Neeraj K. Saxena

Exendin-4, a Glucagon-Like Protein-1 (GLP-1) Receptor Agonist, Reverses Hepatic Steatosis in ob/ob Mice

Nonalcoholic fatty liver disease (NAFLD) represents a burgeoning problem in hepatology, and is associated with insulin resistance. Exendin‐4 is a peptide agonist of the glucagon‐like peptide (GLP) receptor that promotes insulin secretion. The aim of this study was to determine whether administration of Exendin‐4 would reverse hepatic steatosis in ob/ob mice. Ob/ob mice, or their lean littermates, were treated with Exendin‐4 [10 μg/kg or 20 μg/kg] for 60 days. Serum was collected for measurement of insulin, adiponectin, fasting glucose, lipids, and aminotransferase concentrations. Liver tissue was procured for histological examination, real‐time RT‐PCR analysis and assay for oxidative stress. Rat hepatocytes were isolated and treated with GLP‐1. Ob/ob mice sustained a reduction in the net weight gained during Exendin‐4 treatment. Serum glucose and hepatic steatosis was significantly reduced in Exendin‐4 treated ob/ob mice. Exendin‐4 improved insulin sensitivity in ob/ob mice, as calculated by the homeostasis model assessment. The measurement of thiobarbituric reactive substances as a marker of oxidative stress was significantly reduced in ob/ob‐treated mice with Exendin‐4. Finally, GLP‐1–treated hepatocytes resulted in a significant increase in cAMP production as well as reduction in mRNA expression of stearoyl‐CoA desaturase 1 and genes associated with fatty acid synthesis; the converse was true for genes associated with fatty acid oxidation. In conclusion, Exendin‐4 appears to effectively reverse hepatic steatosis in ob/ob mice by improving insulin sensitivity. Our data suggest that GLP‐1 proteins in liver have a novel direct effect on hepatocyte fat metabolism. (HEPATOLOGY 2006;43:173–181.)

Concomitant Activation of the JAK/STAT, PI3K/AKT, and ERK Signaling Is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells

Various epidemiologic studies have shown that obesity is associated with hepatocellular carcinoma. Leptin, the key player in the regulation of energy balance and body weight control, also acts as a growth factor on certain organs in both normal and disease states. It is plausible that leptin acts to promote hepatocellular carcinogenesis directly affecting malignant properties of liver cancer cells. However, a direct role for leptin in hepatocellular carcinoma has not been shown. In this study, we analyzed the role of leptin and the mechanism(s) underlying its action in hepatocellular carcinoma cells, which express both short and long isoforms of leptin receptors. Treatment with leptin resulted in increased proliferation of both HepG2 and Huh7 cells and involves activation of signal transducers and activators of transcription 3 (STAT3), AKT, and extracellular signal-regulated kinase (ERK) signaling pathways. Leptin-induced phosphorylation of ERK and AKT was dependent on Janus-activated kinase (JAK)/STAT activation. Intriguingly, we also found that leptin potently induces invasion of hepatocellular carcinoma cells in Matrigel invasion and electric cell-substrate impedance-sensing assays. Leptin-stimulated invasion was effectively blocked by pharmacologic inhibitors of JAK/STAT and, to a lesser extent, by ERK and phosphatidylinositol 3-kinase (PI3K) inhibition. Importantly, leptin also induced the migration of both HepG2 and Huh7 cells on fibronectin matrix. Inhibition of JAK/STAT, ERK, and PI3K activation using pharmacologic inhibitors effectively blocked leptin-induced migration of HepG2 and Huh7 cells. Taken together, these data indicate that leptin promotes hepatocellular carcinoma growth, invasiveness, and migration and implicate the JAK/STAT pathway as a critical mediator of leptin action. Our findings have potential clinical implications for hepatocellular carcinoma progression in obese patients.

