KR20240022999A - Composition for the prevention or treatment of metabolic inflammatory diseases comprising an eIF2α and its downstream signaling pathway modulator as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of metabolic inflammatory diseases, which contains as an active ingredient a regulator of eIF2α phosphorylation and the downstream signaling pathways resulting therefrom. It was confirmed that ATF4, which is related to eIF2α phosphorylation, is highly related to integrated stress response and metabolic inflammation in eIF2α phosphorylation and the resulting downstream signaling pathway. In addition, it was confirmed that ATF4 deficiency suppresses weight gain and regulates the polarization of macrophages, and eIF2α phosphorylation, which inhibits ATF4, and the resulting regulator of downstream signaling pathways are effective in metabolic inflammatory diseases such as obesity. confirmed. In addition, it was confirmed that eIF2α phosphorylation and the resulting regulator of downstream signaling pathways improved macrophage imbalance by regulating the increased expression of inflammatory macrophages M1 and decreased anti-inflammatory macrophage M2 expression in obesity. , it was confirmed that eIF2α phosphorylation and its regulation of downstream signaling pathways can improve metabolic inflammatory diseases.

Description

Composition for the prevention or treatment of metabolic inflammatory diseases comprising an eIF2α and its downstream signaling pathway modulator as an active ingredient}

The present invention relates to a composition for preventing or treating metabolic inflammatory diseases, which contains as an active ingredient a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway.

When cells are stressed, cellular homeostasis is disrupted, and the integrated stress response is a response induced as a defense mechanism to restore homeostasis. The integrated stress response is a eukaryotic protein synthesis initiation factor mediated by four phosphorylating enzymes: PERK (PKR-like ER kinase), GCN2 (general control nonderepressible 2), PKR (protein kinase R), and HRI (heme-regulated inhibitor kinase). Signaling begins when eukaryotic initiation factor 2 alpha (eIF2α) is phosphorylated, and phosphorylated elF2α inhibits the overall protein synthesis process within the cell, allowing the cell time to respond to stress. In situations where most proteins cannot be synthesized, the synthesis of a transcriptional regulator called ATF4 (activating transcription factor 4) is promoted, and the induced ATF4 moves to the nucleus and increases the transcription of several downstream target genes. Downstream genes of ATF4 include GADD34 (Growth arrest and DNA damage-inducible 34) and tRNA syntheases. By resuming protein synthesis suppressed by elF2α phosphorylation, it induces cells that have overcome stress to synthesize new proteins. do. However, when cellular stress is not resolved and worsens, the cell dies due to increased protein synthesis caused by the above genes and cell death genes including CHOP (C/EBP homologous protein). Therefore, the integrated stress response is called a double-edged sword, and it is important to efficiently control the excessive stress response (hypersensitized) and the insufficient stress response (desensitized).

Meanwhile, as the obesity problem becomes more serious, awareness that obesity itself is a disease is spreading. The progression of obesity can typically be judged by whether or not the person is overweight, and specifically by an increase in body fat in a specific area (for example, abdominal obesity, etc.). When classified as obese, that is, obese people have a higher morbidity and mortality rate compared to other diseases, and compared to people with normal weight, the death rate due to diabetes is four times, the death rate due to mild liver disease is twice, and the death rate due to cerebrovascular disease is four times higher than that of people with normal weight. The mortality rate is reported to be 1.6 times higher and the death rate due to coronary artery disease to be 1.8 times higher.

Recently, research reports have been accumulating that metabolic diseases such as obesity are caused by dysfunction of insulin target cells (liver cells, muscle cells, adipocytes, etc.) caused by chronic inflammation. In the case of metabolic diseases caused by chronic inflammation, the role of macrophages among inflammatory cells is known to be very important.

Accordingly, the present inventors confirmed that the integrated stress response acting on macrophages is involved in the pathological mechanism of metabolic inflammation caused by stress such as obesity, and that controlling the integrated stress response can improve metabolic inflammatory diseases. Upon confirmation, the present invention was completed.

The purpose of the present invention is to provide a composition for preventing or treating metabolic inflammatory diseases, which includes eIF2α phosphorylation and a regulator of downstream signaling pathways resulting therefrom as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving metabolic inflammatory diseases, which contains eIF2α phosphorylation and the resulting downstream signaling pathway regulator as an active ingredient.

Another object of the present invention is to increase the expression of anti-inflammatory macrophage M2 (anti-inflammatory M2) containing eIF2α phosphorylation and the resulting downstream signaling pathway regulator as an active ingredient, or to increase the expression of pro-inflammatory macrophage M1 (pro-inflammatory M1). To provide a composition for suppressing expression.

In order to achieve the above object, a composition for preventing or treating metabolic inflammatory diseases is provided, which includes a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving metabolic inflammatory diseases, which includes eIF2α phosphorylation and a regulator of downstream signaling pathways resulting therefrom as an active ingredient.

In addition, a composition for increasing the expression of anti-inflammatory macrophage M2 (anti-inflammatory M2) or inhibiting the expression of pro-inflammatory macrophage M1 (pro-inflammatory M1) containing eIF2α phosphorylation and the resulting downstream signaling pathway regulator as an active ingredient. to provide.

The present invention has identified that eIF2α phosphorylation and the resulting downstream signaling pathways are related to metabolic inflammatory diseases, and that ATF4, which is related to eIF2α phosphorylation in eIF2α phosphorylation and the resulting downstream signaling pathways, is also related to integrated stress response and metabolic inflammation. It was confirmed that was high. In addition, it was confirmed that ATF4 deficiency suppresses weight gain and regulates the polarization of macrophages, and eIF2α phosphorylation, which inhibits ATF4, and the resulting regulator of downstream signaling pathways are effective in metabolic inflammatory diseases such as obesity. confirmed. In addition, it was confirmed that eIF2α phosphorylation and the resulting regulator of downstream signaling pathways improved macrophage imbalance by regulating the increased expression of inflammatory macrophages M1 and decreased anti-inflammatory macrophage M2 expression in obesity. , eIF2α phosphorylation and the resulting regulator of downstream signaling pathways can improve metabolic inflammatory diseases and can be usefully used in the treatment of metabolic inflammatory diseases such as obesity.

