 mice, which received delayed onset NAC treatment after 16 and 32 weeks on HFD, respectively (FIG.13). Furthermore, NAC treatment was not associated with reduced caloric intake, as the average food consumption rates were comparable between NAC-treated HFD and ND mice and untreated HFD controls (FIG.14). Thus, the observed NAC-mediated improvements in body weights, white adipose tissue mass, and hepatic steatosis confirm that NAC treatment causes an overall negative energy balance in HFD mice where caloric output significantly surpasses caloric intake. These results are consistent with the lipidomic profiling of liver tissue from the four experimental groups of Example 1, which demonstrated that of the 21 classes of lipids analyzed only triacylglycerols (TAGs) were significantly reduced in HFD + NAC mice when compared to untreated HFD mice (FIG.8). Further lipidomic analysis highlighted that of the 277 TAGs analyzed 149 were significantly decreased in HFD + NAC mice when compared to untreated HFD mice (FIG.15). These results suggested that NAC causes negative energy balance and enhanced caloric output in HFD mice due to loss of TAGs. [00143] HFD mice treated with NAC developed fatty and greasy hair as early as 1 week after treatment initiation (FIGS.16 and 17), suggesting that NAC treatment causes negative energy balance and adipose loss under HFD conditions through enhanced secretion of fat including TAGs through the skin. To test this hypothesis, hair samples were collected from HFD and HFD + NAC mice for hair lipid isolation and subsequent thin layer chromatography analysis. HFD mice treated with NAC demonstrated significantly increased hair lipid mass in comparison to untreated HFD controls (FIG.18, Panel A). Thin layer chromatography further revealed that the enhanced lipid mass in HFD + NAC mice was mainly due to an increase in TAGs and a large fraction of sebum specific wax esters (FIG.18, Panel B), confirming that sebaceous glands are involved in enhanced secretion of lipids upon NAC treatment. Taken together, these results corroborate that NAC treatment causes adipose loss in HFD mice through enhanced lipid secretion through the skin. [00144] ND mice treated with NAC did not exhibit a greasy and fatty fur phenotype (FIG. 16, Panel B) and did not show weight loss in comparison to ND controls (FIG.19, Panel A). Moreover, ND mice treated with NAC showed no difference in hair lipid mass compared to untreated ND controls (FIG.19, Panel B). Thin-layer chromatography confirmed that ND + NAC mice did not display elevated levels of TAGs or wax esters relative to untreated ND controls (FIG.19, Panel C). Collectively, these results suggest that sebaceous glands contribute to the enhanced lipid secretion upon NAC treatment under HFD but not ND conditions. Example 3: NAC affects sebaceous gland morphology in HFD mice [00145] The impact of NAC on sebaceous gland morphology was investigated in B6 wildtype mice. Four experimental groups were provided food and water ad libitum: normal chow diet (ND) (n=8), ND + NAC (n=13), 60% high-fat diet (HFD) (n=20), and HFD + NAC (n=25). NAC drinking water (10 g/L) was given to the ND + NAC and HFD + NAC groups. After six weeks of treatment, mice aged 14-16 weeks were euthanized and skin samples were collected from their backs. The samples were fixed in formalin, paraffin- embedded, and sectioned into five-micron slices, which were then subjected to hematoxylin and eosin (H&E) staining. The H&E-stained slides were scanned at 40x magnification using an Aperio GT450 slide scanner, and image analysis was conducted using QuPath software. Sebaceous glands in each skin sample were identified and navigated at magnifications ranging from 10x to 40x, manually annotated, and classified as either transverse (T) or longitudinal (L) based on histological features determined by the sample's orientation and cut. QuPath software measured the area of each annotation, corresponding to sebaceous glands, in square micrometers. [00146] Histological examination of the cutaneous tissue in HFD mice subjected to the NAC treatment demonstrated a reduction in sebaceous gland size in comparison to untreated HFD mice (see FIGS.20 and 21). This finding suggests a potential mechanism, which may involve an increase in holocrine secretion. Consequently, sebaceous gland expansion is hindered, culminating in a reduced sebaceous gland morphology. Example 4: NAC causes adipose loss in HFD mice through enhanced lipid secretion in the skin across a range of NAC dosing [00147] The dose response of NAC treatment on enhancement of lipid secretion through the skin and protection against HFD-induced obesity and obesity-related complications was investigated in B6 wildtype mice. Seven experimental groups were provided food and water ad libitum, including normal chow diet (ND) (n=8), ND + NAC 0.1% (n=15), and ND + NAC 1% (n=13) as well as 60% high-fat diet (HFD) (n=9), HFD + NAC 0.1% (n=15), HFD + NAC 0.2% (n=10), and HFD + NAC 1% (n=15). NAC drinking water at various concentrations (10 g/L (1%), 2 g/L (0.2%), or 1 g/L (0.1%)) was administered to the respective ND + NAC and HFD + NAC groups. Mice underwent treatment for