Faridoddin Mirshahi

Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated state in advanced liver cirrhosis

Advanced cirrhosis is associated with generalized vasodilation of unknown origin, which contributes to mortality. Cirrhotic patients are endotoxemic, and activation of vascular cannabinoid CB1 receptors has been implicated in endotoxin-induced hypotension. Here we show that rats with biliary cirrhosis have low blood pressure, which is elevated by the CB1 receptor antagonist SR141716A. The low blood pressure of rats with CCl4-induced cirrhosis was similarly reversed by SR141716A, which also reduced the elevated mesenteric blood flow and portal pressure. Monocytes from cirrhotic but not control patients or rats elicited SR141716A-sensitive hypotension in normal recipient rats and showed significantly elevated levels of anandamide. Compared with non-cirrhotic controls, in cirrhotic human livers there was a three-fold increase in CB1 receptors on isolated vascular endothelial cells. These results implicate anandamide and vascular CB1 receptors in the vasodilated state in advanced cirrhosis and indicate a novel approach for its management.

Increased Hepatic Synthesis and Dysregulation of Cholesterol Metabolism Is Associated with the Severity of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular and liver-related mortality. NAFLD is characterized by both triglyceride and free cholesterol (FC) accumulation without a corresponding increment in cholesterol esters. The aim of this study was to evaluate the expression of cholesterol metabolic genes in NAFLD and relate these to disease phenotype. NAFLD was associated with increased SREBP-2 maturation, HMG CoA reductase (HMGCR) expression and decreased phosphorylation of HMGCR. Cholesterol synthesis was increased as measured by the circulating desmosterol:cholesterol ratio. miR-34a, a microRNA increased in NAFLD, inhibited sirtuin-1 with downstream dephosphorylation of AMP kinase and HMGCR. Cholesterol ester hydrolase was increased while ACAT-2 remained unchanged. LDL receptor expression was significantly decreased and similar in NAFLD subjects on or off statins. HMGCR expression was correlated with FC, histologic severity of NAFLD and LDL-cholesterol. These data demonstrate dysregulated cholesterol metabolism in NAFLD which may contribute to disease severity and cardiovascular risks.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells.

Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gα(s) and caused an increase in intracellular cAMP and Ca(2+). OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gα(s) inhibitor) or U73122 (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, U73122 or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G(s)/cAMP/Ca(2+) pathway. 8-pCPT-2-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.

Background & Aims The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development. Methods A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains. Results Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4–8 weeks), steatohepatitis (16–24 weeks), progressive fibrosis (16 weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD. Conclusions We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH. Lay summary We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

Activation of the GP130-STAT3 axis and its potential implications in nonalcoholic fatty liver disease

The status of the GP130-STAT3 signaling pathway in humans with nonalcoholic fatty liver disease (NAFLD) and its relevance to disease pathogenesis are unknown. The expression of the gp130-STAT3 axis and gp130 cytokine receptors were studied in subjects with varying phenotypes of NAFLD including nonalcoholic steatohepatitis (NASH) and compared with lean and weight-matched controls without NAFLD. Gp130 and its downstream signaling element (Tyk2 and STAT3) expression were inhibited in obese controls whereas they were increased in NAFLD. IL-6 levels were increased in NASH and correlated with gp130 expression (P < 0.01). Palmitate inhibited gp130-STAT3 expression and signaling. IL-6 and palmitate inhibited hepatic insulin signaling via STAT3-dependent and independent mechanisms, respectively. STAT3 overexpression reversed palmitate-induced lipotoxicity by increasing autophagy (ATG7) and decreasing endoplasmic reticulum stress. These data demonstrate that the STAT3 pathway is activated in NAFLD and can worsen insulin resistance while protecting against other lipotoxic mechanisms of disease pathogenesis.

The multifaceted role of natriuretic peptides in metabolic syndrome

Due to globalization and sophisticated western and sedentary lifestyle, metabolic syndrome has emerged as a serious public health challenge. Obesity is significantly increasing worldwide because of increased high calorie food intake and decreased physical activity leading to hypertension, dyslipidemia, atherosclerosis, and insulin resistance. Thus, metabolic syndrome constitutes cardiovascular disease, type 2 diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD) and recently some cancers are also considered to be associated with this syndrome. There is increasing evidence of the involvement of natriuretic peptides (NP) in the pathophysiology of metabolic diseases. The natriuretic peptides are cardiac hormones, which are produced in the cardiac atrium, ventricles of the heart and the endothelium. These peptides are involved in the homeostatic control of body water, sodium intake, potassium transport, lipolysis in adipocytes and regulates blood pressure. The three known natriuretic peptide hormones present in the natriuretic system are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and c-type natriuretic peptide (CNP). These three peptides primarily function as endogenous ligands and mainly act via their membrane receptors such as natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C) and regulate various physiological and metabolic functions. This review will shed light on the structure and function of natriuretic peptides and their receptors and their role in the metabolic syndrome.