Figure 1 is a schematic diagram of the present invention regarding eIF2α phosphorylation and the resulting downstream signaling pathway regulator. Due to stress, eIF2α phosphorylation is induced and a transcriptional regulator called ATF4 is induced, and the transcription of downstream genes is sequentially regulated, thereby activating the integrated stress response. The polarization of macrophages is controlled through the regulator that controls this activation, alleviating inflammation. This diagram shows the process of alleviating inflammation by restoring M2 macrophages, ultimately improving liver fibrosis, a metabolic inflammatory disease, and increasing the activation of brown/beige fat.
Figure 2 schematically illustrates the process of obesity according to the specific gravity of macrophages. In normal conditions, the proportion of M2 among macrophages is relatively high, but when stress is induced, the proportion of M2 decreases and the proportion of M1 increases. Due to the strengthening of the pro-inflammatory function of M1 and the weakening of the anti-inflammatory function of M1, metabolic As the inflammatory response worsens, metabolic inflammatory disease appears. In addition, as the proportion of M2 decreases, the activity of brown and beige fat that consumes surplus energy decreases, so the surplus energy cannot be consumed and accumulates, falling into a vicious cycle that leads to obesity and further worsens metabolic inflammatory diseases. At this time, eIF2a phosphorylation and downstream signaling pathways play a major role, and when this signaling pathway is controlled, the proportion of M2 increases and the proportion of M1 decreases, improving the metabolic inflammation situation, and at the same time, brown and beige fat due to increased M2. This diagram shows that the function is activated again and consumes more energy, thereby alleviating obesity.
Figure 3 is a diagram confirming changes in mouse obesity phenotype according to high-fat diet feeding.
A: Check abdominal fat and organ tissue
B: Confirmation of liver, epididymal white adipose tissue (epWAT) and subcutaneous white adipose tissue (scWAT)
C: Quantification of weight change
D: Check blood sugar levels
E: Check glucose tolerance
F: Confirmation of insulin resistance
Figure 4 is a diagram showing the inflammatory response of mouse adipose tissue following high-fat diet feeding with a crown-like structure (CLS) (A: confirmation of epWAT, B: confirmation of scWAT).
Figure 5 is a diagram confirming the expression of inflammatory factors and ER stress markers in total adipose tissue following high-fat diet feeding (A: epWAT confirmed, B: scWAT confirmed).
Figure 6 is a diagram confirming the expression of inflammatory factors and ER stress markers in the stromal vascular fraction (SVF) according to high-fat diet feeding (A: epWAT confirmed, B: scWAT confirmed).
Figure 7 is a diagram confirming macrophage polarization according to high-fat diet feeding.
A: Confirmation of epWAT M1 macrophage polarization
B: Confirmation of M1 and M2 macrophage factor expression in epWAT
C: Confirmation of scWAT M1 macrophage polarization
D: Confirmation of M1 and M2 macrophage factor expression in scWAT
Figure 8 is a diagram confirming M1 and M2 macrophage polarization according to treatment with tunicamycin (TM), an ER stress inducer.
A: M1 macrophage polarization flow cytometry results
B: Confirmation of expression of M1 macrophage-related factors
C: M2 macrophage polarization flow cytometry results.
D: Confirmation of expression of M2 macrophage-related factors
Figure 9 is a diagram confirming the recovery of M2 polarization of the three lower pathway inhibitors of the unfolded protein response (UPR) in the ER stress response.
A: Confirmation of M2 polarization promotion by ISRIB, an eIF2α-ATF4 pathway inhibitor.
B: Confirmation of M2 polarization promotion by 4μ8C, an inhibitor of the IRE1α-XBP1 pathway.
C: Confirmation of promotion of M2 polarization by Ceapin A7, an ATF6α pathway inhibitor.
Figure 10 is a diagram confirming the obesity phenotype according to the administration of ISRIB in high-fat diet-induced obese mice (A: quantification of organ weight, B: confirmation of blood sugar level, C: quantification of blood lipids).
Figure 11 is a diagram confirming the metabolic phenotype according to the administration of ISRIB in high-fat diet-induced obese mice (A: confirmation of abdominal fat and organs, B: quantification of body weight change, C: confirmation of glucose tolerance, D: confirmation of insulin resistance).
Figure 12 is a diagram confirming liver tissue and liver toxicity following administration of ISRIB in high-fat diet-induced obese mice (A: confirmation of fatty tissue in the liver, B: quantification of liver toxicity factors).
Figure 13 is a diagram confirming CLS following administration of ISRIB in high-fat diet-induced obese mice (A: confirmation and quantification of epWAT CLS, B: confirmation and quantification of scWAT CLS).
Figure 14 is a diagram confirming M1 and M2 macrophage polarization following administration of ISRIB in high-fat diet-induced obese mice by flow cytometry (A: confirmation of eqWAT polarization, B: confirmation of scWAT polarization).
Figure 15 is a diagram confirming the expression of inflammation and ER stress factors in adipose tissue following administration of ISRIB in high-fat diet-induced obese mice.
A: Confirmation of expression of inflammatory factors and ER stress factors in epWAT
B: Confirmation of macrophage polarization and stress factor expression in epWAT
C: Confirmation of expression of inflammatory factors and ER stress factors in scWAT
D: Confirmation of macrophage polarization and stress factor expression in scWAT
Figure 16 is a diagram confirming the amount of food, rectal temperature, and activity according to the administration of ISRIB in high-fat diet-induced obese mice (A: Quantification of food amount, B: Checking rectal temperature, C: Checking oxygen consumption, D: Checking activity) .
Figure 17 is a diagram confirming the expression of thermogenic proteins following administration of ISRIB in high-fat diet-induced obese mice by immunohistochemical staining.
A: Check for brown and beige adipose tissue (BAT)
B: Check scWAT
Figure 18 is a diagram confirming the expression of thermogenic genes following administration of ISRIB in high-fat diet-induced obese mice (A: confirmed in BAT, B: confirmed in scWAT).
Figure 19 is a diagram confirming the expression of thermogenic protein and tyrosine hydroxlase (TH) protein according to administration of ISRIB in high-fat diet-induced obese mice (A: confirmation of BAT, B: confirmation of scWAT).
Figure 20 is a diagram confirming the amount of norepinephrine following administration of ISRIB in high-fat diet-induced obese mice.
Figure 21 is a diagram showing the production method and bone marrow-specific ATF4 deletion of a myeloid ATF4 deletion mouse (A: Schematic illustration of the production method, B: Confirmation of bone marrow-specific ATF4 deletion).
Figure 22 is a diagram confirming the obesity phenotype according to high-fat diet feeding in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ).
A: Check abdominal fat and organs
B: Quantification of weight change
C: Check glucose tolerance
D: Confirmation of insulin resistance
Figure 23 is a diagram confirming the metabolic phenotype according to high-fat diet feeding to myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ).
A: Organ weight quantification
B: Check epWAT and scWAT
C: Confirmation of fat vacuoles in the liver
D: Blood lipid analysis
Figure 24 is a diagram confirming CLS according to high-fat diet feeding in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ) (A: confirmation of CLS in epWAT, B: CLS in scWAT check).
Figure 25 is a diagram confirming M1 and M2 macrophage polarization in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ) by flow cytometry (fed with normal diet).
Figure 26 is a diagram confirming M1 and M2 macrophage polarization according to high-fat diet feeding in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ) (A: confirmation of epWAT polarization, B : Confirm scWAT polarization).
Figure 27 is a diagram confirming the expression of M1 and M2 macrophage markers and stress factors according to high-fat diet feeding in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ).
A: Confirmation of expression in epWAT
B: Confirmation of expression in epWAT-derived SVF
C: Confirmation of expression in scWAT
D: Confirmation of expression in scWAT-derived SVF
Figure 28 is a diagram confirming M1 macrophage polarization according to TM treatment or overexpression of ATF4 in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control group ( Atf4 ^f/f ).
A: Confirmation of ATF4 and CHOP protein expression
B: Confirmation of M1 macrophage polarization in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and controls ( Atf4 ^f/f )
C: Confirmation of M2 macrophage polarization due to overexpression of ATF4
Figure 29 is a diagram confirming M2 macrophage polarization according to TM treatment in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: flow cytometry results and quantification, B : Confirmation of M2 macrophage gene expression, C: Confirmation of M2 macrophage marker protein expression).
Figure 30 is a diagram confirming M2 macrophage polarization due to overexpression of ATF4.
A: Flow cytometry results and quantification
B: Confirmation of M2 macrophage gene expression
C: Confirmation of IL-4 receptor alpha (IL-4ra) expression following TM treatment in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and control ( Atf4 ^f/f ) mice
D: Confirmation of IL-4ra expression following ATF4 overexpression
Figure 31 is a diagram confirming the mechanism by which ATF4 protein inhibits M2 macrophages.
A: Confirmation of changes in protein expression related to M2 polarization signaling following TM treatment
B: Confirmation of liver-enriched inhibitory protein (LIP) expression following TM treatment
C: Confirmation of LIP protein expression after TM treatment and ATF4 deletion and re-expression
Figure 32 is a diagram confirming the amount of food, rectal temperature, and activity according to ATF4 deletion in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: Quantification of food amount, B : check rectal temperature, C: check oxygen consumption, D: check activity).
Figure 33 is a diagram confirming the expression of heat protein in BAT due to ATF4 deletion in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: Confirmation of UCP1 expression, B: UCP1 and TH expression confirmed, C: heat protein gene expression confirmed).
Figure 34 is a diagram confirming heat protein expression in scWAT due to ATF4 deletion in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: Confirmation of UCP1 expression, B: UCP1 and TH expression confirmed, C: heat protein gene expression confirmed).
Figure 35 is a diagram confirming the amount of norepinephrine due to ATF4 deletion in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and control ( Atf4 ^f/f ) mice.
Figure 36 is a diagram confirming the heat metabolism phenotype according to cold stress in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and control ( Atf4 ^f/f ) mice.
A: Check for changes in body temperature
B: Confirmation of UCP1 expression in BAT (immunohistochemical staining)
C: Confirmation of UCP1 and TH expression in BAT
D: Confirmation of heat protein gene expression in BAT
Figure 37 is a diagram quantifying body weight and organ weight according to cold stress in myeloid ATF4 deletion mice and control mice (A: body weight quantification, B: organ weight quantification).
Figure 38 is a diagram confirming the formation of fat vacuoles in adipose tissue due to cold stress in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: Confirmation of BAT vacuoles; B: scWAT fear confirmation).
Figure 39 is a diagram confirming heat protein expression in scWAT according to cold stress in myeloid ATF4 deletion mice ( Atf4 ^f/f ; lyz2-Cre ) and control ( Atf4 ^f/f ) mice (A: Confirmation of UCP1 expression, B: UCP1 and TH expression confirmed, C: heat protein gene expression confirmed).
Figure 40 is a diagram confirming the amount of norepinephrine according to cold stress in myeloid ATF4 deletion mice ( Atf4 ^f/f ;lyz2-Cre ) and control ( Atf4 ^f/f ) mice.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

The present invention provides a composition for preventing or treating metabolic inflammatory diseases, comprising a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway as an active ingredient.