up to eight weeks and were euthanized at 12-16 weeks of age. Animal weights and food consumption rates as defined by food intake in grams per day and cage were measured weekly. At the endpoint, inguinal fat pad, and epididymal fat pad white adipose tissues were collected and weighed. Hair samples were collected from selected groups of mice (HFD and HFD + 0.2% NAC) for hair lipid isolation and subsequent thin layer chromatography analysis. [00148] The results demonstrate that NAC treatment at the highest concentration of 1% caused a substantial reduction in weight gain in HFD mice when compared to untreated HFD controls, as shown by terminal body weight analysis (see FIG.22). Notably, NAC treatment at lower concentrations of 0.1% and 0.2% also elicited significant decreases in weight gain among HFD mice. Furthermore, the tested NAC doses were not associated with reduced caloric intake, as the average food consumption rates were comparable between various NAC-treated HFD groups and untreated HFD controls (see FIG.23). [00149] In comparison to HFD controls, NAC treatment resulted in a significant decrease in white adipose tissue mass in HFD mice, even at lower concentrations of 0.1% and 0.2%, as determined by analysis of inguinal and epididymal fat pad weights (see FIG.24). These results suggest that NAC treatment induces an overall negative energy balance in HFD mice where caloric output significantly surpasses caloric intake, even at lower concentrations of 0.1% and 0.2%. [00150] Importantly, HFD mice treated with NAC at concentrations of 0.1%, 0.2%, and 1% exhibited fatty and greasy hair as early as 1 week after treatment initiation (see FIG.25), suggesting that NAC treatment causes a negative energy balance and adipose loss under HFD conditions through enhanced lipid secretion through the skin and/or sebaceous glands, even at lower NAC concentrations of 0.1% and 0.2%. To substantiate this, hair samples from HFD and HFD + NAC 0.2% mice were collected for hair lipid isolation and subsequent thin layer chromatography (TLC) analysis. HFD mice treated with 0.2% NAC exhibited a significantly increased hair lipid mass in comparison to untreated HFD controls (see FIG.26). TLC further revealed that the enhanced lipid mass in HFD + 0.2% NAC mice was mainly due to a significant increase in triglycerides and a large proportion of wax and cholesterol esters (see FIG.26), confirming the involvement of skin and/or sebaceous glands in the enhanced lipid secretion on NAC treatment, even at lower treatment doses. [00151] In summary, these results corroborate the notion that NAC treatment induces adipose loss in HFD mice through the enhanced secretion of lipids via the skin across a range of treatment doses. Example 5: Underlying mechanism of NAC-mediated lipid secretion is independent of adaptive T and B cells and TSLP [00152] Tbet-/-RAG and RAG-deficient mice, both of which lack T and B cells, were used to understand the role that adaptive T and B cells and thymic stromal lymphopoietin (TSLP) may play in the effects of NAC in two separate studies. In the first study, three experimental groups of Tbet-/-RAG mice, which are double KO mice that lack T and B cells and responsiveness to the T cell-mediated TSLP mechanism, were provided food and water ad libitum: normal chow diet (ND), HFD, and HFD + NAC. NAC drinking water (10 g/L) was given to the HFD + NAC group starting three weeks after the HFD was started. The results show that HFD Tbet-/-RAG mice treated with NAC displayed similar adipose loss as compared to wildtype mice treated with NAC, as indicated by comparable reduction in weight gain (compare HFD Tbet-/-RAG mice results shown in FIG.27, Panel A with wildtype results shown in FIG.11) and presence of greasy fur changes (compare HFD Tbet- /-RAG mice results shown in FIG.27, Panel B with wildtype results shown in FIGS.16 and 17). [00153] In the second study, three experimental groups of RAG- deficient mice, are only deficient for the RAG gene (not T-bet) but also lack T and B cells and responsiveness to T cell-mediated TSLP effects, were provided food and water ad libitum: normal chow diet (ND) (n=3), HFD (n=4), and HFD + NAC (n=4). NAC drinking water (10 g/L) was given to the HFD + NAC group starting three weeks after the HFD was started. [00154] The NAC-treated HFD RAG-deficient mice showed less weight gain and significantly reduced white adipose tissue mass as compared to untreated HFD RAG- deficient mice (see FIGS.27-29). In fact, body weight and food consumption of the treated HFD RAG-deficient mice resembled that of the ND RAG-deficient mice (see FIG.28). [00155] The results from these studies demonstrate that the underlying mechanism of NAC- mediated lipid secretion from skin and/or sebaceous glands is independent of adaptive T and B cells and TSLP. REFERENCES Cotter TG and Rinella M, Nonalcoholic fatty liver disease 2020: the state of the disease, Gastroenterology, 2020, 158: 1851-1864. Kwak MS and Kim D, Long-term outcomes of nonalcoholic fatty liver disease, Current Hepatology Reports, 2015, 14: 69–76. Pirillo A, et al., Global epidemiology of dyslipidaemias, Nature Review Cardiology, 2021, 18: 689-700.

