Glucagon-Like Peptide Receptor Agonist Inhibits Angiotensin II-Induced Proliferation and Migration in Vascular Smooth Muscle Cells and Ameliorates Phosphate-Induced Vascular Smooth Muscle Cells Calcification.

BACKGRUOUND: Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system. METHODS: To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide. RESULTS: Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs. CONCLUSION: These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

BACKGRUOUND: Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. METHODS: Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. RESULTS: SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. CONCLUSION: These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

Docosahexanoic Acid Attenuates Palmitate-Induced Apoptosis by Autophagy Upregulation via GPR120/mTOR Axis in Insulin-Secreting Cells.

BACKGRUOUND: Polyunsaturated fatty acids (PUFAs) reportedly have protective effects on pancreatic ß-cells; however, the underlying mechanisms are unknown. METHODS: To investigate the cellular mechanism of PUFA-induced cell protection, mouse insulinoma 6 (MIN6) cells were cultured with palmitic acid (PA) and/or docosahexaenoic acid (DHA), and alterations in cellular signaling and apoptosis were examined. RESULTS: DHA treatment remarkably repressed caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive red dot signals in PA-treated MIN6 cells, with upregulation of autophagy, an increase in microtubule- associated protein 1-light chain 3 (LC3)-II, autophagy-related 5 (Atg5), and decreased p62. Upstream factors involved in autophagy regulation (Beclin-1, unc51 like autophagy activating kinase 1 [ULK1], phosphorylated mammalian target of rapamycin [mTOR], and protein kinase B) were also altered by DHA treatment. DHA specifically induced phosphorylation on S2448 in mTOR; however, phosphorylation on S2481 decreased. The role of G protein-coupled receptor 120 (GPR120) in the effect of DHA was demonstrated using a GPR120 agonist and antagonist. Additional treatment with AH7614, a GPR120 antagonist, significantly attenuated DHA-induced autophagy and protection. Taken together, DHA-induced autophagy activation with protection against PA-induced apoptosis mediated by the GPR120/mTOR axis. CONCLUSION: These findings indicate that DHA has therapeutic effects on PA-induced pancreatic ß-cells, and that the cellular mechanism of ß-cell protection by DHA may be a new research target with potential pharmacotherapeutic implications in ß-cell protection.

The intracellular Ca2+ channel TRPML3 is a PI3P effector that regulates autophagosome biogenesis.

Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca2+-dependent manner. The source of Ca2+ and the molecular mechanism by which Ca2+ is provided for this process are not known. The intracellular Ca2+ permeable channel transient receptor potential mucolipin 3 (TRPML3) localizes in the autophagosome and interacts with the mammalian autophagy-related protein 8 (ATG8) homolog GATE16. Here, we show that lipid-regulated TRPML3 is the Ca2+ release channel in the phagophore that provides the Ca2+ necessary for autophagy progress. We generated a TRPML3-GCaMP6 fusion protein as a targeted reporter of TRPML3 compartment localization and channel function. Notably, TRPML3-GCaMP6 localized in the phagophores, the level of which increased in response to nutrient starvation. Importantly, phosphatidylinositol-3-phosphate (PI3P), an essential lipid for autophagosome formation, is a selective regulator of TRPML3. TRPML3 interacted with PI3P, which is a direct activator of TRPML3 current and Ca2+ release from the phagophore, to promote and increase autophagy. Inhibition of TRPML3 suppressed autophagy even in the presence of excess PI3P, while activation of TRPML3 reversed the autophagy inhibition caused by blocking PI3P. Moreover, disruption of the TRPML3-PI3P interaction abolished both TRPML3 activation by PI3P and the increase in autophagy. Taken together, these results reveal that TRPML3 is a downstream effector of PI3P and a key regulator of autophagy. Activation of TRPML3 by PI3P is the critical step providing Ca2+ from the phagophore for the fusion process, which is essential for autophagosome biogenesis.

Dulaglutide Ameliorates Palmitic Acid-Induced Hepatic Steatosis by Activating FAM3A Signaling Pathway.