The term “prevention” used in the present invention refers to any action that suppresses the symptoms or delays the progression of a specific disease by administering the composition of the present invention.

The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.

“eIF2α phosphorylation and its resulting downstream signaling pathway” of the present invention is a cellular stress response conserved in eukaryotic cells and refers to a series of reactions that downregulate protein synthesis and upregulate specific genes in response to internal or environmental stress. . eIF2α phosphorylation and the resulting activation of downstream signaling pathways can be induced within cells by exogenous or intrinsic conditions, and hypoxia, amino acid deficiency, glucose deficiency, viral infection, and the presence of oxidants can act as extrinsic factors. , intrinsic factors may include endoplasmic reticulum stress due to the unfolded protein response (UPR) and mitochondrial stress due to inhibition of mitochondrial homeostasis. Stress signals can phosphorylate a subunit of a protein complex called eIF2α, activating the ATF4 gene and affecting the expression of various genes. When eIF2α is phosphorylated, the activity of the complex decreases, leading to a decrease in translation initiation and protein synthesis, which then promotes the expression of the ATF4 gene. The unfolded protein response (UPR) includes PKR-like ER kinase (PERK), Inositol-requiring enzyme 1α (IRE1α), and Activating Transcription Factor 6α (ATF6α). It is initiated by the activation of three ER transmembrane proteins, and continued activation of PERK leads to phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) and C/EBP homologous protein (CHOP). refers to a series of reactions that induce the expression of

In addition, induction of phosphorylated eIF2α and CHOP leads to inhibition of adipogenesis, and the IREα-Xbp1 pathway and ATF6 are known to promote white adipocyte differentiation, respectively. For example, CHOP is known to inhibit white fat production by forming a complex with C/EBPβ and blocking the transcription of target genes responsible for white fat differentiation.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

According to one embodiment of the present invention, the downstream signaling pathway may be ATF4, and phosphorylation of eIF2α may increase the expression of ATF4 (activating transcription factor 4).

According to one embodiment of the present invention, the signal transduction pathway regulator may be a compound represented by the following formula (1).

[Formula 1]

The compound of Formula 1 of the present invention is an integrated stress response inhibitor, and may be a compound of CAS number 1597403-47-8. In the present invention, the compound of Formula 1 or its pharmaceutically acceptable It may further contain salt.

The term "pharmaceutically acceptable salt" means that, within the scope of sound medical judgment, it is intended for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions and the like, and in a reasonable advantage/disadvantage ratio. These salts are meant to be proportional. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or It is a salt of an amino group formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, and cyclopentaneprop. Cypionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- Hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, Oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluene sulfo. nate, undecanoate, valerate salt, and the like.

Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, pharmaceutically acceptable salts include, when appropriate, non-toxic ammoniums, quaternary ammoniums, and counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. Contains amine cations formed using.

According to one embodiment of the present invention, the composition may regulate macrophage polarization, and the macrophages are pro-inflammatory macrophage M1 (pro-inflammatory M1) or anti-inflammatory macrophage M2 (anti-inflammatory M2). It could be.

According to one embodiment of the present invention, controlling macrophage polarization may include suppressing the expression of pro-inflammatory macrophage M1, and suppressing the expression of pro-inflammatory macrophage M1 may include IL-1β (Interleukin 1 beta). , it may inhibit the expression of IL-6 (Interleukin 6) or Nos2 (Nitric oxide synthase 2).

According to one embodiment of the present invention, controlling the macrophage polarization may mean increasing the expression of anti-inflammatory macrophage M2, and increasing the expression of anti-inflammatory macrophage M2 may include Arg1 (arginase-1). ), Ym1 (chitinase 3-like 3), or Fizz1 (resistin like beta) expression.

Referring to Figure 2 of the present invention, in a normal state, the proportion of M2 among macrophages is relatively high, but when stress is induced, the proportion of M2 decreases and the proportion of M1 increases, and the pro-inflammatory function of M1 is strengthened and M1 Due to the weakening of the anti-inflammatory function, the metabolic inflammatory response worsens, resulting in metabolic inflammatory disease. In addition, as the proportion of M2 decreases, the activity of brown and beige fat that consumes surplus energy decreases, so the surplus energy cannot be consumed and accumulates, falling into a vicious cycle that leads to obesity and further worsens metabolic inflammatory diseases. At this time, eIF2a phosphorylation and downstream signaling pathways play a major role, and when this signaling pathway is controlled, the proportion of M2 increases and the proportion of M1 decreases, improving the metabolic inflammation situation, and at the same time, brown and beige fat due to increased M2. The function is activated again, consuming more energy and alleviating obesity.

According to one embodiment of the present invention, the composition may inhibit metabolic inflammation of adipose tissue, and the adipose tissue is inguinal white adipose tissue (iWAT), epididymal white adipose tissue , eWAT) or liver tissue.

According to one embodiment of the present invention, the metabolic inflammation may be the formation of a crown-like structure (CLS) of adipose tissue.

According to one embodiment of the present invention, the composition may increase energy metabolism, and the increase in energy metabolism may increase the formation of brown adipocytes or beige adipocytes, It may increase oxygen consumption.

The “brown adipocytes cells” of the present invention are also called brown fat, and are distinguished from white adipose tissue, which is a common storage fat, due to their brown color. It is composed of cells containing a large amount of mitochondria and oil droplets, and has many sympathetic nerve fibers, so it has high metabolic activity, especially fat decomposition and fatty acid oxidation ability, and is classified as an acid-heating organ for regulating body temperature. It is present in large quantities in newborn animals, and usually degenerates slowly as the thermoregulatory function develops, but it is sometimes found in significant amounts in adults, and increases with cold acclimatization. Brown adipose tissue has the characteristic of rapidly generating a large amount of heat due to the action of noradrenalin, but in the oxidation process of fatty acids, a mechanism that inhibits ATP production is activated due to the low P/O ratio.

The “beige adipocytes cells” of the present invention are fat cells that generate heat to maintain body temperature from external low temperatures, exist within white fat, and possess a large number of mitochondria within the cells. It refers to adipocytes that express developmental regulatory proteins and form polytropic fat. Beige adipocytes exhibit different characteristics from white adipocytes. While white adipocytes are cells that store energy, beige adipocytes are adipocytes that consume energy to generate heat. Beige fat has been reported to be produced from white adipocytes through a pathway called browning, and has different characteristics from white adipocytes, making it a cell that is attracting attention as a treatment target for various metabolic diseases, including obesity.

Referring to Figure 1 of the present invention, eIF2a phosphorylation is induced by various stresses, which induces a transcriptional regulator called ATF4, and the transcription of downstream genes is sequentially regulated, thereby activating the integrated stress response. Through the regulator that controls this activation, It relieves inflammation by controlling the polarization of macrophages. By restoring M2 macrophages, inflammation is alleviated, which ultimately improves liver fibrosis, a metabolic inflammatory disease, and also increases the activation of brown/beige fat, leading to weight loss.

According to one embodiment of the present invention, the increase in energy metabolism may increase body temperature, and increasing the body temperature may increase the expression of a thermogenic protein or a thermogenic gene, and the thermogenic protein is separated. uncoupling protein 1 (UCP1) or It may be tyrosine hydroxylase (TH), and the thermogenic gene consists of UCP1, Prdm16 (PR domain containing 16), Cox8b (Cytochrome c oxidase subunit 8 B), and Pgc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha). It may have been chosen by the military.

