KR20170025909A - Pharmaceutical composition for preventing or treating metabolic dysfunction comprising sphingosine 1-phosphate or a Sphk2 expression-elevating agent - Google Patents

Pharmaceutical composition for preventing or treating metabolic dysfunction comprising sphingosine 1-phosphate or a Sphk2 expression-elevating agent Download PDF

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KR20170025909A
KR20170025909A KR1020150123024A KR20150123024A KR20170025909A KR 20170025909 A KR20170025909 A KR 20170025909A KR 1020150123024 A KR1020150123024 A KR 1020150123024A KR 20150123024 A KR20150123024 A KR 20150123024A KR 20170025909 A KR20170025909 A KR 20170025909A
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sphk2
sphingosine
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박태식
심순미
이수연
맹효진
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가천대학교 산학협력단
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Abstract

The present invention relates to a pharmaceutical composition, food composition or cosmetic composition for preventing or treating metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate synergist and an Sphk2 expression-enhancing agent, and a method for screening a material for preventing or treating metabolic dysfunction using the above mechanism. The pharmaceutical composition, food composition or cosmetic composition according to the present invention is useful for preventing or treating metabolic dysfunction, particularly for preventing or treating fatty liver, such as non-alcoholic fatty liver, dyslipidemia such as hyperlipidemia or hypertriglyceridemia and obesity. The screening method using the above mechanism is useful for screening a material for preventing or treating metabolic dysfunction.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating metabolic dysfunction, which comprises a sphingosine 1-phosphate or a substance which enhances the expression of Sphk2.

The present invention relates to a pharmaceutical composition for preventing or treating metabolic dysfunction, and more particularly, to a pharmaceutical composition for preventing or treating metabolic dysfunction, which comprises a sphingosine 1-phosphate, a sphingosine 1-phosphate ascorbate and a sphk2 expression enhancer And to a pharmaceutical composition for preventing or treating metabolic dysfunction.

Obesity due to excessive nutrient intake is a major cause of metabolic dysfunctions such as insulin resistance, hypertension and cardiovascular disease (Non-Patent Documents 1-3). Elevated plasma free fatty acid (FFA) leads to abnormal fatty acid (FA) uptake in the liver, adipose tissue and heart, which causes fatty liver, inflammation and cardiac dysfunction, respectively (Non-Patent Document 4-6 ). Increased tissue uptake of FFA under obesity conditions leads to the synthesis of triglycerides (TG), phospholipids and sphingolipids. In this process, the synthesis of certain lipid metabolites is known to provide signaling molecules that regulate the metabolic function system (Non-Patent Document 7). Lipid-mediated regulation of the cell's insulin signaling pathway is associated with the activation of serine / threonine kinase, leading to the progression of insulin function in insulin-sensitive tissues (Non-Patent Documents 8-10).

Endoplasmic reticulum (ER) stress is induced by a variety of cellular injuries, including glucose depletion, inflammatory conditions, and destruction of calcium homeostasis (Non-Patent Document 11). Since ER is a major organ in the cell that is responsible for protein folding and maturation as well as protein transport to other cell compartments, when the ER function becomes abnormal due to accumulation of unfolded protein, it is referred to as unfolded protein response (UPR) And thereby relieves the ER-related stress (Non-Patent Documents 11 to 13). UPR is the release of GRP78 from the ER and (i) the pathway of protein kinase RNA-like ER associated kinase (PERK), (ii) the inositol-requiring protein 1? IRE1? Pathway, and (iii) the activating transcription factor 6 (ATF6) pathway (Non-Patent Document 14). The PERK pathway is activated in diet-induced obese (DIO) mice, while chemical inducers that induce acute ER stress, such as tunicamycin and thapsigargin, All of the UPR signaling pathways are induced, suggesting that the respective pathways are distinct from each other under different conditions of physiological stress (Non-Patent Document 15-17). These transcription factors are involved in the expression of UPR genes, including chaperones and lipid-producing genes.

Sphingosine kinase (SphK) catalyzes phosphorylation of sphingosine to synthesize sphingosine 1-phosphate (S1P), a lysophospholipid (Non-Patent Document 18). SphK is a 49 kDa protein which has been purified and confirmed from rat kidney, and includes Sphk1a and Sphk1b as a result of cloning and characterization (Non-Patent Document 19, 20). The second isoform, Sphk2, is highly homologous to Sphk1, and both isoforms have five conserved domains found in lipid kinases (Non-Patent Document 19). Sphk1 is highly expressed in lung, spleen, kidney, and blood, while Sphk2 is expressed mainly in liver, kidney, and heart (Non-Patent Documents 20 and 21). The S1P level is precisely controlled by the synthesis by SphK catalyst, the irreversible decomposition by S1P degrading enzyme (lyase) and the formation of sphingosine by dephosphorylation by S1P phosphatase (Non-Patent Document 22). S1P is a ligand for the specific receptor G protein (G protein receptor, S1P 1 -S1P 5) family. S1P functions through secretion and binding to S1P G-protein receptors, a process referred to as " inside-out " signaling (Non-Patent Document 22). Despite their similarity and biochemical function, Sphk1 is pro-apoptotic, while Sphk2 has an anti-apoptotic role as opposed to this (Non-Patent Document 23).

However, little is known about the regulation of Sphk2 and the role of Sphk2 in liver glucose / lipid metabolism, and the use of Sphk2-related mechanisms in chronic metabolic diseases has not been reported.

Korean Patent Registration No. 2008-0108523 Korean Patent Laid-Open Publication No. 2013-0115311 European Patent Registration No. 0652755

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The present inventors investigated whether ER stress induces Sphk2 expression and elevate liver S1P. Sphk2 is upregulated by acute ER stress, and Sphk2, which is expressed in liver, is involved in proximal insulin signaling Independently activating AKT (also called serine / threonine-specific protein kinase, PKB, AKT) leads to upregulation of oxidative genes and increased FA oxidation resulting in a decrease in lipid droplet accumulation and And the effect of improving glucose sensitivity. These results confirmed that the UPR pathway regulates Sphk2 under specific physiological conditions and that S1P production alleviates ER stress-mediated abnormalities through activation of FA oxidation.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate ascender, and Sphk2 expression enhancer, food composition , A cosmetic composition and a method for screening a substance for preventing or treating metabolic dysfunction using the above-mentioned mechanism.

According to one aspect of the present invention there is provided a pharmaceutical composition for preventing or treating metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate ascensor, and Sphk2 expression enhancer / RTI >

In one embodiment, the Sphk2 may be liver specific Sphk2.