BACKGROUND: Dulaglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), has been shown to reduce body weight and liver fat content in patients with type 2 diabetes. Family with sequence similarity 3 member A (FAM3A) plays a vital role in regulating glucose and lipid metabolism. The aim of this study was to determine the mechanisms by which dulaglutide protects against hepatic steatosis in HepG2 cells treated with palmitic acid (PA). METHODS: HepG2 cells were pretreated with 400 µM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide for 24 hours. Hepatic lipid accumulation was determined using Oil red O staining and triglyceride (TG) assay, and the expression of lipid metabolism-associated factor was analyzed using quantitative real time polymerase chain reaction and Western blotting. RESULTS: Dulaglutide significantly decreased hepatic lipid accumulation and reduced the expression of genes associated with lipid droplet binding proteins, de novo lipogenesis, and TG synthesis in PA-treated HepG2 cells. Dulaglutide also increased the expression of proteins associated with lipolysis and fatty acid oxidation and FAM3A in PA-treated cells. However, exendin-(9-39), a GLP-1R antagonist, reversed the expression of FAM3A, and fatty acid oxidation-associated factors increased due to dulaglutide. In addition, inhibition of FAM3A by siRNA attenuated the reducing effect of dulaglutide on TG content and its increasing effect on regulation of fatty acid oxidation. CONCLUSION: These results suggest that dulaglutide could be used therapeutically for improving nonalcoholic fatty liver disease, and its effect could be mediated in part via upregulation of FAM3A expression through a GLP-1R-dependent pathway.

Clusterin Protects Lipotoxicity-Induced Apoptosis via Upregulation of Autophagy in Insulin-Secreting Cells.

BACKGROUND: There is a great need to discover factors that could protect pancreatic ß-cells from apoptosis and thus prevent diabetes mellitus. Clusterin (CLU), a chaperone protein, plays an important role in cell protection in numerous cells and is involved in various cellular mechanisms, including autophagy. In the present study, we investigated the protective role of CLU through autophagy regulation in pancreatic ß-cells. METHODS: To identify the protective role of CLU, mouse insulinoma 6 (MIN6) cells were incubated with CLU and/or free fatty acid (FFA) palmitate, and cellular apoptosis and autophagy were examined. RESULTS: Treatment with CLU remarkably upregulated microtubule-associated protein 1-light chain 3 (LC3)-II conversion in a doseand time-dependent manner with a significant increase in the autophagy-related 3 (Atg3) gene expression level, which is a mediator of LC3-II conversion. Moreover, co-immunoprecipitation and fluorescence microscopy experiments showed that the molecular interaction of LC3 with Atg3 and p62 was markedly increased by CLU. Stimulation of LC3-II conversion by CLU persisted in lipotoxic conditions, and FFA-induced apoptosis and dysfunction were simultaneously improved by CLU treatment. Finally, inhibition of LC3-II conversion by Atg3 gene knockdown markedly attenuated the cytoprotective effect of CLU. CONCLUSION: Taken together, these findings suggest that CLU protects pancreatic ß-cells against lipotoxicity-induced apoptosis via autophagy stimulation mediated by facilitating LC3-II conversion. Thus, CLU has therapeutic effects on FFA-induced pancreatic ß-cell dysfunction.

Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling.

Vascular calcification increases the risk of developing cardiovascular disease, and it is closely associated with metabolic disorders such as diabetes mellitus and non-alcoholic fatty liver disease. We investigated whether the activators of AMP-activated protein kinase (AMPK), metformin, resveratrol, and exendin-4, improved inorganic phosphate (Pi)-induced vascular calcification in rat vascular smooth muscle cells (VSMCs) and whether these effects were via AMPK. Pi increased calcium deposition in a dose-dependent manner, and metformin, resveratrol, and exendin-4 significantly decreased calcium deposition in the Pi-treated VSMCs. Moreover, metformin and exendin-4 increased the expression of a SMC marker gene, α-smooth muscle actin, and Ampk and reduced the receptor activator of nuclear factor kappa-Β ligand (Rankl)/osteoprotegerin ratio. Metformin, resveratrol, and exendin-4 reduced the expression of osteoblast differentiation-associated factors, such as runt-related transcription factor 2, bone morphogenic protein-2, p-small mothers against decapentaplegic 1/5/8, and Rankl. Inhibition of AMPK by siRNA adversely affected the anti-calcification effects of metformin, resveratrol, and exendin-4 and reversed the reduction of the expression of Rankl by metformin and exendin-4 in the Pi-treated VSMCs. These data suggest that metformin, resveratrol, and exendin-4 ameliorate Pi-induced vascular calcification by inhibiting osteoblast differentiation of VSMCs, which is mediated by AMPK.

