Jamie E. Mells

Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway.

Glucagon‐like peptide 1 (GLP‐1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP‐1 functions as an incretin and stimulates glucose‐mediated insulin production by pancreatic β cells. In this study, we demonstrate that exendin‐4/GLP‐1 has a cognate receptor on human hepatocytes and that exendin‐4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon‐like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP‐1. Exendin‐4 increased the phosphorylation of 3‐phosphoinositide‐dependent kinase‐1 (PDK‐1), AKT, and protein kinase C ζ (PKC‐ζ) in HepG2 and Huh7 cells. Small interfering RNA against GLP‐1R abolished the effects on PDK‐1 and PKC‐ζ. Treatment with exendin‐4 quantitatively reduced triglyceride stores compared with control‐treated cells. Conclusion: This is the first report that the G protein–coupled receptor GLP‐1R is present on human hepatocytes. Furthermore, it appears that exendin‐4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease. (HEPATOLOGY 2010)

GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy.

Background Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis. Methodology/Principal Findings Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide. Conclusions/Significance GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis.

Obesity is rapidly becoming a pandemic and is associated with increased carcinogenesis. Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity‐related carcinogenesis. Recently, we reported the oncogenic role of leptin including its potential to increase tumor invasiveness and migration of hepatocellular carcinoma (HCC) cells. In the present study we investigated whether adiponectin could antagonize the oncogenic actions of leptin in HCC. We employed HCC cell lines HepG2 and Huh7, the nude mice‐xenograft model of HCC, and immunohistochemistry data from tissue‐microarray to demonstrate the antagonistic role of adiponectin on the oncogenic actions of leptin. Adiponectin treatment inhibited leptin‐induced cell proliferation of HCC cells. Using scratch‐migration and electric cell‐substrate impedance‐sensing‐based migration assays, we found that adiponectin inhibited leptin‐induced migration of HCC cells. Adiponectin treatment effectively blocked leptin‐induced invasion of HCC cells in Matrigel invasion assays. Although leptin inhibited apoptosis in HCC cells, we found that adiponectin treatment induced apoptosis even in the presence of leptin. Analysis of the underlying molecular mechanisms revealed that adiponectin treatment reduced leptin‐induced Stat3 and Akt phosphorylation. Adiponectin also increased suppressor of cytokine signaling (SOCS3), a physiologic negative regulator of leptin signal transduction. Importantly, adiponectin significantly reduced leptin‐induced tumor burden in nude mice. In HCC samples, leptin expression significantly correlated with HCC proliferation as evaluated by Ki‐67, whereas adiponectin expression correlated significantly with increased disease‐free survival and inversely with tumor size and local recurrence. Conclusion: Collectively, these data demonstrate that adiponectin has the molecular potential to inhibit the oncogenic actions of leptin by blocking downstream effector molecules. (HEPATOLOGY 2010

Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet

The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase β-oxidation.

Adiponectin activation of AMPK disrupts leptin‐mediated hepatic fibrosis via suppressors of cytokine signaling (SOCS‐3)

Adiponectin is an adipocytokine that was recently shown to be anti‐fibrogenic in hepatic fibrosis. Leptin, on the other hand, promotes hepatic fibrosis. The purpose of the present study was to elucidate a mechanism (or mechanisms) whereby adiponectin dampens leptin signaling in activated hepatic stellate cells (HSCs), and prevents excess extracellular matrix production. Activated HSCs, between passages 2 and 5, were cultured and exposed to recombinant human adiponectin and recombinant leptin. Immunoblot analysis for SOCS‐3, TIMP‐1, and the phosphorylated species of Stat3 and adenosine monophosphate‐activated protein kinase (AMPK) were conducted. We also examined MMP‐1 activity by immunosorbant fluorimetric analysis. In HSCs, adiponectin‐induced phosphorylation of AMPK, and subsequently suppressed leptin‐mediated Stat3 phosphorylation and SOCS‐3 induction. Adiponectin also blocked leptin‐stimulated secretion of TIMP‐1, and significantly increased MMP‐1 activity, in vitro. To extend this study, we treated adiponectin knockout mice (Ad−/−) daily with 5 mg/kg recombinant leptin and/or carbon tetrachloride (2 ml/kg) for 6 weeks. Post‐necropsy analysis was performed to examine for inflammation, and histological changes in the Ad−/− and wild‐type mice. There was no significant difference in inflammation, or aminotransferases, between mice receiving carbon tetrachloride and leptin versus carbon tetrachloride alone. As anticipated, the combination of leptin and CCl4 enhanced hepatic fibrosis in both wild‐type and Ad−/− mice, as estimated by amount of collagen in injured livers, but wild‐type mice had significantly higher levels of SOCS‐3 and significantly lower levels of TIMP‐1 mRNA and protein than did adiponectin KO mice exposed to both CCl4 and leptin. We therefore conclude that the protective effects of adiponectin against liver fibrosis require AMPK activation, and may occur through inhibition of the Jak‐Stat signal transduction pathway. J. Cell. Biochem. 110: 1195–1207, 2010. Published 2010 Wiley‐Liss, Inc.