In one embodiment, the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia, and obesity, wherein the fatty liver may be a nonalcoholic fatty liver, wherein the dyslipidemia is hyperlipidemia or hypertriglyceridemia .

In one embodiment, the agent that raises the expression of Sphk2 may be DNA or RNA.

In one embodiment, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or additives.

According to one aspect of the present invention, there is provided a food composition for metabolic dysfunction improvement comprising any one selected from the group consisting of a sphingosine 1-phosphate, a sphingosine 1-phosphate ascertant, and a Sphk2 expression enhancer / RTI >

In one embodiment, the food composition may be any one selected from the group consisting of dietary supplements, health functional foods, and food additives.

According to one aspect of the present invention, there is provided a cosmetic composition for improving metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate ascorbate, and Sphk2 expression enhancer / RTI >

Further, according to one aspect of the present invention, there is provided a method of preparing a cell or tissue of a high fat dietary animal, comprising: (a) preparing a cell or tissue of a high fat dietary animal; (b) contacting the test substance to the cell or tissue of step (a); (c) measuring the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in the cell or tissue of step (b); And (d) selecting a test substance exhibiting an increase in the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein compared to a control group in which the test substance is not treated. Is provided.

As a result of overexpression of hepatocyte Sphk2 by injecting adenovirus containing Sphk2 gene into high-fat diet feeding mice, Sphk2 overexpression in liver and S1P increase resulting in upregulation of oxidative gene and activation of fatty acid oxidation It has been shown that it alleviates ER stress - mediated anomalies by decreasing the local accumulation of insulin by increasing the insulin sensitivity and insulin resistance by activating the insulin signaling system through the increase of pAKT. In addition, when Sphk2 overexpression was observed, the expression of fatty acid oxidases and the activation of fatty acid oxidation (increased) decreased triglyceride and oxygen uptake increased with time, indicating that increased expression of Sphk2 increased hepatic glucose and fatty acid metabolism It was found to improve.

Therefore, the composition comprising any one selected from the group consisting of a sphingosine 1-phosphate, a sphingosine 1-phosphate ascender and a substance that enhances the expression of Sphk2 of the present invention is useful as a metabolic dysfunction, particularly a non- Food compositions and cosmetic compositions for the prevention or treatment of dyslipidemia and obesity such as hyperlipidemia, hyperlipidemia or hypertriglyceridemia, and the screening method using the above mechanism may be used for preventing or treating metabolic dysfunction It can be usefully used as a screening method for screening substances.