Resveratrol, an activator of SIRT1, improves ER stress by increasing clusterin expression in HepG2 cells.

Endoplasmic reticulum stress (ER stress) is involved in lipid metabolism and lipotoxicity and can lead to apoptosis. Resveratrol, a sirtuin 1 (SIRT1) agonist, prevents ER stress and improves ER stress-induced hepatic steatosis and cell death. Clusterin is a secreted chaperone and has roles in various physiological processes. However, changes in the expression of clusterin upon ER stress and the connection between SIRT1 and clusterin in protection against ER stress are not well known. In cells treated with tunicamycin, resveratrol increased the expression of clusterin mRNA and protein and the secreted clusterin protein level in conditioned medium. Resveratrol decreased protein expression of the ER stress markers, p-PERK, p-IRE1α, and CHOP, and increased the expression of the ER-associated degradation (ERAD) factors, SEL1L and HRD1, in tunicamycin-treated cells. However, no changes in the expression of these genes were observed in clusterin siRNA-transfected cells. Moreover, increased LAMP2 and LC3 expression and decreased Rubicon expression were observed in cells treated with resveratrol or secreted clusterin. These data suggest that SIRT1 activation by resveratrol attenuates ER stress by promoting protective processes such as ERAD and autophagy pathways and that these protective effects are mediated by clusterin.

Exendin-4 improves ER stress-induced lipid accumulation and regulates lipin-1 signaling in HepG2 cells.

Lipin-1 performs dual function during lipid metabolism, i.e., it functions as a transcriptional coactivator and as a phosphatidate phosphatase during triglyceride biosynthesis. We investigated whether exendin-4 prevented endoplasmic reticulum (ER) stress-induced hepatic steatosis and whether the protective effects of exendin-4 were associated with lipin-1 signaling. Tunicamycin and thapsigargin, ER stress inducers, increased triglycerides (TG) content and expression of genes encoding lipid droplet surface proteins. Exendin-4 decreased the expression of ER stress markers phosphorylated PKR like ER kinase (PERK), phosphorylated inositol-requiring enzyme 1 alpha (IRE1α), and glucose-regulated protein 78 kDa (GRP78) proteins and spliced X-box binding protein 1 (XBP-1s) mRNA and increased the expression of genes encoding lipolytic enzymes hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL) and VLDL assembly-associated proteins microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB) in tunicamycin-pretreated cells. Moreover, exendin-4 significantly decreased lipin-1ß/α ratio by increasing SFRP10 and increased lipin-1 nuclear localization. The decrease in lipin-1ß/α ratio was also observed in SIRT1 and AMPK agonist-treated cells. These data suggest that exendin-4 improves ER stress-induced hepatic lipid accumulation by increasing lipolysis and VLDL assembly, which is partially mediated by the regulation of lipin-1 signaling.

Exendin-4 Inhibits Hepatic Lipogenesis by Increasing ß-Catenin Signaling.

The aim of this study is to investigate whether the beneficial effect of exendin-4 on hepatic steatosis is mediated by ß-catenin signaling. After the HepG2 human hepatoma cells were treated with PA for 24 hours, total triglycerides levels were increased in a dose-dependent manner, and the expression levels of perilipin family members were upregulated in cells treated with 400 µM PA. For our in vitro model of hepatic steatosis, HepG2 cells were treated with 400 µM palmitic acid (PA) in the presence or absence of 100 nM exendin-4 for 24 hours. PA increased the expression of lipogenic genes, such as sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPARγ), stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) and triglyceride synthesis-involved genes, such as diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) in HepG2 cells, whereas exendin-4 treatment significantly prevented the upregulation of SREBP-1c, PPARγ, SCD1, FAS, ACC, DGAT1 and DGAT2. Moreover, exendin-4 treatment increased the expression of phosphorylated glycogen synthase kinase-3 beta (GSK-3ß) in the cytosolic fraction and the expression of ß-catenin and transcription factor 4 (TCF4) in the nuclear fraction. In addition, siRNA-mediated inhibition of ß-catenin upregulated the expression of lipogenic transcription factors. The protective effects of exendin-4 on intracellular triglyceride content and total triglyceride levels were not observed in cells treated with the ß-catenin inhibitor IWR-1. These data suggest that exendin-4 treatment improves hepatic steatosis by inhibiting lipogenesis via activation of Wnt/ß-catenin signaling.