According to one embodiment of the present invention, the metabolic inflammatory diseases include obesity, hepatic fibrosis, fatty liver, alcoholic liver disease, and type 1 diabetes. , may be selected from the group consisting of type 2 diabetes, hypoglycemia, hypercholesterinemia, hyperlipidemia, hemochromatosis, amyloidsis, and porphyria, , preferably obesity or liver fibrosis, but is not limited thereto.

In addition, the present invention provides a food composition for preventing or improving metabolic inflammatory diseases, which contains a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway as an active ingredient.

As used herein, the term “improvement” refers to any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a normal food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition to the extract as an active ingredient, the food composition contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating metabolic inflammatory diseases. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high-quality pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agent. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

In addition, the present invention increases the expression of anti-inflammatory macrophage M2 (anti-inflammatory M2) or inhibits the expression of pro-inflammatory macrophage M1 (pro-inflammatory M1), which contains eIF2α phosphorylation and the resulting downstream signaling pathway regulator as an active ingredient. Provides a composition for

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation Example 1> Preparation for confirmation of correlation between eIF2α-ATF4 signaling and obesity

<1-1> Animal model

To produce bone marrow-specific Atf4-knockout mice ( Atf4 ^f/f ;Lyz2-Cre ) of the present invention, Cre recombinase is activated under the control of the lysozyme 2 gene promoter/enhancer element. Lyz2-Cre mice expressing the gene (LysM-Cre) were transformed into Atf4 ^f/f, in which exons 2 and 3 of the Atf4 allele are flanked by the LoxP region. By crossing with mice, Atf4-knockout mice ( Atf4 ^f/f ;Lyz2-Cre ) were created. Afterwards, the mice were fed a normal control diet (NCD or ND). To create diet-induced obesity (DIO) mice, they were fed a high fat diet (HFD) for 16 weeks. In addition, in order to test DIO-induced obesity and to confirm the effect of administering ISRIB (integrated stress response inhibitor, Apexbio, B3699), a drug known to control the integrated stress response, C57BL/6J mice were purchased from Jackson laboratry. ISRIB used in the present invention is a compound of formula 1 below.

[Formula 1]

For the low-temperature exposure experiment, 10-week-old male mice were exposed to a low temperature environment of 4°C, and a control group at room temperature (RT) was placed in an incubator at 25°C. All animal models were reared under a 12-hour dark/light cycle, and all animal care experiments and procedures were performed in accordance with the protocols and guidelines approved by the Animal Care Committee of Soonchunhyang University.

<1-2> Confirmation of metabolic phenotype

For the glucose tolerance test (GTT), mice were fasted for 6 hours and then intraperitoneally injected with 1 g of glucose per kg of body weight. For the insulin tolerance test (ITT), 1 unit/kg of insulin (Lilly, HI0210) was injected using an insulin syringe (BD science, BD328820) in mice that had been fasted for 6 hours. Blood sugar levels were checked with a glucometer (Barogen) at 0, 30, 60, and 120 minutes after injection.

In addition, to inject ISRIB intraperitoneally, ISRIB was administered daily at a concentration of 2.5 mg/kg to C57BL/6J mice fed NCD or HFD. ISRIB was a solvent mixed with DMSO and polyethylene glycol at a volume ratio of 1:1. It was dissolved and used.

Indirect calorimetry measurements were then performed using the Comprehensive Laboratory Animal Monitoring System (CLAMS, TSE Systems). Mice were acclimated to CLAMS for 24 hours before measuring oxygen consumption, and were subject to physical activity for 24 hours. was monitored. Oxygen consumption (VO2), food intake, and physical activity were recorded at 40-minute intervals, and oxygen consumption was corrected using mouse body weight. Additionally, mouse rectal temperature was measured using a probe digital thermometer (Harvard Apparatus).

<1-3> Isolation of stromal vascular fraction from adipose tissue

Mouse epididymal and subcutaneous adipose tissues were isolated, and 1 mg/ml of collagenase type 1 (Corning, H3375) supplemented with 0.5% BSA (Bioshop, ALB001.100) was added. Collagenase type 1, Worthington, LS004196) was added and incubated with shaking at 37°C for 30 minutes to react. After completion of the reaction, 5 mM EDTA was added and treated for 5 minutes to terminate the reaction. Afterwards, the digested tissue was filtered through a 100 μm mesh and centrifuged at 500xg for 5 minutes. Afterwards, the floating adipocytes and stromal vascular fraction (SVF) pellets were resuspended in red-blood-cell lysis buffer (Gibco, A10492-01) for further experiments. It was used for.

<1-4> Plasma biochemical analysis

Blood sugar concentration was measured using a blood glucose meter (glucose stripe, Barogen). Plasma triglyceride, total cholesterol, free fatty acid, alanine transaminase, and aspartate aminotransferase were measured using each kit (Wako, 632- 50991; Wako, 635-50981; Wako, 294-63601; Biovision, K562-100; and Biovision, K552-100).

<1-5> Histological analysis

Liver and adipose tissue were fixed for 18 hours using 4% paraformaldehyde (GeneAll, CNP001-1000) at 4°C. Afterwards, it was treated with ethanol and xylene using a tissue processor (Thermo fisher scientific) and then embedded in paraffin using a paraffin embedder (Thermo fisher scientific). Afterwards, it was sectioned at 4 μm using a microtome (Leica) and stained with hematoxylin & eosin (H&E). For immunohistochemistry (IHC) staining, rehydrated tissue sections were immersed in sodium citrate buffer (pH6.0) at 15 psi for 20 minutes for antigen retrieval, and then incubated in 3% hydrogen peroxide. Processed. Afterwards, treated with Anti-F4/80 (Bio-Rad, MCA497RT), Anti-TNFα (CST, #3707), Anti-ARG1 (CST, #9819) and Anti-UCP1 (Abcam, ab10983), incubated at 4℃ 18 It was reacted for some time. Afterwards, it was treated with secondary antibodies derived from mouse and rabbit (Jackson lab, 115-035-003, 111-035-003), reacted for 2 hours, and visualized using a DAB staining kit (Vector laboratories, SK-4100). did. After DAB staining, hematoxylin was used for counterstaining, and after dehydration with xylene, the section slides were mounted with mounting solution (Fisher chemicals, SP15-100). The IHC staining results were confirmed with a BX61 microscope (Olympus), and for immunofluorescence (IF) expression, Alexa 594 was reacted with a secondary antibody. IF results were confirmed using an Eclipse Ti-U fluorescence microscope (Nikon).

<1-6> Flow cytometry

Isolated SVF was blocked using anti-mouse CD16/32 antibody (Fc block, Biolegend, 101302) at 4°C for 15 minutes. Then FITC-anti-F4/80 (Biolegend, 123108), APC-Cy7-anti-CD45 (Biolegend, 103116), PE-anti-CD11c (Biolegend, 117308), APC-anti-CD206 (Biolegend, 141708) and It was treated with a fluorescent dye-conjugated antibody containing Pacific blue-anti-Annexin V (Biolegend, 640926) and reacted at 4°C for 30 minutes. Afterwards, it was washed three times with phosphate-buffered saline (PBS) and fixed with PBS and 0.1% paraformaldehyde. Afterwards, flow cytometry was performed using BD FACS Canto (BD science), and data were analyzed using Flowjo (BD science).

<1-7> Bone marrow-derived macrophage differentiation analysis

A leg bone obtained from a mouse was dissected, capillaries were obtained using cold RPMI (Corning, 10-040-CV), and these were washed to obtain bone marrow. The obtained bone marrow was centrifuged at 500xg for 5 minutes to obtain a cell pellet. The obtained cell pellet was resuspended in RPMI and filtered through 100 μM mesh to remove extracellular clumps. Isolated bone marrow cells were cultured in differentiation medium [150 ml L929 conditioned medium, 500 ml RPMI, 1% penicillin streptomycin (Corning, 30-002-CI), 1% Primocin (Invivogen, ant-pm-1), 10% cow. Differentiation was achieved by culturing in fetal serum (Equiptech, SFBM30) and 1% non-essential amino acids (Corning, 25-025-CI) and 450 μl 2-mercaptoethanol (Gibco, 21985-023)]. After 7 days of differentiation, bone marrow derived macrophages (BMDM) were used for further experiments, and L929 conditioned medium was cultured in RPMI, 1% penicillin streptomycin, and 10% fetal bovine serum for 7 days in a T75 flask. Manufactured. Culture fluid at each time point was collected using a filter bottle (Corning, 430517).