FIG. 1 shows the relative levels ((A) and (B)) of the mRNA of the sphingolipid biosynthetic genes in ER stress induced mice with tunicamycin, the expression ratio (C) of Sphk2 mRNA with time, And the amount of Sphk2 protein (D). Specifically, DMSO or tunicamycin (2.5 μg / g body weight, n = 5 per group) was injected intraperitoneally ((A) and (B)) into wild type (WT) C57Bl / 6 mice Liver was separated and quantified by quantitative PCR analysis (n = 5, mean ± SEM, * p <0.05). Primary mouse hepatocytes were harvested after treatment with tunicamycin (1.25 [mu] g / ml) or DMSO for the indicated times. Quantitative RT-PCR analysis was performed to confirm Sphk2 expression (C) (n = 3, mean ± SEM, * p <0.05 compared to control). Primary mouse hepatocytes were harvested after treatment with tunicamycin (1.25 μg / ml) or DMSO for 12 hours. The amount of Sphk2 protein was determined by SDS-PAGE and immunoblotting (D). CerS2-5 (Ceramide synthase2-5, ceramide synthase 2-5); AcCer (Acid ceramidase, acid ceramidase); Acer1-3 (Alkaline ceramidase 1-3, Alkaline ceramidase 1-3); ATF4 (activating transcription factor 4, transcription activation factor 4); ATF6 (activating transcription factor 6, transcription activation factor 6); CHOP (C / EBP homologous protein, C / EBP homologous protein); NCer (neutral ceramidase, neutral ceramidase); Smpd1 (acid sphingomyelinase, acid sphingomyelin degrading enzyme); Smpd2 (neutral sphingomyelinase 1, neutral sphingomyelinase 1); Smpd3 (neutral sphingomyelinase 2, neutral sphingomyelinase 2); Sphk1-2 (sphingosine kinase 1-2, sphingosine kinase 1-2); sXBP1 (spliced XBP1); uXBP1 (unsliced XBP1).
Figure 2 shows that hyperlipidemia and inflammatory ER stress differently regulate Sphk2. WT C57B1 / 6 mice were fed with either normal diet or 60 kcal% high fat diet for 4 weeks and liver samples were taken and quantitative RT-PCR was performed on liver RNA (A). Immunoblotting analysis was performed on tissue lysates between mice (B). Saline or LPS was injected intraperitoneally into WT C57B1 / 6 mice and samples were taken 8 hours after injection. Quantitative RT-PCR using liver mRNA was performed to measure gene expression (C). Western blot analysis was performed on tissue lysates between mice (D). n = 6, mean + -SEM, * p < 0.05.
Figure 3 shows the expression of ER stress genes in the liver after 8 weeks (A) and 12 weeks (B) feeding of high fat diets (n = 6, mean ± SEM, * p <0.05).
Figure 4 shows the results of a high fat diet changing ceramide and sphingomyelin levels in plasma and liver (ceramide (A, D), sphingomyelin (B, E), and sphinganine, sphingosine, (C, F) containing 1-phosphate, n = 6, mean ± SEM. * P <0.05 vs normal dietary group).
FIG. 5 shows the result of Sphk2 expression measurement after co-transfection of the reporter construct comprising the Sphk2 promoter with ATF4, sXBP1 or CHOP, respectively. Specifically, pcDNA 3.0 (0.2 μg or 0.4 μg) containing the Sphk2 reporter construct and ATF4, sXBP1 or CHOP was co-transfected into HepG2 cells. The promoter activity was measured by the luciferase assay. (A) (n = 5, mean ± SEM, * p <0.05 vs. control group pcDNA3.0 control, #p <0.05 vs. 0.2 μg transfected group). Primary mouse hepatocytes were infected with AdGFP, AdATF4 or AdsXBP1 as a control for 24 hours. Immunoblotting analysis was performed on cell lysates (B). HepG2 cells were transfected with control or ATF4 siRNA and then treated with tunicamycin (1.25 μg / ml) for 6 hours. Cells were then harvested and mRNA expression was measured by quantitative RT-PCR. n = 3, mean + -SEM, * p < Control siRNA (C). Western blot analysis of cell lysates (D).
FIG. 6 shows the expression levels (A), sphingoid base levels (B), ceramide levels (C) and (C) levels of each protein after infection with primary mouse liver cells with adenovirus expressing human Sphk2 (AdSphk2) (D) of Sphingomyelin (SM), and the expression level (E) of each protein after administration of HNMPA to Sphk2-overexpressing cells. Specifically, primary mouse hepatocytes were infected with AdGFP or AdSphk2 in a gene-concentration-dependent manner for 24 hours. Cells were harvested and cell lysates were analyzed by immunoblotting (A). Three independent experiments were performed and the results were shown. Primary mouse hepatocytes were infected with AdGFP or AdSphk2 for 24 hours at 5 MOI. Sphingoid base (B), ceramide (C), and sphingomyelin (SM) (D) were measured by LC-MS / MS (n = 3, mean ± SEM, * p <0.05 vs. AdGFP, sphinganine (SA), sphingosine (SO), sphingosine 1-phosphate (S1P), ceramide (Cer), sphingomyelin (SM). HepG2 cells were infected with AdGFP or AdSphk2 for 24 h at 5 MOI and treated with insulin (50 nM) for 10 min. Another group of cells was treated with HNMPA- (AM) 3 for 6 h and then with insulin for 10 min. After harvesting the cells, the cell lysates were analyzed by immunoblotting (E).
Figure 7 shows that Sphk2 overexpression changes the plasma ceramide (A), dihydroceramide (B), sphingosine (C), sphingosine 1-phosphate (C), sphingomyelin (D) and glucosylceramide . Specifically, WT C57Bl / 6 mice were fed a normal diet or a 60 kcal% high fat diet for 4 weeks and then infected with AdGFP or AdSphk2 (1 X 10 9 PFU / ml) into the tail vein. Plasma was collected 7 days after injection. (N = 7, mean ± SEM) of ceramide (A), dihydroceramide (B), sphingoid base (C), sphingomyelin (D) and glucosyl ceramide . * p = 0.05 vs. Ad-GFP virus-injected mice).
FIG. 8 shows that Sphk2 overexpression induces the expression of ceramide (A), dihydroceramide (B), sphinganine (C), sphingosine (C), sphingosine 1-phosphate (C), sphingomyelin (E) and cholesterol / triglyceride (F). Specifically, WT C57Bl / 6 mice fed with high fat diet (60 kcal% fat) for 4 weeks were infected with AdGFP or AdSphk2 (1 X 10 9 PFU / ml) by a tail vein injection. The mouse liver was isolated 14 days after injection. The ceramide (A), dihydroceramide (B), sphingoid base (C), SM (D), and glucosyl ceramide (E) in the liver were measured by LC-MS / MS. After lipid extraction from liver tissue, total cholesterol and TG were measured by colorimetric method (F) (n = 7-8, mean ± SEM. * P <0.05 vs. AdGFP-injected mice).
9 is a graph showing the expression (A) of the lipid-producing gene or the FA oxidation-related gene, the level of protein expressed from the gene (B), the lipid (C) in the liver, the level of plasma? -Hydroxybutyrate And the oxygen consumption rate (OCR) (E) in the presence of palmitic acid. Specifically, AdGFP or AdSphk2 (1 X 10 9 PFU) was injected into the tail vein in WT C57bl6 / J mice fed high fat diets (60 kcal% fat). Mouse liver and plasma were isolated 14 days after injection. FA biosynthesis and oxidation-related gene expression were confirmed by quantitative RT-PCR (A) (n = 7, mean ± SEM. * P <0.05). Immunoblot analysis was performed on liver lysates at 14 days after injection (B). After the liver was separated and frozen, the frozen section samples were stained with oil red O (top) (C) and hematoxylin and eosin (H & E) (bottom) (C) (40x magnification photograph). (D) (n = 7, mean + -SEM. * P < 0.05) in plasma was measured for? -Hydroxybutyrate (ketone body). Primary mouse hepatocytes were infected with AdGFP and AdSphk2 at 5 MOI, treated with the BSA-palmitate complex and incubated for 2 hours, and the oxygen consumption rate was measured at the indicated time (E) (n = 5, mean ± SEM . * p < 0.05).
10 shows the expression levels of plasma glucose (A), plasma insulin level (B), plasma glucose (C), glycogen synthase gene expression (D) and AKT phosphorylation level in skeletal muscle / adipose tissue in Sphk2 overexpressing mice E). Specifically, AdGFP or AdSphk2 (1 X 10 9 ) was injected into the tail vein in WT C57Bl / 6 mice fed high fat diets (60 kcal% fat) for 4 weeks. Experiments were performed 7 days after adenovirus injection. The mice fasted for 16 hours were injected with 1 g / kg body weight of glucose and plasma glucose was measured at the indicated time (A). Plasma insulin levels were measured during the glucose tolerance test (B). Mice were fasted for 4 hours and insulin (1 unit / kg body weight) was injected intraperitoneally and plasma glucose was measured at the indicated times (AdGFP (n = 7) and AdSphk2 (n = 8), mean ± SEM. * p < 0.05 vs. AdGFP (C)). Mice were divided into two groups, one group being the control group and the other group being injected intraperitoneally with insulin (0.5 unit / kg body weight) for 10 minutes. Western blot analysis of insulin signal transduction proteins was performed by separating liver (D). The skeletal muscle (E) and fat cells (F) were separated and Western blot analysis was performed. Two independent experiments were performed to show the results (n = 6 per group).

The present invention provides a pharmaceutical composition for preventing or treating metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate ascorbate, and Sphk2 expression enhancer.

In one embodiment, the Sphk2 may be, but is not limited to, a liver-specific Sphk2.

In one embodiment, the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia, and obesity, wherein the fatty liver may be a nonalcoholic fatty liver, wherein the dyslipidemia is hyperlipidemia or hypertriglyceridemia .

In one embodiment, the agent that raises the expression of Sphk2 may be DNA or RNA.

In one embodiment, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or additives. That is, the pharmaceutical composition of the present invention can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method . The pharmaceutically acceptable carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It also includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Solid form preparations for oral use include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like, and may include lubricants such as magnesium stearate and talc. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like. Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, ethyl And injectable esters such as oleate. As a suppository base, witepsol, macrogol, tween, cacao butter, laurin, glycerogelatin and the like can be used.

