Serum Markers of Hepatocyte Death and Apoptosis Are Non Invasive Biomarkers of Severe Fibrosis in Patients with Alcoholic Liver Disease

BACKGROUND: Quantification of hepatocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics. METHODOLOGY/PRINCIPAL FINDINGS: CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFβ assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively. CONCLUSION/SIGNIFICANCE: Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies

Tpl2 Kinase Is Upregulated in Adipose Tissue in Obesity and May Mediate Interleukin-1β and Tumor Necrosis Factor-α Effects on Extracellular Signal–Regulated Kinase Activation and Lipolysis

International audienceOBJECTIVE Activation of extracellular signal–regulated kinase-(ERK)-1/2 by cytokines in adipocytes is involved in the alterations of adipose tissue functions participating in insulin resistance. This study aims at identifying proteins regulating ERK1/2 activity, specifically in response to inflammatory cytokines, to provide new insights into mechanisms leading to abnormal adipose tissue function. RESEARCH DESIGN AND METHODS Kinase activities were inhibited with pharmacological inhibitors or siRNA. Lipolysis was monitored through glycerol production. Gene expression in adipocytes and adipose tissue of obese mice and subjects was measured by real-time PCR. RESULTS IκB kinase-(IKK)-β inhibition prevented mitogen-activated protein (MAP) kinase kinase (MEK)/ERK1/2 activation in response to interleukin (IL)-1β and tumor necrosis factor (TNF)-α but not insulin in 3T3-L1 and human adipocytes, suggesting that IKKβ regulated a MAP kinase kinase kinase (MAP3K) involved in ERK1/2 activation induced by inflammatory cytokines. We show that the MAP3K8 called Tpl2 was expressed in adipocytes and that IL-1β and TNF-α activated Tpl2 and regulated its expression through an IKKβ pathway. Pharmacological inhibition or silencing of Tpl2 prevented MEK/ERK1/2 activation by these cytokines but not by insulin, demonstrating its involvement in ERK1/2 activation specifically in response to inflammatory stimuli. Importantly, Tpl2 was implicated in cytokine-induced lipolysis and in insulin receptor substrate-1 serine phosphorylation. Tpl2 mRNA expression was upregulated in adipose tissue of obese mice and patients and correlated with TNF-α expression. CONCLUSIONS Tpl2 is selectively involved in inflammatory cytokine–induced ERK1/2 activation in adipocytes and is implicated in their deleterious effects on adipocyte functions. The deregulated expression of Tpl2 in adipose tissue suggests that Tpl2 may be a new actor in adipose tissue dysfunction in obesity

Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation, Insulin Resistance and Hepatic Steatosis

BACKGROUND: Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver. METHODOLOGY: Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 -/-). PRINCIPAL FINDINGS: In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 -/- mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 -/- mice. CONCLUSION/SIGNIFICANCE: These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorder

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent

Liquid Chromatography-Mass Spectrometry-Based Parallel Metabolic Profiling of Human and Mouse Model Serum Reveals Putative Biomarkers Associated with the Progression of Nonalcoholic Fatty Liver Disease

Keywords: NAFLD, steatosis, NASH, metabolomics, biomarkers. FOOTNOTE

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

mTor, acting mainly via mTORC1, controls dystrophin transcription in a raptor- and rictor-independent mechanism

A Serum Factor Induces Insulin-Independent Translocation of GLUT4 to the Cell Surface which Is Maintained in Insulin Resistance

In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor

The Osteopontin Level in Liver, Adipose Tissue and Serum Is Correlated with Fibrosis in Patients with Alcoholic Liver Disease

<div><h3>Background</h3><p>Osteopontin (OPN) plays an important role in the progression of chronic liver diseases. We aimed to quantify the liver, adipose tissue and serum levels of OPN in heavy alcohol drinkers and to compare them with the histological severity of hepatic inflammation and fibrosis.</p> <h3>Methodology/Principal Findings</h3><p>OPN was evaluated in the serum of a retrospective and prospective group of 109 and 95 heavy alcohol drinkers, respectively, in the liver of 34 patients from the retrospective group, and in the liver and adipose tissue from an additional group of 38 heavy alcohol drinkers. Serum levels of OPN increased slightly with hepatic inflammation and progressively with the severity of hepatic fibrosis. Hepatic OPN expression correlated with hepatic inflammation, fibrosis, TGFβ expression, neutrophils accumulation and with the serum OPN level. Interestingly, adipose tissue OPN expression also correlated with hepatic fibrosis even after 7 days of alcohol abstinence. The elevated serum OPN level was an independent risk factor in estimating significant (F≥2) fibrosis in a model combining alkaline phosphatase, albumin, hemoglobin, OPN and FibroMeter® levels. OPN had an area under the receiving operator curve that estimated significant fibrosis of 0.89 and 0.88 in the retrospective and prospective groups, respectively. OPN, Hyaluronate (AUROC: 0.88), total Cytokeratin 18 (AUROC: 0.83) and FibroMeter® (AUROC: 0.90) estimated significance to the same extent in the retrospective group. Finally, the serum OPN levels also correlated with hepatic fibrosis and estimated significant (F≥2) fibrosis in 86 patients with chronic hepatitis C, which suggested that its elevated level could be a general response to chronic liver injury.</p> <h3>Conclusion/Significance</h3><p>OPN increased in the liver, adipose tissue and serum with liver fibrosis in alcoholic patients. Further, OPN is a new relevant biomarker for significant liver fibrosis. OPN could thus be an important actor in the pathogenesis of this chronic liver disease.</p> </div