<1-8> Macrophage polarization analysis

To polarize M1 macrophages in BMDM, 1 μg/ml of LPS and 10 unit/ml of INFγ were treated for 24 hours. Additionally, for M2 polarization, 10 units/ml of IL-4 was treated for 24 hours. All cytokines were used after being diluted in processing medium (RPMI, 10% FBS, 1% PS, 1% NEAA, 0.1% 2-ME).

<1-9> Chemical treatment of macrophages

For ER stress-inducing, 500 ng/ml of tunicamycin was treated for 24 hours under conditions without L929 conditioned medium. Then, for pharmacological inhibition experiments, BMDMs were treated with 100 nM ISRIB and 1 μg/ml CeapinA7 (Merck, SML2330) for 24 hours.

<1-10> Adenovirus infection

The genetic components of the adenovirus vector construct were cloned and inserted into the adenovirus type 5 (ad5) pVQ-K-NpA shuttle vector. Afterwards, the accuracy of the gene sequence was verified using automated DNA sequencing. ViraQuest was used to perform the virus generation and propagation process, and viruses were used to infect cells at a rate of 100 viruses per cell. The viral core used was an adenovirus vector core (University of Michigan) designed to express β-galactosidase. Afterwards, 10 ² viral particles/ml of ATF4 adenovirus and β-galactosidase were treated with BMDM at a cell concentration of 10 ⁶ cells/wll in a 6 well plate, and reacted for 24 hours.

<1-11> Quantitative RT-PCR

Cells and tissues were dissolved using a reagent mixed with TRIzol reagent (Geneall, 301-001) and chloroform at a volume ratio of 1:6. Afterwards, the supernatant was obtained by centrifugation at 13,000 rpm for 15 minutes at 4°C. The obtained supernatant was mixed 1:1 with isopropanol (Sigma, I9516-500ML), and incubated at room temperature for 10 minutes before and after the reaction. Afterwards, RNA was precipitated by centrifugation at 13,000 rpm, and 70% ethanol was added. Afterwards, the RNA pellet was resuspended in distilled water and left at 55°C for 10 minutes. The isolated RNA was quantified using Nano-drop (Thermo Fisher Scientific). cDNA was synthesized from the isolated RNA using a reverse transcriptase kit (Toyobo, TOFSQ-201), the cDNA was diluted 1:40, and quantitative real-time PCR was performed. Quantitative real-time PCR (Bio-Rad) was performed using SYBR Green reagent (Enzynomics, RT501M) and mouse primers, and all target primers were normalized to 18s rRNA primers.

<1-11> Immunoblotting

Cells and tissues were incubated in RIPA buffer (1% NP-40, 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 50mM Tris-HCl, pH 7.4) containing phosphatase/phosphatase inhibitors (Thermo fisher Scientific, 78442). was dissolved for 30 minutes. The dissolved sample was then centrifuged at 12,000 rpm for 20 minutes at 4°C, the supernatant was stored in a new tube, and quantified using a protein quantitative analysis kit (Thermo fisher Scientific, 23225). Afterwards, the quantified sample was heated at 100°C for 5 minutes using 4X laemmli sample buffer (Bio-Rad, BR1610747) and 2-mercaptoethanol (Sigma, M6250-100ML). Afterwards, electrophoresis was performed on an SDS-PAGE gel using an electrophoresis equipment (Bio-Rad) using Tris-glycine buffer (with SDS). After electrophoresis, the separated proteins were transferred to a methanol-activated polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membrane using Tris-glycine buffer (containing 20% methanol, no SDS) using transfer equipment (Bio-Rad). ) Protein was transferred to. Afterwards, blocking was performed with 5% skim milk (BD science, BD232100) for 1 hour, followed by anti-phospho-eIF2α (Abcam, Ab32157), anti-eIF2α (CST, #9722), and anti-ATF4 (CST, #11815). , anti-CHOP (Santacruz, sc-575), anti-α-Tubulin (Sigma, T9026), anti-IL4RA (Santacruz, sc-165974), anti-phospho-STAT6 (CST, #9361), anti-STAT6 (Santacruz, sc-271213), anti-UCP1 (Abcam, ab10983), anti-ARG1 (CST, #9819) and Anti-TNFα (CST, #3707) were added to the membrane and shaken for 18 hours at 4°C. I ordered it. Afterwards, HRP-conjugated mouse and rabbit secondary antibodies (Jackson lab, 115-035-003, 111-035-003) were added and reacted for 1 hour, and Supersignal west pico chemiluminescence substrate (Thermo fisher Scientific, 34080) was added. It was visualized using. Afterwards, it was developed using a developer (Agfa). In all washing steps, Tris-buffered saline with 0.1% Triton-X was used.

<Example 1> Confirmation of macrophage polarization and ER stress induction due to high-fat diet-induced obesity

We confirmed whether ER stress induces obesity-related chronic inflammation in adipose tissue. Specifically, compared to NCD mice, HFD mice had significantly increased body weight and organ weight due to significantly increased blood sugar levels and liver steatosis (Figures 3A to 3D). In addition, HFD mice had decreased glucose tolerance and insulin resistance, confirming that their metabolic phenotype was impaired (Figures 3E and 3F).

Since the development of chronic low-grade metabolic inflammation is induced in white adipose tissue (WAT) in obesity, the inflammatory status of subcutaneous (sc) and epididymal (ep) WAT was checked, and the results showed that scWAT and epWAT In histological analysis, the number of crown-like structures (CLS) was significantly increased in the WAT of HFD obese mice, confirming that immune cells infiltrate adipose tissue (Figures 4A and 4B). . Additionally, the expression levels of inflammatory marker genes, including Adgre1 (F4/80), monocyte chemoattractant protein 1 (Mcp1), and tumor necrosis factor alpha (Tnfa), were significantly increased in scWAT and epWAT of HFD obese mice ( Figures 5A and Figure 5B).

As a result of confirming the inflammatory response of immune cells infiltrating adipose tissue, the stromal vascular fraction (SVF), which represents infiltrated immune cells, showed the expression of inflammatory marker genes in scWAT and epWAT of HFD obese mice, similar to the overall WAT. This significantly increased (Figures 6A and 6B). From the above results, it was confirmed that adipose tissue induces inflammatory stress in HFD obesity.

<Example 2> Confirmation of ER stress correlation in M1/M2 imbalance and HFD obesity

Among the infiltrated immune cells, macrophages are known to be an important factor in obesity-related chronic inflammation in adipose tissue. In particular, M1/M2 imbalance due to an increase in M1 type macrophages and a decrease in M2 type macrophages is a characteristic of metabolic inflammation. It is known. Accordingly, we confirmed whether HFD-induced obesity was related to obesity-related adipose tissue macrophage (ATM) polarization. As a result, in flow cytometry analysis of the SVF of scWAT and epWAT, the number of total macrophages (F4/80+) was increased in HFD mice compared to NCD mice. Compared to NCD mice, in HFD mice, the ratio of M1 ATM (F4/80+;CD11c+) was confirmed to be significantly increased compared to M2 ATM (F4/80+;CD206+) in the SVF of epWAT. On the other hand, there was no significant difference in scWAT (Figures 7A and 7C). Additionally, the mRNA expression of M1-specific genes, including Nos2 (Nitric oxide synthase 2) and IL-6 (Interleukin 6), was significantly increased in the SVF of obese scWAT and epWAT, but Arg1 (arginase-1) and Fizz1 ( It was confirmed that the mRNA expression of M2-specific genes, including resistin-like-α, was significantly reduced (Figures 7B and 7D). In addition, expression of Chop (C/EBP homologous protein), Atf6α (activating transcription factor 6 alpha), and spliced It was confirmed that ER stress markers increased (Figures 5 and 6). The above results confirmed that HDF-induced obesity induces chronic inflammation with an imbalance in the M1/M2 ratio and increased ER stress.