The dosage of sphingosine 1-phosphate, sphingosine 1-phosphate, and the substance that enhances the expression of Sphk2 contained in the pharmaceutical composition of the present invention depends on the condition and body weight of the patient, the degree of disease, the drug form, Depending on the duration, it may be suitably selected by those skilled in the art. For example, the substance that elevates the expression of sphingosine 1-phosphate, the sphingosine 1-phosphate ascender and the expression of Sphk2 is 0.0001 to 1000 mg / kg, preferably 0.01 to 1000 mg / kg per day The above administration may be administered once a day or divided into several times. In addition, the pharmaceutical composition of the present invention may contain 0.001 to 50% by weight of the sphingosine 1-phosphate, sphingosine 1-phosphate ascorbate, and Sphk2 expression enhancer based on the total weight of the composition.

The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes, for example, oral, intraperitoneal, rectal or intravenous, intramuscular, subcutaneous, intrauterine, intracerebroventricular &Lt; / RTI &gt;

Further, according to one aspect of the present invention, there is provided a food composition for improving metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate ascorbate and a substance which raises the expression of Sphk2, A cosmetic composition is provided.

In one embodiment, the food composition may be any one selected from the group consisting of dietary supplements, health functional foods, and food additives.

In one embodiment, the Sphk2 may be, but is not limited to, a liver-specific Sphk2.

In one embodiment, the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia, and obesity, wherein the fatty liver may be a nonalcoholic fatty liver, wherein the dyslipidemia is hyperlipidemia or hypertriglyceridemia .

In one embodiment, the agent that raises the expression of Sphk2 may be DNA or RNA.

According to one aspect of the present invention, there is provided a cosmetic composition for improving metabolic dysfunction, which comprises any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate ascorbate, and Sphk2 expression enhancer / RTI &gt;

Further, according to one aspect of the present invention, there is provided a method of preparing a cell or tissue of a high fat dietary animal, comprising: (a) preparing a cell or tissue of a high fat dietary animal; (b) contacting the test substance to the cell or tissue of step (a); (c) measuring the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in the cell or tissue of step (b); And (d) selecting a test substance exhibiting an increase in the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein compared to a control group in which the test substance is not treated. Is provided.

In one embodiment, the Sphk2 may be, but is not limited to, a liver-specific Sphk2.

In one embodiment, the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia, and obesity, wherein the fatty liver may be a nonalcoholic fatty liver, wherein the dyslipidemia is hyperlipidemia or hypertriglyceridemia .

In one embodiment, the test material may be DNA or RNA.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

< Example >

1. Materials

C18: 0, C18: 1), sphingomyelin (SM 16: 0, C18: 0, C18: 1), ceramides (C14: 0, C16: 0, C18: 0, C18: (SA), sphingosine 1-phosphate (S1P), and glucosyl ceramide (C16: 0, C18: 0, C18: 1, C24: 1) were purchased from Avanti Polar Lipids, Alabaster, AL). Acetonitrile, methanol and chloroform were purchased from Fisher Scientific (Pittsburgh, Pa.). Insulin was purchased from Eli Lilly (Humulin), and tunicamycin was purchased from Sigma-Aldrich (St. Louis, Mo.). HNMPA- (AM) 3 was purchased from Abcam (Cambridge, Mass.) And K145 was purchased from Sigma-Aldrich (St. Louis, Mo.). ATF4-specific siRNA and control siRNA were purchased from Qiagen (Boston, Mass.).

2. Animal experiments

An 8-week-old male C57Bl / 6 mouse was purchased from Charles River Laboratories and fed and fed free of water and diets in a pathogen-free animal breeding facility (usually diet: Lab Diet Inc.). ) Picolab rodent diet 20, high fat diet: 60 kcal% fat diet for 4 weeks; Research Diet Inc. D12492). Recombinant adenovirus (1 × 10 9 PFU) was injected into the tail vein. For the glucose tolerance test, mice were fasted for 16 hours and then glucose (2 g / kg body weight) was injected into the peritoneal cavity (Non-Patent Document 41). For my insulin function test, the mice were fasted for 4 hours and insulin (1 Unit / kg body weight) was injected intraperitoneally. Blood was collected from the tail vein at a given time and blood glucose was measured. To determine the insulin response, insulin (0.5 unit / kg body weight) was injected intraperitoneally for 10 minutes prior to liver isolation. ER stress was induced by intraperitoneal injection of tunicamycin (2.5 μg / g body weight for 2 hours) and lipid polysaccharide (LPS, 3 μg / g body weight, for 8 hours). For gene expression and lipid metabolism analysis, mice were fasted for 6 hours and sacrificed to separate and collect blood and tissues.

3. Cell culture

HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 10 units / ml penicillin and 10 μg / ml streptomycin at 37 ° C and 5% CO 2 . Five MOIs (multiplicity of infection) of GFP and Sphk2 adenovirus were infected with HepG2 cells for 24 hours. Insulin (50 nM) was treated for 10 minutes and the cells were harvested. Another cell was treated with DMSO or HNMPA- (AM) 3 (10 uM) for 6 h and then treated with insulin for 10 min. After adenoviral infection or transfection of the over-expression vector, the cells were cultured in insulin-free serum-free medium for 16 hours to measure expression and insulin effect.

4. Primary hepatocyte preparation and culture

8 weeks old wild-type (WT) C57BL6 male mice were anesthetized and the liver was isolated. Primary hepatocytes were prepared as described below according to the method described in the prior art (Non-Patent Document 24). Cells were cultured in M199 medium supplemented with 10% FBS, 10 units / ml penicillin, 10 ug / ml streptomycin, and 10 nM dexamethasone (Welgene, Korea). After cells were attached, cells were infected with each adenovirus for 16-24 hours. In addition, the cells were maintained in serum-free medium for one day and then treated with tunicamycin (1.25 μg / ml) for a defined period of time. For ATF4 knockdown experiments, control and ATF4 siRNA oligomers (Qiagen) were transfected with lipofectamine (Invitrogen, Carlsbad, Calif.). After 48 hours of incubation, tunicamycin (1.25 μg / ml) was incubated in serum-free conditions.

5. Plasmids and recombination Adenovirus  Produce

The promoter sequence of Sphk2 (-2050 / -1) was amplified from mouse genomic DNA by PCR and inserted into pGL3 basic vector (Promega) to construct a pSphk2-pGL3 reporter construct. pcDNA3-flag-ATF4 and pcDNA-flag-sXBP1 were obtained from fellow researchers (Dr. Kobayashi, Korea University, Seoul, Korea). Recombinant adenovirus was prepared using a vector system (AdEasy Adenoviral Vector System, Stratagene, La Jolla, CA, USA) and a vector (pAdTrack CMV vector) (Non-Patent Document 25). Mouse Sphk2 cDNA was cloned into AdTrack CMV vector and recombined using cells (BJ5183-AD-1 Electroporation Competent cells, Stratagene, Calif.). After confirming recombination, the total adenovirus genome was isolated and transfected into HEK293 cells. CsCl concentration gradient centrifugation, dialyzed overnight at 4 ° C in sucrose buffer (10 mM Tris-HCl pH 8.0, 2 mM MgCl 2, 4% sucrose) and the adenovirus was purified. The purified adenovirus was stored at -80 DEG C until use (Non-Patent Document 42). As a control, adenovirus expressing green fluorescent protein was prepared and used by the method described above.