AMP-activated protein kinase suppresses the expression of LXR/SREBP-1 signaling-induced ANGPTL8 in HepG2 cells.

ANGPTL8 is a liver-derived secretory protein that leads to elevated serum triglyceride and the level of circulating ANGPTL8 is strongly associated with obesity and diabetes. Here we investigated the mechanisms of activation and inhibition of ANGPTL8 expression in hepatocytes. The expression of ANGPTL8 was significantly increased in HepG2 cells exposed to palmitic acid, tunicamycin, or T0901317, and was reversed in cells treated with AICAR. Palmitic acid, tunicamycin, and T0901317 increased LXRα and SREBP-1c mRNA expression. The inhibitory effect of AICAR on the expression of T0901317-induced ANGPTL8 was most strongly evident in cells that were transfected with SREBP-1 siRNA. AICAR increased phosphorylation of PPARα and the effect of AICAR was not observed in cells treated with PPARα inhibitor. Metformin had a similar effect on ANGPTL8 expression to that of AICAR. These data suggest that AMPK can suppress the expression of LXR/SREBP-1 signal-induced ANGPTL8 in HepG2 cells.

Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steatosis.

OBJECTIVE: Hepatokine fibroblast growth factor (FGF) 21 takes part in the regulation of lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF21 in the liver, and whether the effects of exendin-4 on the regulation of FGF21 expression are mediated via silent mating type information regulation 2 homolog (SIRT) 1 or SIRT6. MATERIALS/METHODS: The C57BL/6J mice were fed a low fat diet, high fat diet, or high fat diet with 1 nmol/kg/day exendin-4 intraperitoneal injection for 10 weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4 mM) with or without exendin-4 (100 nM) and FGF21 (50 nM) for 24 hours. The change of FGF21 and its receptors expression by exendin-4 were measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF21, SIRT1 and SIRT6, by 10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic lipid metabolism. RESULTS: Exendin-4 increased the expression of FGF21 and its receptors in high fat diet-induced obese mice. In addition, recombinant FGF21 treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF21 siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF21 mRNA, whereas decreased SIRT1 expression by inhibition of FGF21 was not observed. CONCLUSIONS: These data suggest that exendin-4 could improve fatty liver by increasing SIRT1-mediated FGF21.

Exendin-4 attenuates endoplasmic reticulum stress through a SIRT1-dependent mechanism.

Accumulation of excess hepatic lipids contributes to insulin resistance and liver disease associated with endoplasmic reticulum (ER) stress. Exendin-4 is an agonist of the glucagon-like peptide 1 receptor and plays a role in improving insulin resistance and liver disease by increasing silent mating type information regulation 2 homolog (SIRT) 1. However, the effects and mechanism of action of exendin-4 on responses to palmitic acid (PA)-induced ER stress in hepatocytes have not been clearly defined. We investigated whether exendin-4 attenuates PA-induced ER stress via SIRT1 in HepG2 cells. PA treatment induced increased expression of PRKR-like endoplasmic reticulum kinase, inositol-requiring kinase 1α (IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) mRNA. Exendin-4 decreased the expression of P-IRE1α, ATF6, X-box binding protein-1 and CHOP, and increased the expression of SERCA2b. A significant decrease in the hepatic expression of PUMA, BAX, cytochrome c, and cleaved caspase-3 were observed in hepatocytes treated with exendin-4. The TUNEL assay consistently showed that exendin-4 reversed hepatocyte apoptosis induced by treatment with PA. Inhibition of SIRT1 by nicotinamide and siRNA significantly increased the expression of ER stress marker genes in cells treated with both PA and exendin-4. In conclusion, increased SIRT1 by exendin-4 attenuates PA-induced ER stress and mitochondrial dysfunction in hepatocytes.