<Example 3> Confirmation of M2 polarization and ER stress-mediated association of eIF2α-ATF4 pathway

Since it is known that ER stress can be a factor in obesity-related metabolic dysfunction, the present invention specifically confirmed that ER stress is induced in ATM caused by WAT in HFD-induced obese mice (FIGS. 5 and 6). Therefore, we confirmed whether obesity-induced ER stress affects macrophage polarization. Specifically, mouse bone marrow derived macrophages (BMDM), in which M1 or M2 polarization was induced by treatment with INFγ and LPS or IL-4, respectively, were used, and ER stress was used as an N-glycosylation inhibitor during the period of macrophage polarization. It was induced by treatment with tunicamycin (TM). As a result, TM treatment increased the number of F4/80+;CD11c+ cells in BMDMs during M1 polarization (Figure 8A), but decreased the number of F4/80+;CD206+ cells in BMDMs during M2 polarization (Figure 8B). ). In addition, the mRNA expression of M1 marker genes IL-1b (Interleukin 1 beta), IL-6, and Nos2 was significantly increased by TM treatment in BMDMs during M1 polarization (Figure 8C), but in contrast, M2 marker genes Arg1 and Ym1 were significantly increased by TM treatment. (chitinase 3-like 3) and Fizz1 expression were significantly decreased by TM treatment in BMDMs during M2 polarization (Figure 8D), indicating that ER stress promotes polarization of M1 macrophages, but polarization of M2 macrophages It was confirmed that it decreased.

Subsequently, in the present invention, among the three pathways of unfolded protein response (UPR), the pathway related to ER stress-mediated inhibition of M2 macrophage polarization was identified. Specifically, to inhibit the eIF2α/ATF4 pathway, IRE1α/XBP1 pathway, and ATF6α pathway, which are UPR pathways related to ER stress, ISRIP, 4μ8c, and Ceapin A7 were treated, respectively. As a result, treatment with ISRIP and 4μ8c significantly increased M2 macrophage markers, which were reduced by TM treatment, confirming that the eIF2α/ATF4 pathway and IRE1α/XBP1 pathway are ER stress-mediated M2 polarization inhibition pathways (Figure 9), it was confirmed that eIF2α/ATF4 signaling is the most important pathway for suppressing M2 polarization in vitro.

<Example 4> Confirmation of decreased metabolic inflammation and M1/M2 ratio due to inhibition of eIF2α-ATF4 pathway

It was determined whether inhibition of the eIF2α-ATF4 pathway could restore suppressed M2 ATM polarization in HFD obese mice. ISRIB was injected intraperitoneally at a concentration of 2.5 mg/Kg into C57BL6 mice fed ND or HFD for 10 weeks, and DMSO was administered to the Vehicle group. As a result, there was no significant difference in body weight or body fat in the ND mouse group compared to the Vehicle group in the mice administered ISRIB. However, in HFD-induced obese mice, it was confirmed that organ weight and blood sugar were decreased in mice administered ISRIB compared to the Vehicle group (Figures 10A and 10B), and body weight was confirmed to be reduced (Figures 11A and 11B). . In addition, it was confirmed that triglyceride (TG), cholesterol, and NEFA levels were significantly reduced (Figure 10C).

In addition, ISRIB-treated obese mice had decreased lipid content in the liver (Figure 12A) and decreased glucose tolerance and insulin resistance (Figures 11C and 11D), suggesting that eIF2α-ATF4 signaling is responsible for obesity in vivo. It was confirmed that metabolic dysfunction was induced and treatment with ISRIB, an inhibitor of eIF2α-ATF4, improved metabolic dysfunction.

In addition, the expression of aspartate transaminase (AST) and alanine transaminase (ALT), which are liver damage factors, did not increase with ISRIB treatment, making ISRIB a safe substance that does not induce liver damage. This was confirmed (Figure 12B).

Afterwards, it was confirmed whether the metabolic phenotype improved by ISRIB administration affects chronic inflammation and macrophage polarization in HFD-fed mice. As a result, it was confirmed that the CLS structure was significantly reduced in both epWAT and scWAT in the ISRIB group compared to the Vehicle group in HFD obese mice, confirming that inflammation in adipose tissue was improved by ISRIB treatment (Figures 13A and 13B). . In addition, we examined whether the reduction of inflammation by ISRIB under HFD feeding was correlated with macrophage polarization. As a result, the number of F4/80+;CD11c+ macrophages in SVF derived from epWAT or scWAT of DMSO- or ISRIB-treated mice in HFD obese mice was It was confirmed that the number of F4/80+;CD206+ macrophages was significantly increased (FIGS. 14A and 14B), confirming that ISRIB suppresses M1 polarization and increases M2 polarization in HFD obese mice. In addition, in adipose tissue, the expression of inflammatory genes Tnfa and Mcp1, and UPR genes Chop, sXBP1, and Atf6α were confirmed to be significantly reduced by treatment with ISRIB (Figures 15A and 15C), and M1-specific genes It was confirmed that the mRNA expression of IL-6 and Nos2 was significantly decreased, and the mRNA expression of M2-specific genes Arg1 and Fizz1 was significantly increased by administration of ISRIB (Figures 15B and 15D). As a result of the above results, it was confirmed that when eIF2α-ATF4 signaling in vivo is inhibited by ISRIB administration, obesity and related metabolic symptoms are improved.

<Example 5> Confirmation of brown and beige adipocyte differentiation due to inhibition of eIF2α-ATF4 pathway

In Example 4, it was confirmed that the administration of ISRIB delayed weight gain in HFD obese mice, and since ISRIB is known to affect brain regions that can affect food intake, the administration of ISRIB decreased the weight gain in mice. We checked whether there were any changes in food intake. As a result, it was confirmed that there was no significant difference in the dietary amount according to the administration of DMSO or ISRIB in ND mice or HFD obese mice (FIG. 16A). However, in HFD obese mice, an increase in rectal body temperature was confirmed in the ISRIB-administered group compared to the Vehicle group, and there was no significant difference in core body temperature in ND-fed mice (FIG. 16B). In addition, when ISRIB was administered to HFD obese mice, oxygen consumption (VO ₂ ) was confirmed to significantly increase (Figure 16C), and significant differences in physical activity were confirmed between the Vehicle group and the ISRIB group in ND mice and HFD obese mice. (Figure 16D), it was confirmed from the above results that inhibiting the eIF2α-ATF4 pathway with ISRIB improves diet-induced obesity by increasing energy consumption without changing the amount of food or activity.

Brown and beige adipose tissue (BAT) is a special cell that plays an important role in energy consumption through heat generation. It generates heat by dissociating uncoupling protein 1 (UCP1), thereby promoting mitochondrial respiration. This is a process that involves the separation of cells, which convert stored energy into heat to naturally regulate body temperature and energy metabolism. Accordingly, the present invention confirmed whether inhibition of eIF2α-ATF4 signaling could increase the thermogenic activity of brown and beige adipocytes. Expression of UCP1 was significantly increased in BAT and scWAT of both ND mice and HFD obese mice administered ISRIB (Figures 17A and 17B), and in BAT and scWAT of HFD obese mice, fever was observed in the ISRIB group compared to the Vehicle group. It was confirmed that the mRNA expression of developmental genes Ucp1, Prdm16 (PR domain containing 16), Cox8b (Cytochrome c oxidase subunit 8 B), and Pgc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) was significantly increased (Figure 18A and Figure 18B). In addition, it was confirmed that the amounts of UCP1 protein and tyrosine hydroxylase (TH) in BAT and scWAT increased with the administration of ISRIB in HDF obese mice (Figures 19A and 19B). Additionally, it was confirmed that administration of ISRIB increased the level of norepinephrine, a neurotransmitter released from sympathetic nerve terminals (FIG. 20). Norepinephrine is known to activate thermogenesis in brown and beige adipocytes, and from the above results, it can be seen that the eIF2α-ATF4 pathway reduces thermogenesis in brown and beige adipocytes, but the heat was reduced by treatment with ISRIB. It was confirmed that production was increased and energy metabolism was promoted.