6. Trait injection and Luciferase  Reporter Measurements

When HepG2 cells (4 x 10 5 cells / ml) were confluent at 80-90% in 10% FBS, 10 units / ml penicillin, and 10 μg / ml streptomycin supplemented culture medium (Ham's F12 medium) Lt; RTI ID = 0.0 &gt; 24-well &lt; / RTI &gt; Using the transfection reagent (FuGENE® HD transfection reagent, Roche, Mannheim, Germany) according to the manufacturer's instructions, the cells were transfected with pSphk2-pGL3 reporter construct; And co-transfected with an ER stress-marker cloning vector containing ATF4, ATF6 or sXBP1-pcDNA3.0, respectively. Transfection efficiency was standardized and corrected using pTK-RL, an expression vector for Renilla luciferase. After incubation for 24 hours, the cells were washed with PBS, dissolved in a buffer (Passive Lysis buffer), and analyzed with a fluorescence detector (Dual-Luciferase Assay System (Promega, Madison, Wis.), CentroXS3 LB930 (Berthold Technologies GmbH & )) Was used to measure fluorescence.

7. RNA production and quantitative real-time PCR (real-time PCR )

Total RNA was isolated from HepG2 cells and primary mouse hepatic cells using an RNA extraction kit (easy-spin Total RNA Extraction Kit, Intron Biotechnology, Korea). Liver tissues were homogenized and mRNA was isolated using a separation reagent (TRIzol reagent, Invitrogen) according to the manufacturer's instructions. First strand cDNA was synthesized using a cDNA synthesis kit (iScript ™ cDNA Synthesis Kit, Bio-Rad, Hercules, Calif.). Real-time PCR analysis was performed using a real-time PCR system (ABI7300 equipment (Applied Biosystems Inc, Carlsbad, CA) and SYBR Green Master Mix (Takara, Japan)). Expression of the gene was normalized and corrected for? -Actin or cyclophilin. Primer sequences are as follows.

Figure pat00001

Figure pat00002

8. Western Blot  analysis

Total cellular proteins were lysed with lysis buffer containing 10 mM Tris (pH 7.8), 1 mM EDTA, 150 mM NaCl, a phosphatase inhibitor and a protease inhibitor. Using 30 μg of protein, immunoblotting was performed according to the method described in the prior art (Non-Patent Document 26). actin, AKT, phospho-AKT (Ser473), insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), phosphor-tyrosine ), Sphk2, glucose 6-phosphatase (G6Pase), PEPCK, ATF4, PPARα (peroxisome proliferation activated receptor α), carnitine palmitoyltransferase 1 (CPT1), ACOX1 (acyl CoA oxidase 1, Abcam, Cambridge, MA) , And XBP-1 (Santa Cruz Biotechnology, Santa Cruz, Calif.). The blot was developed with a chemiluminescent substrate (Millipore, Bilecica, Calif.) And probed with a fluorescence image analyzer (LAS4000 luminescent image analyzer, Fujifilm, Japan).

9. Sphingoid query  LC / MS / MS analysis

To analyze the sphingolipids, cells were harvested and lysed in PBS. A known amount of C17: 0-ceramide (internal standard) was added to the cell extract containing 1 mg of protein, and chloroform / methanol (2: 1, v / v) containing 0.01% butylated hydroxytoluene Were used to extract sphingolipids. KOH was added and the phospholipids were saponified at 37 ° C for 2 hours. The extract was neutralized by the addition of acetic acid, and the organic phase was separated and dried under nitrogen gas. (C16: 0, C18: 0, C24: 0, C24: 1), sphinganine, sphingosine, S1P, SM (C16: 0, C16: 0, C18: 0, C18: (XTerra C18, 3.5 [mu] m, 2.1 * 50 mm) was separated by HPLC on a C18 column (C18: 1) and glucosylceramide (C16: 0, C18: 0, C18: 1, C24: 43) and ionized in a positive electrospray ionization mode. MS-MS (MS / MS) system was used for the quantitative analysis of multiple reaction monitoring (MRM) using an API 4000 Q-trap (Applied biosystem, Framingham, Mass.) And interfaced with an electrospray ionization source The [M +] / product ion from the relevant sphingolipid metabolite was measured.

10. Metabolite  Measure

Blood glucose was measured under basal conditions in the glucose tolerance test using an automatic glucose monitor (One Touch, Lifescan). Plasma and liver triglyceride, cholesterol, HDL, LDL, and NEFA were measured with a colorimetric kit (Wako, Wako Pure Chemical Industries, Ltd., Japan). Insulin was measured using a mouse insulin ELISA kit (ALPCO). ALT and AST activity were measured by activity assay kit (Sigma Aldrich). Plasma ketone (β-hydroxybutyrate) was measured by colorimetric assay kit (Cayman chemical, Ann Arbor, Mich.).

11. Measurement of oxygen consumption rate

The oxygen consumption rate (OCR) was measured using an extracellular Flux Analyzer (XF-24, Seahorse Biosciences, North Billerica, MA, USA). Primary mouse hepatocytes were inoculated into 24-well plates (2 x 10 &lt; 4 &gt; / plate) and incubated overnight in M199 medium with 10% FBS. The cells were washed with the medium, infected with adenovirus containing GFP or Sphk2, and then cultured for 24 hours. Buffer solution in the same buffer for one hour at (Krebs-Henseleit buffer, 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO 4, 1.2 mM Na 2 HPO 4, 2.5 mM glucose and 0.5 mM carnitine) to wash the cells, 37 ℃ Lt; / RTI &gt; The cells were then loaded into an analyzer (XF24 analyzer) and the oxygen consumption rate was measured at short and repeated intervals. BSA (17 μM) or BSA complexed palmitic acid (final concentration 150 uM). The mixing, atmospheric and measurement times were 3, 2, and 3 minutes, respectively.

12. Histology

For tissue analysis, the liver was separated and fixed in 10% buffered formalin for 24 hours, or frozen in OCT insertion medium. The 5-μm sections of the liver inserted in paraffin were stained with hematoxylin and eosin (Sigma-Aldrich). The frozen slice samples were stained with oil red O. Images were obtained directly using a slide scanner.