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)

The roles of leptin and adiponectin : A novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology

Although leptin is a key adipokine promoting liver fibrosis, adiponectin may prevent liver injury. To determine the role of these adipokines in liver fibrosis and to understand their expression in vivo, fa/fa rats and their lean littermates were subjected to bile duct ligation (BDL). Histomorphometry for collagen and alpha-smooth muscle actin (alpha-SMA) revealed that lean rats, but not fa/fa littermates, had significant fibrosis with abundant hepatic stellate cell (HSC) activation. The lean-BDL rats had significantly higher leptin concentrations in the hepatic vein than lean sham-operated, fa/fa BDL, or fa/fa sham-operated rats. Co-localization of leptin and alpha-SMA in activated HSCs was observed by immunohistochemistry. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis confirmed the presence of leptin and alpha-SMA in activated, but not quiescent, HSCs, whereas only quiescent HSCs synthesized adiponectin mRNA and protein. Adiponectin overexpression in activated HSCs reduced proliferation, augmented apoptosis, and reduced expression of alpha-SMA and proliferating cell nuclear antigen. Adiponectin receptors (AdipoR1 and AdipoR2) were detected in both activated and quiescent HSCs, but only activated HSCs produced significant apoptosis after treatment with either globular or full-length adiponectin. Adiponectin may act to reverse HSC activation, maintain HSC quiescence, or significantly, may have important therapeutic implications in liver fibrosis.

Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation

A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cell (HSC) phenotype by both proliferation and inhibition of apoptosis. We provide evidence that leptin is a potent HSC mitogen and dramatically inhibits stellate cell apoptosis. Leptin proved to be as potent an HSC mitogen as platelet‐derived growth factor (PDGF) as assessed by bromodeoxyuridine (BrdU) incorporation in isolated primary HSCs; data using fluorescent propidium iodide (PI) uptake revealed that leptin, like PDGF, increased HSC populations in the S‐ and G2/M‐phases of the cell cycle. Leptin resulted in a robust increase in cyclin D1 expression. Using the chemical inhibitor of Janus kinase 2 (Jak2) activity, AG 490, and overexpression of the suppressor of cytokine signaling 3 (SOCS‐3), we show that blockade of leptin receptor (Ob‐Rb) phosphorylation blocks leptin‐induced HSC proliferation. Leptin‐associated phosphorylation of both extracellular regulated kinase (p44/p42, Erk) and Akt is also prohibited. Further, the PI‐3 kinase inhibitor LY294002 and MAPK inhibitor PD98059 were found to significantly reduce leptin‐induced HSC proliferation, thereby indicating that leptin induced HSC proliferation is Akt‐ and Erk‐dependent. Akt was also protective against HSC apoptosis. Leptin abolished both cycloheximide‐induced and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis, demonstrated by reduced caspase‐3 activity, HSC‐TUNEL staining, and DNA fragmentation. We conclude that leptin acts as a direct hepatic stellate cell survival agonist. Importantly, we have demonstrated that leptin‐induced HSC proliferation and survival by Ob‐Rb phosphorylation are both Erk‐ and Akt‐dependent.

Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Obesity is an independent risk factor for breast cancer, and obese breast cancer patients exhibit a higher risk for larger tumor burden and increased metastasis. Obesity, as associated with metabolic syndrome, results in an increase in circulating insulin-like growth factor (IGF), which acts as a mitogen. In addition, higher plasma level of adipocytokine leptin is associated with obesity. In the present study, we show that cotreatment with leptin and IGF-I significantly increases proliferation as well as invasion and migration of breast cancer cells. We found a novel bidirectional crosstalk between leptin and IGF-I signaling; IGF-I induced phosphorylation of leptin receptor (Ob-Rb) and leptin induced phosphorylation of IGF-I receptor (IGF-IR), whereas cotreatment induced synergistic phosphorylation and association of Ob-Rb and IGF-IR along with activation of downstream effectors, Akt and extracellular signal-regulated kinase. Leptin increased phosphorylation of IGF signaling molecules insulin-receptor substrate (IRS)-1 and IRS-2. Interestingly, we found that leptin and IGF-I cotreatment synergistically transactivated epidermal growth factor receptor (EGFR), depending on the proteolytic release of EGFR ligands, as the broad-spectrum matrix metalloproteinase inhibitor GM6001 could inhibit this effect. Using clinically relevant EGFR inhibitors, erlotinib and lapatinib, we found that inhibition of EGFR activation effectively inhibited leptin- and IGF-I-induced invasion and migration of breast cancer cells. Taken together, these data suggest a novel bidirectional crosstalk between leptin and IGF-I signaling that transactivates EGFR and promotes the metastatic properties as well as invasion and migration of breast cancer cells. Our findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the procancerous effects of leptin and IGF-I in breast cancers.

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.

Leptin-induced Growth Stimulation of Breast Cancer Cells Involves Recruitment of Histone Acetyltransferases and Mediator Complex to CYCLIN D1 Promoter via Activation of Stat3

Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.

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.