<Example 6> Confirmation of decreased metabolic function and inflammation due to ATF4 deficiency

From the above examples, it was confirmed that the eIF2α-ATF4 pathway suppresses M2 macrophage polarization and reduces thermogenic activity in HFD obese mice. However, since the administration of ISRIB can also affect various organs of the body, the use of ISRIB It was specifically confirmed whether administration inhibited the eIF2α-ATF4 pathway. As ATF4 is a key downstream regulator in the eIF2α pathway, mice expressing CRE recombinase under the control of the lysozyme promoter (Lyz2-cre) were crossed with Atf4 to generate myeloid-specific Atf4-deficient ( Atf4 ^f/f ;Lyz2-cre ) mice. It was manufactured (Figure 21A). ATF4 protein was selectively deleted in BMDM and peritoneal macrophages, and was found to be expressed normally in epWAT and scWAT, confirming myeloid ATF4 deletion (Figure 21B). Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f There were no significant changes in the phenotype that occurred when mice were fed an ND diet, and no significant differences were observed in body weight, serum lipid content, liver lipid accumulation, glucose, and insulin resistance (Figures 22A to 22D, Figure 23A to Figure 23D). However, when HFD was fed to Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f mice, it was confirmed that body weight was significantly reduced in Atf4 ^f/f ;Lyz2-cre mice compared to Atf4f ^f/f mice (Figure 22A and Figure 22B). In addition, it was confirmed that lipid levels were reduced in the liver and serum (Figures 23C and 23D), and glucose tolerance and insulin resistance were improved (Figures 22C and 22D). The sizes of epWAT and scWAT were significantly reduced in Atf4 f ^/f ;Lyz2-cre mice compared to Atf4f f ^/f mice when fed on a HFD (Figure 23B), consistent with the results found in mice administered ISRIB. Compared to Atf4f ^f/f mice, CLS was significantly reduced in epWAT and scWAT of Atf4 ^f/f ;Lyz2-cre mice (Figures 24A and 24B). From the above results, it was confirmed that deletion of Atf4 reduces inflammation in adipose tissue.

<Example 7> Confirmation of M1/M2 polarization correction effect in ATF4 bone marrow-specific deletion mice

We determined whether myeloid-specific deletion of Atf4 affects macrophage polarization. Specifically, flow cytometry was performed using SVF isolated from epWAT or scWAT of Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f mice fed ND and HFD, and Atf4 ^f/f ;Lyz2 fed ND. There was no significant difference in F4/80+;CD11c+ and F4/80+;CD206+ macrophages in SVF derived from epWAT and scWAT of -cre and Atf4f ^f/f mice (Figure 25). However, compared to Atf4f ^f/f mice fed HDF, F4/80+;CD11c+ cells were significantly reduced in SVF derived from epWAT and scWAT of Atf4 ^f/f ;Lyz2-cre , and F4/80+;CD206+ cells were significantly reduced. was confirmed to be significantly increased (Figures 26A and 26B), confirming that myeloid ATF4 deletion suppresses M1 polarization induced by HFD and improves suppression of M2 polarization.

Additionally, to further confirm the effect of Atf4 deletion on macrophage polarization, macrophages were analyzed in adipose tissue as well as in the SVF of scWAT and epWAT of ND- or HFD-fed Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f mice. As a result of confirming the expression of mRNA related to polarization, compared to Atf4f ^f/f mice fed a HFD, mRNA expression of M1-specific genes IL-6 and Nos2 was decreased in Atf4 ^f/f ;Lyz2-cre mice in all tissues. It was confirmed that the expression of M2-specific genes Arg1 and Fizz1 was significantly increased (Figures 27A and 27C), and the same expression was also confirmed in SVF derived from scWAT and epWAT (Figures 27B and 27D). . In particular, compared with Atf4f ^f/f mice, the level of Chop was significantly reduced in the SVF of Atf4 ^f/f ;Lyz2-cre , indicating that the eIF2α-ATF4 signaling pathway was activated in macrophages of Atf4 ^f/f ;Lyz2-cre mice. This suggests that it has been effectively removed from .

<Example 8> Confirmation of the association of ER stress inhibition with M2 polarization of ATF4

We confirmed whether ATF4 regulates macrophage polarization in a cell-autonomous manner under ER stress conditions. BMDM isolated from Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f mice were treated with 10 ng/ml of INFγ or IL-4 and LPS (10 ng/ml). Afterwards, M1 and M2 polarization according to TM treatment was confirmed (Figure 28A). As a result, as in the results of the above example, ER stress increases the number of F4/80+;CD11c+ cells and the number of F4/80+;CD206+ cells in BMDM derived from Atf4f ^f/f mice upon treatment with TM. was decreased, but no significant difference was observed in the numbers of F4/80+;CD11c+ cells and F4/80+;CD206+ cells in BMDM of Atf4 ^f/f ;Lyz2-cre mice (Figures 28B and 29A). In addition, it was confirmed that the mRNA expression of M2-specific genes Arg1, Ym, and Fizz1, which were decreased upon TM treatment, was reversed in ATF4-deficient BMDM (Figure 29B), and the expression of ARG1 protein was also increased following Atf4 deletion in BMDM (Figure 29B). 29C).

In addition, to further confirm the role of ATF4, adenoviruses expressing ATF4 (Ad-ATF4) or β-gal protein (Ad-β-gal) under the control of the CMV (cytomegalovirus) promoter were used, and M2 polarization vs. The number of phagocytes (F4/80+; CD206+) was significantly decreased by Ad-ATF4 infection, but in Ad-β-gal infection, there was no significant difference in expression (Figure 30A), and the High expression of ATF4 increased the number of F4/80+;CD11c+ cells (Figure 28C). In addition, high-expression induction of ATF4 significantly increased the mRNA expression of the Chop gene and significantly decreased the mRNA expression of the M2-specific genes Arg1, Ym1, and Fizz1 (Figure 30B). The above results confirmed that ATF4 contributes to the inhibition of M2 polarization under ER stress conditions.

Afterwards, the mechanism by which ATF4 suppresses M2 polarization was confirmed. Polarization of macrophages toward the M2 subtype involves binding of IL-4 to its receptor (IL-4 receptor, IL-4R), starting with subsequent phosphorylation of STAT6 and activating the expression of the M2 gene, a component of the IL-4R. Since it was confirmed that the mRNA expression of the encoding IL-4rα gene was significantly reduced by ER stress in mouse embryonic fibroblast (MEF), the effect of ATF4 on IL-4rα expression was confirmed. As a result, it was confirmed that treatment with TM significantly reduced IL-4rα mRNA expression in Atf4f/f BMDMs compared to Atf4f/f;Lyz2-cre BMDMs (Figure 30C), and the induction of high expression of ATF4 resulted in an increase in IL-4rα gene expression. mRNA expression was suppressed (Figure 30D).

Additionally, administration of TM significantly reduced the expression of IL-4rα protein in Atf4f/f mice, but there was no significant change in expression in Atf4f/f;Lyz2-cre mice. In addition, the phosphorylated form of STAT6 (pSTAT6), a downstream pathway of IL-4 receptor signaling, was induced by treatment with IL-4, but was decreased by treatment with TM in Atf4f ^f/f f mice. However, in Atf4 ^f/f ;Lyz2-cre mice, no significant difference in pSTAT6 expression was observed due to TM treatment (Figure 31A).

Afterwards, we confirmed how the eIF2α-ATF4 pathway suppresses IL-4rα gene expression. The C/EBPβ gene can produce three isoforms, including liver-enriched activator protein (LAP), LPA-2, and liver-enriched inhibitory protein (LIP). It is known that, among isoform proteins, LIP increases significantly upon ER stress in an eIF2α-ATF4-dependent manner. Additionally, LIP induced by ER stress can inhibit the transcription of the IL-4rα gene in MEFs. As a result, LIP protein expression was significantly increased in Atf4f ^f/f BMDMs, but LIP protein expression was significantly decreased in Atf4 ^f/f ;Lyz2-cre BMDMs (Figure 31B). In addition, when the expression of ATF4 was overexpressed by infection with Ad-ATF4, the LIP protein level increased in Atf4 ^f/f ;Lyz2-cre BMDM (Figure 31C), and as a result, LIP was associated with ER stress and ATF4 expression. It was confirmed that it acts as a transcriptional repressor that suppresses the expression of IL-4rα. Therefore, it was confirmed that activation of the eIF2α-ATF4 pathway and subsequent induction of LIP suppresses IL-4rα gene expression, leading to impairment of IL-4/IL-13 signaling required for M2 polarization.