13. Statistical Analysis

Results were expressed as mean ± SEM. Comparisons between different groups were performed with a two-tailed unpaired Student's t-test. P values less than 0.05 (P < 0.05) were considered statistically significant.

14. Results

(1) ER stress is induced in mouse primary hepatocytes and In vivo Sphk2  Expression Upwards

Recent studies have shown that ER stress activates liver lipid biosynthesis by upregulating major lipid-producing genes such as DGAT2 (Dacylglycerol acyltransferase 2), SREBP1c (sterol responsive element binding protein 1c), and LIPIN2 (Non-Patent Document 27) . Since sphingophyll biosynthesis is a non-oxidative FA pathway, we decided to investigate whether ER stress regulates the biosynthesis of sphingolipid, a signaling lipid of metabolic control disorder. In order to examine the above mechanism in vivo, the transfection of the liver with the WT mouse was performed by administering an acute ER stress inducing substance, tunicamycin, to the mouse, and the expression of the sphingolipid biosynthetic gene was measured.

As shown in Fig. 1 (A), among the sphingolipid biosynthetic genes, CerS3, Acer2, and Acer3 were upregulated. In addition, UPR gene expression including ATF4, ATF6, CHOP, sXBP1, and uXBP1 was all upregulated (see Fig. 1 (B)). As a result, it was confirmed that Sphk2, which is the end point of upregulation in the sphingolipid biosynthetic pathway, was induced by tunicamycin globally at 6 hours after the administration of the tunicamycin.

Sphk2 mRNA and protein levels were increased in a time-dependent manner when primary mouse hepatic cells were treated with tunicamycin (see Figures 1 (C) and 1 (D)). On the other hand, Sphk1, another isotype of SphK, was scarcely found under any conditions (see Fig. 1 (B)). These results indicate that Sphk2 is globally regulated by acute ER stress due to tunicamycin.

(2) high fat diets and Endotoxin  ER stress Activate , Sphk2  Expression is regulated differently

Since tunicamycin induces the expression of all UPR genes, we have examined which ER stress-dependent signaling pathways are directly associated with Sphk2 upregulation. To test whether Sphk2 is regulated by ER stress in a physiological environment, mice were fed high fat diets (HFD) for 4 weeks to produce hyperlipemic ER stress conditions. As a result, it was found that the expression of liver Sphk2 mRNA and protein was inhibited in the liver of mice fed with HFD as compared with the control mice (see Figs. 2 (A) and 2 (B)).

Liver sXBP-1 activation was found in 4-week HFD fed mice, while prolonged HFD feeding (see HFD feeding at 8 and 12 weeks, Figures 3 (A) and 3 (B) , ATF6 did not change and ATF4 expression was inhibited (see Figs. 2 (A) and 2 (B)). HFD elevated ceramide and sphingomyelin (SM) levels in plasma and liver, but S1P was reduced (no statistical significance, see FIG. 4).

Another lipid polysaccharide (LPS), an ER stress activator, induced UPR genes such as ATF4 and CHOP (C / EBP homologous proteins), but did not induce sXBP1 (see Figures 2 (C) and 2 (D)). Although most of the lipogenesis genes were down-regulated, Sphk2 mRNA and protein levels were elevated (see Figures 2 (C) and 2 (D)). These results indicate that LPS-mediated ER stress induces Sphk2, but that the regulation of Sphk2 is dependent on the pathophysiological conditions for ER stress induction.

(3) ATF4  Activation Sphk2  Across the board Upwards

To study the different regulation of Sphk2 by ATF4 and sXBP1, Sphk2 expression was measured after co-transfection of reporter constructs containing the Sphk2 promoter with ATF4, sXBP1 or CHOP, respectively. As a result, sXBP1 inhibited Sphk2 expression, whereas ATF4 increased the induction of Sphk2 by 6-fold (see Fig. 5 (A)). We also examined the effect of overexpressing CHOP, a direct, transcriptional target of ATF4, on the expression of Sphk2. However, Sphk2 promoter activity was not changed, confirming that Sphk2 expression was induced only by ATF4 activation. Consistent with these results, adenovirus overexpression of ATF4 in primary mouse hepatic cells increased the level of Sphk2 protein, but showed no change in sXBP1 overexpression (see FIG. 5 (B)). To confirm that Sphk2 is a direct sub-target of ATF4, ATF4 siRNA was transfected. Downregulation of ATF4 resulted in the suppression of Sphk2 mRNA and protein levels (see Figures 5 (C) and 5 (D)). These results confirmed that Sphk2 was globally regulated by ATF4.

(4) Sphk2 -medium S1P  Creation AKT  Phosphorylation Activate

The effects of ceramide and S1P on the regulation of the insulin signaling pathway have been reported (non-patent documents 28, 29). S1P, the product of Sphk, is known to improve glucose homeostasis in diabetic animal models (Non-Patent Document 29). Adenoviruses expressing human Sphk2 (AdSphk2) were prepared and used to infect primary mouse hepatic cells to ascertain whether elevated Sphk2 expression regulates ER stress-dependent signaling pathways.

As a result, phosphorylation of AKT was increased in a gene-concentration-dependent manner by AdSphk2 infection (see Fig. 6 (A)) (Non-Patent Document 29). However, there was no change in the level of pIRS1 or pIRS2, and the increased pAKT was not associated with IRS1 or IRS2, the proximal insulin signaling intermediate protein. Thus, increased pAKT was found to be independent of tyrosine phosphorylation of IRS. Sphk2 expression did not alter sphingolipid metabolites such as sphinganine (SA), sphingosine (SO), ceramide (Cer) and sphingomyelin (SM), but only the cell level of S1P was elevated 6 (B) to 6 (D)).

To investigate whether high-level insulin signaling is involved in phosphorylation of AKT in Sphk2-overexpressing cells, we examined the insulin response by administering HNMPA, an insulin receptor (IR) antagonist. As a result, insulin-induced pAKT levels were not changed by IR inhibition in Sphk2 overexpressing cells (see Fig. 6 (E)). On the other hand, phosphorylation of AKT by insulin was reduced in the control group by HNMPA-mediated IR inhibition. These results suggest that Sphk2-induced elevation of liver S1P activates AKT independently of the insulin signaling pathway.

(5) Sphk2  The expression of lipid profiles in plasma and liver Change

AdSphk2 was injected into the tail vein of HFD fed WT mice for 4 weeks to see if elevated expression of Sphk2 in the liver was indicative of a physiological change. Transduction of recombinant adenoviruses is known to result in preferential targeting of metastatic genes to the liver (Non-Patent Document 30).