Adiponectin inhibits leptin signaling via multiple mechanisms to exert protective effects against hepatic fibrosis

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3-Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad-/- mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also promotes SOCS-3-Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)-MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

The Role of Gastrointestinal Hormones in Hepatic Lipid Metabolism

Hepatocellular accumulation of free fatty acids (FFAs) in the form of triglycerides constitutes the metabolic basis for the development of nonalcoholic fatty liver disease (NAFLD). Recent data demonstrate that excess FFA hepatocyte storage is likely to lead to lipotoxicity and hepatocyte apoptosis. Hence, FFA-mediated hepatocyte injury is a key contributor to the pathogenesis of nonalcoholic steatohepatitis (NASH). Nonalcoholic steatohepatitis, obesity, type 2 diabetes, essential hypertension, and other common medical problems together comprise metabolic syndrome. Evidence suggests that peptide hormones from the L cells of the distal small intestine, which comprise the core of the enteroendocrine system (EES), play two key roles, serving either as incretins, or as mediators of appetite and satiety in the central nervous system. Recent data related to glucagon-like peptide-1 (GLP-1) and other known L-cell hormones have accumulated due to the increasing frequency of bariatric surgery, which increase delivery of bile salts to the hindgut. Bile acids are a key stimulus for the TGR5 receptor of the L cells. Enhanced bile-salt flow and subsequent EES stimulation may be central to elimination of hepatic steatosis following bariatric surgery. Although GLP-1 is a clinically relevant pharmacological analogue that drives pancreatic β-cell insulin output, GLP-1 analogues also have independent benefits via their effects on hepatocellular FFA metabolism. The authors also discuss recent data regarding the role of the major peptides released by the EES, which promote satiety and modulate energy homeostasis and utilization, as well as those that control fat absorption and intestinal permeability. Taken together, elucidating novel functions for EES-related peptides and pharmacologic development of peptide analogues offer potential far-ranging treatment for obesity-related human disease.

Inhibition of ileal bile acid uptake protects against nonalcoholic fatty liver disease in high-fat diet–fed mice