Claims

WHAT IS CLAIMED IS: 1. A method of increasing lipid secretion from sebaceous glands, skin, or a combination thereof, in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of N-acetylcysteine (NAC) to the subject.
2. The method of claim 1, wherein the lipid comprises triacylglycerols.
3. The method of claim 1 or 2, wherein the lipid secretion is increased from sebaceous glands.
4. The method of any one of claims 1-3, wherein the subject has a skin disorder; an age- related condition; obesity; an obesity-related condition; metabolic syndrome; fatty liver disease; dyslipidemia; lipodystrophy; polycystic ovary syndrome; diabetes; cardiovascular disease; kidney disease; wound healing; or inflammation or autoimmune condition.
5. A method of treating a lipid disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of N-acetylcysteine (NAC) to the subject.
6. The method of claim 5, wherein the lipid disorder is a triglyceride-related lipid disorder.
7. The method of claim 5, wherein the lipid disorder is selected from fatty liver disease, hyperlipidemia, obesity, and lipodystrophy.
8. A method of treating a skin disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of N-acetylcysteine (NAC) to the subject.
9. The method of claim 8, wherein the skin disorder is selected from sebaceous hyperplasia; sebostatis; age-related skin changes; atopic dermatitis/eczema; dry skin (xerosis); psoriasis; ichthyosis; Sjogren’s syndrome; skin infections; acne inversa; acne vulgaris; seborrheic dermatitis; dry scalp; hair breakage and damage; hair loss disorders; alopecia; dry hair conditions; wound healing; skin complications and conditions in patients with diabetes mellitus; neurodermatitis; chemotherapy-induced skin complications and conditions; and any combination thereof.
10. A method of reducing adipose tissue in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of N-acetylcysteine (NAC) to the subject.
11. The method of claim 10, wherein the adipose tissue is white adipose tissue.
12. The method of claim 10 or 11, wherein the adipose tissue is epididymal adipose tissue, inguinal adipose tissue, or a combination thereof.
13. A method of inhibiting diet-related weight gain in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of N-acetylcysteine (NAC) to the subject.
14. The method of claim 13, wherein the diet-related weight gain is due to a high-fat diet.
15. The method of any one of claims 1-14, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier.
16. The method of claim 15, wherein the pharmaceutical composition is administered orally.
17. The method of claim 16, wherein the pharmaceutical composition is administered in an amount of about 1 to 200 mg/kg/day.
18. The method of claim 17, wherein the pharmaceutical composition is administered in an amount of about 5 to 100 mg/kg/day.
19. The method of any one of claims 1-18, wherein the pharmaceutical composition comprises an aqueous solution.
20. The method of claim 19, wherein the pharmaceutical composition comprises NAC in a concentration of about 0.05 to 10 g/L.
21. The method of claim 20, wherein the pharmaceutical composition comprises NAC in a concentration of about 1 to 5 g/L.
22. The method of claim 15, wherein the pharmaceutical composition is administered intravenously.
23. The method of claim 22, wherein the pharmaceutical composition is administered in an amount of about 25 to 400 mg/kg/day.
24. The method of claim 23, wherein the pharmaceutical composition is administered in an amount of about 50 to 300 mg/kg/day.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017055924A2 (en) * 2015-09-28 2017-04-06 M.G. Therapeutics Ltd. Thiol and disulfide-containing agents for increasing meibomian gland lipid secretion

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Publication number Priority date Publication date Assignee Title
WO2017055924A2 (en) * 2015-09-28 2017-04-06 M.G. Therapeutics Ltd. Thiol and disulfide-containing agents for increasing meibomian gland lipid secretion

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MA YONGJIE; GAO MINGMING; LIU DEXI: "N-acetylcysteine Protects Mice from High Fat Diet-induced Metabolic Disorders", ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 33, no. 8, 9 May 2016 (2016-05-09), Berlin/Heidelberg, pages 2033 - 2042, XP036002024, ISSN: 0724-8741, DOI: 10.1007/s11095-016-1941-1 *
MARDANI NAFISEH, MOZAFARPOOR SAMANEH, GOODARZI AZADEH, NIKKHAH FARAHNAZ: "A systematic review of N‐acetylcysteine for treatment of acne vulgaris and acne‐related associations and consequences: Focus on clinical studies", DERMATOLOGIC THERAPY, WILEY-BLACKWELL PUBLISHING, INC., US, vol. 34, no. 3, 1 May 2021 (2021-05-01), US , XP093073417, ISSN: 1396-0296, DOI: 10.1111/dth.14915 *
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