Knowledge on osteoporosis in guardians of hip fracture patients.

Treatment gap between the treatment guideline and real clinical practice of osteoporosis has been found to be worldwide. Although insufficient knowledge of guardians on osteoporosis might be one important obstacle to diagnose and treatment of osteoporotic patients, there was not a study on the knowledge of guardians. We evaluated the guardians' knowledge on osteoporosis compared with the knowledge of orthopedic doctors, using a self-administered questionnaire, a modified Facts on Osteoporosis Quiz (FOOQ). In March and April 2012, the knowledge of osteoporosis was measured in 40 guardians of hip fracture patients and 40 orthopedic surgeons using, a modified FOOQ. In terms of treatment and prevention of osteoporosis, the modified FOOQ score of the guardians have inadequate knowledge and understanding about the osteoporosis, compared with orthopedic doctors (p < 0.001). The level of guardians' knowledge on osteoporosis should be considered and improved to achieve satisfactory osteoporosis treatment in hip fracture patients.

National utilization of calcium supplements in patients with osteoporotic hip fracture in Korea.

BACKGROUND: Calcium is prescribed worldwide for patients diagnosed with osteoporosis. However, the national utilization of calcium and compliance with calcium is unclear in Korea. Our purpose is to evaluate Korea's national utilization of calcium and compliance with calcium in patients with osteoporotic hip fracture from 2007 to 2010 using data from the Health Insurance Review and Assessment (HIRA) Service. METHODS: From 2007 to 2011, osteoporotic hip fractures were identified using the International Classification of Diseases, 10th revision (ICD-10) and procedure code form from the nationwide database of the Health Insurance Review and Assessment Service. Compliant users of calcium were defined as the patients' medication possession ratio of 80 or more. We analyzed the compliance of calcium according to age and gender. RESULTS: Among 85,228 patients with hip fracture, 20,800 patients (24.4%) received a prescription of a calcium supplement. Among them, only 1,692 patients (8.1%) were identified as compliant users of calcium. The proportion of compliant users was higher in women than men in all age groups. The proportion of compliant users decreased with age in women. CONCLUSIONS: In Korea, the national utilization of calcium was low and compliance with calcium was unsatisfactory even in patients with osteoporotic hip fracture.

GLP-1 Receptor Agonist and Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), one of the most common liver diseases, is caused by the disruption of hepatic lipid homeostasis. It is associated with insulin resistance as seen in type 2 diabetes mellitus. Glucagon-like peptide-1 (GLP-1) is an incretin that increases insulin sensitivity and aids glucose metabolism. In recent in vivo and in vitro studies, GLP-1 presents a novel therapeutic approach against NAFLD by increasing fatty acid oxidation, decreasing lipogenesis, and improving hepatic glucose metabolism. In this report, we provide an overview of the role and mechanism of GLP-1 in relieving NAFLD.

Repression of sterol regulatory element-binding protein 1-c is involved in the protective effects of exendin-4 in pancreatic ß-cell line.

Exendin-4 (Ex-4), a long-acting agonist of glucagon-like peptide-1 receptor, is a novel anti-diabetic drug that prevents ß-cells against various toxicities. However, the mechanism and molecules mediating the protection procession of Ex-4 are not fully understood. We investigated the protective effect of Ex-4 against lipotoxicity, mediated by a repression of sterol regulatory element-binding protein (SREBP)-1c, a regulator of genes expression involved in fat and cholesterol synthesis. To observe the effect of Ex-4, we evaluated glucose-stimulated insulin secretion (GSIS) and apoptosis in the MIN6 pancreatic ß-cell line, which were cultured in DMEM medium containing 500 µM palmitate, with or without 10 nM Ex-4. We also examined the roles of SREBP-1c in lipotoxicity model by knockdown with si-RNA. Treatment with Ex-4 improved insulin secretion and survival as well as reduced SREBP-1c expression and activity in palmitate-treated MIN6 cells. This improvement was accompanied with an upregulation of PI3K/Akt signaling pathway, and LY294.002, a specific inhibitor of PI3 kinase, abrogated effects of Ex-4 on insulin secretion. Moreover, SREBP-1c in nuclei was increased by the inhibition of PI3 kinase. Lipotoxic effects of palmitate in the insulin secretion and apoptosis were significantly prevented by SREBP-1 knockdown. In conclusion, Ex-4 protects ß-cell against palmitate-induced ß-cell dysfunction and apoptosis, by inhibiting SREBP-1c expression and activity through the PI3K/Akt signaling pathway.

Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice.

The effects of exendin-4 on Sirt1 expression as a mechanism of reducing fatty liver have not been previously reported. Therefore, we investigated whether the beneficial effects of exendin-4 treatment on fatty liver are mediated via Sirt1 in high-fat (HF) diet-induced obese C57BL/6J mice and related cell culture models. Exendin-4 treatment decreased body weight, serum free fatty acid (FA), and triglyceride levels in HF-induced obese C57BL/6J mice. Histological analysis showed that exendin-4 reversed HF-induced hepatic accumulation of lipids and inflammation. Exendin-4 treatment increased mRNA and protein expression of Sirt1 and its downstream factor, AMPK, in vivo and also induced genes associated with FA oxidation and glucose metabolism. In addition, a significant increase in the hepatic expression of Lkb1 and Nampt mRNA was observed in exendin-4-treated groups. We also observed increased expression of phospho-Foxo1 and GLUT2, which are involved in hepatic glucose metabolism. In HepG2 and Huh7 cells, mRNA and protein expressions of GLP-1R were increased by exendin-4 treatment in a dose-dependent manner. Exendin-4 enhanced protein expression of Sirt1 and phospho-AMPKα in HepG2 cells treated with 0.4 mM palmitic acid. We also found that Sirt1 was an upstream regulator of AMPK in hepatocytes. A novel finding of this study was the observation that expression of GLP-1R is proportional to exendin-4 concentration and exendin-4 could attenuate fatty liver through activation of Sirt1.

Essential role of clusterin in pancreas regeneration.

Based on our previous observations that clusterin induction accompanies pancreas regeneration in the rat, we sought to determine if regeneration might be impaired in mice that lacked clusterin. We studied the impact of absent clusterin on morphogenic and functional features of regenerating pancreas. Clusterin induction was accompanied in the regenerating pancreas by a robust development of new lobules with ductules, acini, and endocrine islets in wild type after partial pancreatectomy. In clusterin knock-out mice, however, pancreatectomy resulted in a poor formation of regenerating lobule. In particular, regeneration of beta-cells was also significantly reduced and was associated with persistent hyperglycemia. Duct cells obtained from pancreatectomized clusterin knock-out mice exhibited impaired beta-cell formation in vitro; this was restored by administration of exogenous clusterin. We suggest that clusterin plays a critical role to promote both exocrine and endocrine regeneration following pancreas injury, as well as for in vitro beta-cell regeneration.

Nestin action during insulin-secreting cell differentiation.

Nestin, which was initially identified as a marker of neural stem cells, has been reported in regenerating pancreas as well as in early embryonic stem (ES) cell derivatives. However, little is known about its specific roles in stem cells as a functional regulator. We investigated the source of the action of nestin in ES and adult pancreatic ductal stem (PDS) cells in regard to the neogenesis of insulin-secreting beta-cells. In ES cells, suppression of nestin by gene silencing led to an increased expression of the pluripotency-associated genes, including Oct 4, Nanog, and SSEA-1, before embryoid body (EB) formation, whereas it reduced endodermal and pancreatic transcription factors in EBs. Inhibition of nestin expression in adult PDS cells caused a low expression of pancreatic transcription factors and islet hormones, leading to poor beta-cell development and insulin secretion. These data may indicate not only that nestin is a simple stem cell marker, but also that it constitutes a functional factor at the time of stem cell differentiation. We suggest that nestin plays pivotal roles as an intermediate regulator governing both stemness and differentiation of stem cells in the process of their differentiation into insulin-secreting cells.