Dual-photon microscopy-based quantitation of fibrosis-related parameters (q-FP) to model disease progression in steatohepatitis

There is a need for further refinement of current histological systems for assessment of hepatic fibrosis in nonalcoholic fatty liver disease (NAFLD). This study evaluated hepatic fibrosis in NAFLD using dual‐photon microscopy‐based quantitation of fibrosis‐related parameters (q‐FPs). Fifty test cohort subjects and 42 validation cohort subjects with NAFLD and the full spectrum of fibrosis were studied. q‐FPs were measured in specific predefined regions of interest (general, vessel, perisinusoid, and vascular septa). Seventy q‐FPs had inter‐ and intraobserver concordance ≥0.8 and were related to the NASH Clinical Research Network fibrosis staging. Of these, 16 q‐FPs with the strongest correlations (P < 0.001 for all) were entered in a principal component analysis model (odds ratio [OR] 7.8, P < 0.001), which separated any stage of fibrosis versus no fibrosis, and cirrhosis versus earlier stages with the areas under the receiver operating characteristic curves of 0.88 and 0.93 (P ≤ 0.01 for both), respectively. In an independent multivariable analysis, four q‐FPs—the number of collagen strands (OR 8.5, P = 0.004), strand length (OR 12.0, P = 0.02), strand eccentricity (OR 8.3, P = 0.004), and strand solidity (OR 8.0, P = 0.003)—were independently associated with fibrosis stages and were used to model fibrosis along a continuous linear scale using desirability functions; this linear scale of fibrosis measurement was also related to fibrosis stage (P < 0.0001). The robustness of both the multivariable model and the linear scale of measurement was confirmed in the validation cohort. Conclusion: The q‐FP model provides an accurate reproducible method to evaluate fibrosis in NAFLD along a quantitative and continuous scale. (Hepatology 2017;65:1891‐1903).

Suppression of IGF binding protein-3 by palmitate promotes hepatic inflammatory responses.

IGF‐binding protein‐3 (IGFBP‐3) is a liver‐derived, anti‐inflammatory molecule that is decreased in obesity, a key risk factor for nonalcoholic fatty liver disease (NAFLD). It was not known whether IGFBP‐3 levels were altered in NAFLD, whether such alterations could be the result of lipotoxicity, and whether altered IGFBP‐3 could affect pathways that are involved in hepatic and systemic inflammation. Serum IGFBP‐3 was decreased in patients with NAFLD, whereas liver and circulating IL‐8 levels were increased. Palmitate inhibited IGFBP‐3 secretion by THP‐1macrophages and enhanced IL‐8 expression. Exposure of palmitate‐treated THP‐1macrophages to IGFBP‐3–deficient conditioned medium led to a 20‐fold increase in palmitate‐induced IL‐8 expression by hepatocytes. Conversely, overexpressionof IGFBP‐3 suppressedJNK and NF‐κBactivation andblockedpalmitate‐induced IL‐8 expression in hepatocytes. Silencing IGFBP‐3 in Huh7 cells enhanced JNK and NF‐κB activity and increased palmitate‐induced IL‐8 secretion. These data indicate that IGFBP‐3 serves as an anti‐inflammatory brake in hepatocytes against JNK and NF‐κB and limits their activation and downstream production of proinflammatory cytokines. Under lipotoxic conditions, palmitate inhibits hepatic macrophage secretion of IGFBP‐3, thereby releasing the brake and enhancing palmitate‐induced IL‐8 synthesis and secretion.—Min, H.‐K., Maruyama, H., Jang, B. K., Shimada, M., Mirshahi, F., Ren, S., Oh, Y., Puri, P., Sanyal, A. J. Suppression of IGF binding protein‐3 by palmitate promotes hepatic inflammatory responses. FASEB J. 30, 4071–4082 (2016). www.fasebj.org

Alcohol produces distinct hepatic lipidome and eicosanoid signature in lean and obese

Alcohol- and obesity-related liver diseases often coexist. The hepatic lipidomics due to alcohol and obesity interaction is unknown. We characterized the hepatic lipidome due to 1) alcohol consumption in lean and obese mice and 2) obesity and alcohol interactions. In the French-Tsukamoto mouse model, intragastric alcohol or isocaloric dextrose were fed with either chow (lean) or high-fat, high-cholesterol diet (obese). Four groups (lean, lean alcohol, obese, and obese alcohol) were studied. MS was performed for hepatic lipidomics, and data were analyzed. Alcohol significantly increased hepatic cholesteryl esters and diacylglycerol in lean and obese but was more pronounced in obese. Alcohol produced contrasting changes in hepatic phospholipids with significant enrichment in lean mice versus significant decrease in obese mice, except phosphatidylglycerol, which was increased in both lean and obese alcohol groups. Most lysophospholipids were increased in lean alcohol and obese mice without alcohol use only. Prostaglandin E2; 5-, 8-, and 11-hydroxyeicosatetraenoic acids; and 9- and 13-hydroxyoctadecadienoic acids were considerably increased in obese mice with alcohol use. Alcohol consumption produced distinct changes in lean and obese with profound effects of obesity and alcohol interaction on proinflammatory and oxidative stress-related eicosanoids.