<Example 9> Confirmation of increased brown and beige fat energy consumption in ATF4 bone marrow-specific deletion

It was confirmed that inhibition of eIF2α-ATF4 signaling by ISRIB administration increased energy consumption and brown/beige adipocyte activity, but its direct role in the macrophage pathway was confirmed. In addition, in the above example, it was confirmed that Atf4 ^f/f ;Lyz2-cre mice were resistant to HFD-induced obesity compared to Atf4f ^f/f mice, and similar to the results of ISRIB treatment, Atf4 ^f/f ;Lyz2-cre When ND and HFD were fed to mice and Atf4f ^f/f mice, there was no significant change in food amount (Figure 32A), but when HFD was fed to Atf4 ^f/f ;Lyz2-cre mice, rectal temperature was significantly decreased. An increase was confirmed (Figure 32B). In addition, total oxygen consumption (VO ₂ ), when quantified by lean mass, was significantly increased in Atf4 f/f ;Lyz2-cre mice than in Atf4f ^f/f mice fed HFD (Figure 32C), and was significantly increased in Atf4 ^f/f ; Lyz2-cre mice fed HFD or ND. There was no significant difference in physical activity in each mouse (Figure 32D). Therefore, it was confirmed that the difference in energy expenditure between Atf4 ^f/f ;Lyz2-cre and Atf4f ^f/f mice is not correlated with food intake, total activity, genotype, or amount of food, but is related to thermogenic ability.

Additionally, considering the similarity in phenotype with ISRIB-administered mice, activation of BAT and beige adipocytes in WAT was confirmed. As a result, immunohistochemical staining and immunoblotting analysis showed that the expression of UCP1 protein was significantly increased in the BAT of Atf4 ^f/f ;Lyz2-cre mice compared to Atf4f f ^/f mice fed HFD (Figure 33A and Figure 33B), it was confirmed that the expression of thermogenic proteins Ucp1, Prdm16, Cox8b, and Pcg1α was significantly increased (Figure 33C). In particular, along with UCP1 protein expression in BAT, HFD-fed Atf4 ^f/f ;Lyz2-cre mice had significantly increased UCP1 and TH protein levels in scWAT (Figures 34A and 34B), and thermogenic proteins Ucp1, Prdm16, and Cox8b. And the expression of Pcg1α was also increased in scWAT of HFD-fed Atf4 ^f/f ;Lyz2-cre mice compared to HFD-fed Atf4f ^f/ f mice (FIG. 34C). In addition, similar to ISRIB administration, the level of norepinephrine, a neurotransmitter secreted from sympathetic nerve terminals, also increased (Figure 35), and as a result, ER stress and induction of ATF4 in macrophages suppress energy consumption and obesity. It has been confirmed that it promotes development.

<Example 10> Confirmation of increased cold-induced thermogenesis in ATF4 bone marrow-specific deletion

The effect of bone marrow-specific deletion of ATF4 on adaptive thermogenesis was confirmed. Specifically, mice were exposed to cold stress at 4°C, and as a result, compared to Atf4f ^f/f mice, Atf4 ^f/f ;Lyz2-cre mice were confirmed to maintain body temperature for up to 48 hours (Figure 36A ). Additionally, there was no significant difference in body weight and BAT weight (Figures 37A and 37B). BAT of Atf4 ^f/f ;Lyz2-cre mice had reduced lipid vacuole size (Figure 38A) and increased expression of UCP1 (Figures 36B and 36C) compared to Atf4f ^f/f mice. Expression of thermogenic genes Ucp1, Prdm16, Cox8b, and Pcg1α was increased in BAT of Atf4 f ^/f ;Lyz2-cre mice than in Atf4f ^f/f mice (Figure 36D).

In addition, scWAT from Atf4 ^f/f ;Lyz2-cre mice exposed to a cold environment showed a significant increase in the number of beige adipocytes expressing UCP1 (Figures 38B and 39A), and increased expression of UCP1 and TH. (Figure 39B), the expression of thermogenic genes increased (Figure 39C).

In addition, as in ISRIB-administered and HFD-induced obese Atf4 ^f/f ;Lyz2-cre mice, norepinephrine levels were confirmed to increase in serum and BAT (Figures 40A and 40B), and as a result, ATF4 bone marrow-specific deletion It was confirmed that this increased the thermogenic activity of both BAT and beige fat cells, thereby increasing energy consumption.

Therefore, the present invention has identified that eIF2α phosphorylation and the resulting downstream signaling pathways are related to metabolic inflammatory diseases, and that ATF4, which is related to eIF2α phosphorylation in eIF2α phosphorylation and the resulting downstream signaling pathways, is involved in integrated stress response and metabolic inflammation. It was confirmed that the degree of relevance was high. In addition, it was confirmed that ATF4 deficiency suppresses weight gain and regulates the polarization of macrophages, and eIF2α phosphorylation, which inhibits ATF4, and the resulting regulator of downstream signaling pathways are effective in metabolic inflammatory diseases such as obesity. confirmed. In addition, it was confirmed that eIF2α phosphorylation and the resulting regulator of downstream signaling pathways improved macrophage imbalance by regulating the increased expression of inflammatory macrophages M1 and decreased anti-inflammatory macrophage M2 expression in obesity. , it was confirmed that eIF2α phosphorylation and its regulation of downstream signaling pathways can improve metabolic inflammatory diseases.

Claims (26)

A composition for preventing or treating metabolic inflammatory diseases, comprising as an active ingredient a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway. According to clause 1,
A composition wherein the lower signaling pathway is ATF4. According to clause 1,
A composition wherein the signal transduction pathway regulator is a compound represented by the following formula (1):
[Formula 1]
. According to clause 3,
A composition wherein the compound inhibits ATF4, a downstream signaling pathway of eIF2α. According to clause 1,
The composition regulates macrophage polarization. According to clause 5,
The composition, wherein the macrophages are pro-inflammatory macrophage M1 (pro-inflammatory M1) or anti-inflammatory macrophage M2 (anti-inflammatory M2). According to clause 5,
A composition that regulates macrophage polarization by suppressing the expression of pro-inflammatory macrophage M1. According to clause 7,
A composition that inhibits the expression of pro-inflammatory macrophage M1 by inhibiting the expression of IL-1β (Interleukin 1 beta), IL-6 (Interleukin 6), or Nos2 (Nitric oxide synthase 2). According to clause 5,
A composition that regulates macrophage polarization by increasing the expression of anti-inflammatory macrophage M2. According to clause 9,
A composition wherein increasing the expression of the anti-inflammatory macrophage M2 increases the expression of Arg1 (arginase-1), Ym1 (chitinase 3-like 3), or Fizz1 (resistin-like-α). According to clause 1,
The composition is for suppressing metabolic inflammation of adipose tissue. According to clause 11,
The composition, wherein the adipose tissue is inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), or liver tissue. According to clause 11,
The composition, wherein the metabolic inflammation results in the formation of a crown-like structure (CLS) of adipose tissue. According to clause 1,
The composition increases energy metabolism. According to clause 14,
A composition wherein the increase in energy metabolism increases the formation of brown adipocytes or beige adipocytes. According to clause 14,
The composition, wherein the increase in energy metabolism increases oxygen consumption. According to clause 14,
The composition, wherein the increase in energy metabolism increases body temperature. According to clause 17,
A composition wherein increasing the body temperature increases expression of a thermogenic protein or thermogenic gene. According to clause 18,
A composition wherein the heat generating protein is uncoupling protein 1 (UCP1) or thyroid hormone. According to clause 18,
The composition, wherein the thermogenic gene is selected from the group consisting of UCP1, Prdm16 (PR domain containing 16), Cox8b (Cytochrome c oxidase subunit 8 B), and Pgc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha). According to clause 14,
A composition that increases the energy metabolism by increasing the amount of norepinephrine in the serum. According to clause 1,
The metabolic inflammatory diseases include obesity, hepatic fibrosis, fatty liver, alcoholic liver disease, type 1 diabetes, and type 2 diabetes. ), hypoglycemia, hypercholesterinemia, hyperlipidemia, hemochromatosis, amyloidsis and porphyria. A food composition for preventing or improving metabolic inflammatory diseases, containing eIF2α phosphorylation and a regulator of downstream signaling pathways resulting therefrom as an active ingredient. According to clause 23,
The food composition, wherein the signal transduction pathway regulator is a compound represented by the following formula (1):
[Formula 1]
A composition for increasing the expression of anti-inflammatory macrophage M2 (anti-inflammatory M2) or inhibiting the expression of pro-inflammatory macrophage M1 (pro-inflammatory M1), comprising as an active ingredient a regulator of eIF2α phosphorylation and the resulting downstream signaling pathway. According to clause 25,
The food composition, wherein the signal transduction pathway regulator is a compound represented by the following formula (1):
[Formula 1]
.