AdGFP or AdSphk2 (1 X 10 9 PFU) was injected into the tail vein in wild-type C57bl6 / J mice fed a high fat diet (60 kcal% fat) for 4 weeks. Plasma was isolated on day 14 after injection and hematological parameters were measured and the results are shown in Table 1 (n = 8, mean ± SEM. * P <0.05 vs AdGFP-injected mice).

AdGFP
(n = 8) AdSphk2
(n = 8) Weight (g) 25.4 ± 0.5 26.1 ± 0.5 Glucose (mg / dl) 148.8 ± 6.6 150.0 + - 7.3 TG (mg / dl) 108.9 ± 7.7 99.1 ± 6.1 Cholesterol (mg / dl) 192.7 ± 8.6 162.4 ± 3.1 * HDL-C (mg / dl) 115.3 ± 3.8 113.7 ± 2.2 LDL-C (mg / dl) 38.4 ± 5.3 18.9 ± 0.9 * ALT (U / l) 321.075 + - 48.7 44.7 ± 3.3 * AST (U / l) 189.1 ± 17.3 152.7 ± 20.7 Plasma NEFA (mEq / ml) 1.67 + 0.14 1.46 0.12 Mean ± SEM. * p < AdGFP

As shown in Table 1, plasma total cholesterol, LDL-cholesterol level, and ALT (non-esterified fatty acid) levels did not change, while body weight, plasma glucose level, plasma TG, HDL and NEFA alanine aminotransferase). Thus, it can be seen that the liver function is improved from the reduction of liver enzyme levels such as ALT.

As shown in FIG. 7, Sphk2 overexpression decreased plasma levels of ceramide (A), dihydroceramide (B), sphingosine (C), sphingomyelin (D), and glucosylceramide , And sphingosine 1-phosphate level (C).

As shown in Fig. 8, Sphk2 overexpression increased S1P in the liver but decreased ceramide, SM, glucosyl ceramide, cholesterol and triglyceride (triglyceride, TG). Unlike plasma, the result of a significant reduction in TG in the liver suggests that synthesis or oxidation is regulated by Sphk2. The above lipid profile results show that liver Sphk2 expression affects the regulation of lipid metabolism in HFD fed mice.

(6) Sphk2  Upregulation may lead to liver lipid accumulation Reduce

The results showed that liver lipid metabolism was regulated by Sphk2. Thus, in order to confirm the role of Sphk2 in liver lipid metabolism, expression of genes involved in lipid biosynthesis and FA oxidation .

Despite the absence of changes in lipogenic genes including FAS (fatty acid synthase), SREBP1c, ACC (acyl CoA carboxylase) and DGAT2, the expression of genes related to FA oxidation such as PPARα, CPT1, and ACOX1, (See Fig. 9 (A)). In addition, immunoblot analysis revealed that the protein level from the gene was elevated in the Sphk2-overexpressed liver (see Fig. 9 (B)). In addition, as a result of the oil red O staining of the liver tissues, the lipids were significantly decreased in the liver of AdSphk2-injected mice as compared with the control, which was also in agreement with the reduced liver TG level (Figs. 9 (C) and 8 F)). As a result of increased FA oxidation, plasma [beta] -hydroxybutyrate was elevated in AdSphk2-injected mice (see Fig. 6 (D)).

For further confirmation, primary mouse hepatic cells were infected with AdSphk2 and the oxygen consumption rate (OCR) was measured. This is because the addition of palmitic acid increases the substrate utilization for FA oxidation, and thus the principle of increasing OCR is used. It was found that AdSphk2- infection increases OCR in the presence of palmitic acid in a time-dependent manner compared to AdGFP-infected control or palmitate untreated control (see Fig. 9 (E)). From these results, it can be seen that Sphk2 expression improves HFD-induced fatty liver by activating FA oxidation, decreasing lipid and increasing ketone body.

(7) In the liver Sphk2  Expression of glucose sensitivity Improve

To investigate the role of liver Sphk2 in insulin sensitivity and glucose metabolism, glucose and insulin responses were analyzed in AdSphk2-injected mice. Although there was no change in basal blood glucose levels, glucose sensitivity was improved in Sphk2-overexpressing mice at HFD feeding (see FIG. 10 (A)). Plasma insulin levels were not significantly different in the control and Sphk2 overexpressing mice in the glucose tolerance test and it was found that the improved glucose sensitivity was due to enhanced plasma glucose clearance without changing insulin secretion (Figure 10 (B) Reference). Sphk2 overexpression also improved my insulin function (Fig. 7 (C)). It can be seen that elevated Sphk2 expression from improved glucose sensitivity and insulin response is associated with regulation of insulin signaling to ER stress induced by HFD.

To confirm the effect of Sphk2 upregulation on insulin signaling, an immunoblot analysis of insulin signaling proteins was performed. The basal condition did not change the phosphorylation of AKT and FOXO1, but insulin increased the phosphorylation of AKT and its lower FOXO1 in Sphk2 overexpressing mice (see FIG. 10 (D)).

To determine whether non-liver tissue is associated with improved glucose or insulin function, AKT phosphorylation was measured in skeletal muscle and adipose tissue, but no change in pAKT levels was observed (Fig. 10 (E) and 10 (F) Reference).

15. Discussion

ER is a major cellular organ that is responsible for protein maturation and aqueous to other cell compartments. The accumulation of unfolded proteins acts as stress to the ER, which is associated with the progression of metabolic dysfunction and inflammatory diseases (Non-Patent Document 32). Excessive intake of nutrients and infection-mediated inflammatory responses are a physiological condition that stresses the structure and function of the ER, so that UPR is initiated to maintain ER homeostasis. If ER stress conditions persist, lipid homeostasis is disturbed, and signal transduction of cells such as diacylglycerol (DAG) and FFA disrupts cell signaling. In order to investigate the relationship between ER stress and sphingomyelinin biosynthesis in the present invention, 1) Sphk2-mediated biosynthesis of S1P is activated by inflammatory ER stress in the liver; 2) Sphk2 is upregulated by ATF4; 3) It was confirmed that upregulation of Sphk2 in the liver improves fatty liver and glucose metabolism by activating FA oxidation.