Inhibition of the ileal bile acid transporter treats multiple features of nonalcoholic steatohepatitis in high-fat diet–fed mice. Blocking bile acids to protect the liver Nonalcoholic fatty liver disease, which is associated with the metabolic syndrome, is becoming increasingly common, and there is no specific treatment available. Although the pathogenesis of this disorder is not yet fully understood, it is known that bile acids play key roles in lipid metabolism. Rao et al. have now identified a drug that can be given by mouth and is not systemically absorbed, but inhibits bile acid absorption from the intestine and thereby reduces the severity of fatty liver disease in a mouse model. In addition to its beneficial effects on the liver, the treatment improved glucose tolerance and cholesterol concentrations in the treated animals, suggesting that it may be useful for treating multiple components of the metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and safe and effective therapies are needed. Bile acids (BAs) and their receptors [including the nuclear receptor for BAs, farnesoid X receptor (FXR)] play integral roles in regulating whole-body metabolism and hepatic lipid homeostasis. We hypothesized that interruption of the enterohepatic BA circulation using a luminally restricted apical sodium-dependent BA transporter (ASBT) inhibitor (ASBTi; SC-435) would modify signaling in the gut-liver axis and reduce steatohepatitis in high-fat diet (HFD)–fed mice. Administration of this ASBTi increased fecal BA excretion and messenger RNA (mRNA) expression of BA synthesis genes in liver and reduced mRNA expression of ileal BA-responsive genes, including the negative feedback regulator of BA synthesis, fibroblast growth factor 15. ASBT inhibition resulted in a marked shift in hepatic BA composition, with a reduction in hydrophilic, FXR antagonistic species and an increase in FXR agonistic BAs. ASBT inhibition restored glucose tolerance, reduced hepatic triglyceride and total cholesterol concentrations, and improved NAFLD activity score in HFD-fed mice. These changes were associated with reduced hepatic expression of lipid synthesis genes (including liver X receptor target genes) and normalized expression of the central lipogenic transcription factor, Srebp1c. Accumulation of hepatic lipids and SREBP1 protein were markedly reduced in HFD-fed Asbt−/− mice, providing genetic evidence for a protective role mediated by interruption of the enterohepatic BA circulation. Together, these studies suggest that blocking ASBT function with a luminally restricted inhibitor can improve both hepatic and whole body aspects of NAFLD.

Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis

Previous evidence indicates that adiponectin possesses antifibrogenic activity in inhibiting liver fibrosis. Therapeutic strategies, however, are limited by adiponectin quaternary structure and effective concentrations in circulation. Here we postulate a novel molecular mechanism, whereby adiponectin targets focal adhesion kinase (FAK) activity and disrupts key features of the fibrogenic response. Adiponectin‐null (Ad–/–) mice and wild‐type littermates were exposed to either saline or carbon tetrachloride (CCl4) for 6 wk. CCl4‐gavaged mice were also injected with attenuated adenoviral adiponectin (Ad‐Adn) or Ad‐LacZ for 2 wk. Hepatic stellate cells (HSCs) were treated with or without adiponectin to elucidate signal transduction mechanisms. In vivo delivery of Ad‐Adn markedly attenuates CCl4‐induced expression of key integrin proteins and markers of HSC activation: αv, β3, β1, α2(I) collagen, and α‐smooth muscle actin. Confocal experiments of liver tissues demonstrated that adiponectin delivery also suppressed vinculin and p‐FAK activity in activated HSCs. In vitro, adiponectin induced dephosphorylation of FAK, mediated by a physical association with activated tyrosine phosphatase, Shp2. Conversely, Shp2 knockdown by siRNA significantly attenuated adiponectin‐induced FAK deactivation, and expression of TIMP1 and α2(I) collagen was abolished in the presence of adiponectin and si‐FAK. Finally, we documented that either adiponectin or the synthetic peptide with adiponectin properties, ADP355, suppressed p‐FAK in synthetic matrices with stiffness measurements of 9 and 15 kPa, assessed by immunofluorescent imaging and quantitation. The in vivo and in vitro data presented indicate that disassembly of focal adhesion complexes in HSCs is pivotal for hepatic fibrosis therapy, now that small adiponectin‐like peptides are available.—Kumar, P., Smith, T., Rahman, K., Mells, J. E., Thorn, N. E., Saxena, N. K., Anania, F. A., Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis. FASEB J. 28, 5172–5183 (2014). www.fasebj.org

Erratum to “adiponectin activation of AMPK disrupts leptin-mediated hepatic fibrosis via suppressors of cytokine signaling (SOCS-3)” J Cell Biochem 110: 1195–1207

734 Grant sponsor: NIH Public Health Service; Grant number: DK077137. *Correspondence to: J.A. Handy, DOI 10.1002/jcb.22863 2011 Wiley-Liss, Inc. Published online in Wiley Online Library (wileyonlinelibrary.com). I n J Cell Biochem 110: 1195–1207 an incorrect funding source has been listed. NIH Public Health Service grant DK076742 should be replaced by DK077137 as the final funding source of the article.