ER stress-induced UPR is mediated by three ER-membrane associated proteins, PERK, IRE1a, and ATF6. In this pathway, ER stress-activated transcription factors such as ATF4, XBP1 and ATF6 induce a variety of chaperones to correct functional futures resulting from the accumulation of unfolded proteins. Excessive UPR signaling leads to metabolic disturbances including obesity and fatty liver. Each UPR signaling pathway has a different function in metabolic regulation. For example, XBP1 deficiency leads to the inhibition of new liver lipid biosynthesis leading to reduced plasma TG, cholesterol and free fatty acids (Non-Patent Document 33). On the other hand, ATF4 is activated in DIO mice to activate lipogenesis and PERK-eIF2? (-ATF4) is involved in the activation of inflammatory NF-KB (Non-Patent Documents 34 and 35). Although there is confusion in this UPR classification, metabolic dysregulation and inflammation are the main categories of UPR pathways involved in the stress-signaling pathway.

Non-oxidative FA pathways are involved in the biosynthesis of sphingolipids, and their metabolism leads to biosynthesis of various bioactive lipid metabolites including ceramides, SO and S1P. These sphingolipids metabolites are important signal transducers in metabolic regulation (Non-Patent Documents 36 and 37). Injection of saturated FA leads to elevated ceramide levels in muscle and liver and leads to endo-insulin function (Non-Patent Document 9). The pharmacological and genetic inhibition of new ceramide biosynthesis has been shown to improve glucose sensitivity and insulin response in pre-osteo-treated diet-induced obesity (DIO) mice or heterozygous Sptlc2-deficient mice 38, 39). On the other hand, adenoviral gene transfer of Sphk1 markedly reduced blood glucose levels, as well as TG and cholesterol (Non-Patent Document 29). These results show the association of ceramide and S1P in liver glucose / lipid metabolism for nutritional status.

Recent studies have suggested that lipid metabolism is disturbed by ER stress-activated transcription factors. HFD feeding (4 weeks) resulting in subchronic ER stress activation represents an expression pattern of the UPR protein different from that observed in DIO mice (8 weeks and 12 weeks, see FIG. 3). Activation of sXBP1 and inhibition of ATF4 were observed under these sub-chronic conditions (see Figures 2 (A) and 2 (B)). On the other hand, LPS-induced inflammatory ER stress activated only ATF4, leading to Sphk2 induction, indicating that the onset of ER stress depends on various stimuli (see FIG. 2 (C)). This may be due to different activation of the UPR pathway by hyperlipidemic and inflammatory ER stresses, and each UPR pathway selectively modulates the cause-centered target of stress signaling. For inflammatory ER stresses such as those caused by LPS, hepatocytes may require an urgent response through the combined effects of inflammatory responses and ER stresses primarily induced by ATF4-mediated Sphk2 induction. Both XBP1 and ATF4 explain the liver lipid metabolism, but S1P biosynthesis is regulated at least in the liver. Sphk2 expression is presumed to be regulated differently by UPR transcription factors under different pathophysiological conditions. Further studies of the stress signaling pathway associated with ATF4-mediated Sphk2 upregulation are needed to characterize the role of Sphk2 and its product S1P in metabolic / inflammatory regulation.

Previous studies have suggested that the transfer of the Sphk1 gene improves insulin function and lipid abnormality in KK / Ay diabetic mice by activation of insulin signaling, and that this result is via elevated liver S1P 29). In the present invention, it was determined whether Sphk2, which is a main Sphk isoform for generating S1P, activates the AKT pathway in an IRS-independent manner. This suggests that Sphk contributes to the regulation of hepatic glucose metabolism. The elevated level of S1P supports the possibility that an improvement in glucose sensitivity by hepatic Sphk2 expression in HFD fed mice may act as an activator of insulin signaling under hyperglycemic conditions by increasing pAKT (Non-Patent Document 40). However, adenoviral Sphk2 upregulation did not alter basal basal glucose levels and only altered the insulin response by activating the signaling pathway through increased pAKT. Independently, plasma TG remained unchanged, while liver TG accumulation was reduced by Sphk2 expression. The reduction of liver TG was confirmed to be due to upregulation of FA oxidant gene and increased FA oxidation. AdSphk2-infected hepatocytes increased oxygen consumption and elevated plasma ketone levels, suggesting that Sphk2 expression is an important regulator of FA oxidation. However, pharmacological Sphk2 inhibition does not alter glucose / FA metabolism, suggesting that supplemental mechanisms by liver Sphk2 may exist. The mechanism of Sphk2-mediated activation of liver FA oxidation and the role of S1P requires further study.

In the present invention it has been shown that ER stress-mediated Sphk2 upregulation is mediated via ATF4 activation under inflammatory conditions and improves hepatic glucose and lipid abnormalities. Further investigation is needed in relation to the mechanism by which ER stress-mediated Sphk2 activation improves glucose sensitivity and FA oxidation. One possibility is that the UPR signaling pathway may be an adaptive process for mitigating the stress state of the ER by FA oxidative activation. ATF4-mediated Sphk2 upregulation for inflammatory ER stress regulates cell signaling. As a result, Sphk2 upregulation leads to a decrease in lipid by increased FA oxidation. In addition, Sphk2-mediated activation of AKT improved insulin signaling in pathophysiological conditions. In summary, Sphk2 is regulated by a UPR sensor that is dependent on the cause of ER stress induction, and acts as a molecular messenger to mitigate liver ER stress and maintain glucose and lipid homeostasis.

Claims (11)

Sphingosine 1-phosphate, sphingosine 1-phosphate, and a substance that enhances the expression of Sphk2. 2. A pharmaceutical composition for preventing or treating metabolic dysfunction, comprising: 2. The pharmaceutical composition according to claim 1, wherein the Sphk2 is a liver-specific Sphk2. The pharmaceutical composition according to claim 1, wherein the metabolic dysfunction is any one selected from the group consisting of fatty liver, dyslipidemia and obesity. The pharmaceutical composition according to claim 3, wherein the fatty liver is a non-alcoholic fatty liver. The pharmaceutical composition according to claim 3, wherein the dyslipidemia is hyperlipidemia or hypertriglyceridemia. The pharmaceutical composition according to claim 1, wherein the substance that raises the expression of Sphk2 is DNA or RNA. 7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising at least one pharmaceutically acceptable carrier or additive. Sphingosine 1-phosphate, sphingosine 1-phosphate, and a substance that increases the expression of Sphk2. The food composition according to claim 8, wherein the food composition is any one selected from the group consisting of dietary supplements, health functional foods and food additives. Sphingosine 1-phosphate, sphingosine 1-phosphate, and a substance that increases the expression of Sphk2. (a) preparing a cell or tissue of a high fat dietary animal;
(b) contacting the test substance to the cell or tissue of step (a);
(c) measuring the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in the cell or tissue of step (b); And
(d) selecting a test substance showing an increase in the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in comparison with the control group in which the test substance was not treated
&Lt; / RTI &gt; a method for screening a substance for preventing or treating metabolic dysfunction.
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