Isabel Zvibel

Glucagon-like peptide-1 reduces hepatic lipogenesis via activation of AMP-activated protein kinase

BACKGROUND & AIMS Glucagon-like peptide-1 (GLP-1), a gut-derived peptide degraded by dipeptidyl peptidase-4 (DPP4), stimulates insulin secretion in response to nutrients, yet its direct effect on the liver is controversial. We investigated the effects of GLP-1 on hepatic fat and glucose metabolism and elucidated its mechanism of action. METHODS Hepatic fat metabolism, including lipogenic enzymes and signal transduction regulators, was assessed in livers of DPP4-deficient rats (DPP4-) with chronically elevated GLP-1 and in GLP-1-treated primary hepatocytes. The effect of chronic elevated GLP-1 on insulin sensitivity was measured using the hyperinsulinemic-euglycemic clamp. RESULTS Normal and high fat diet fed DPP4-rats displayed reduced hepatic triglycerides, accompanied by down-regulation of lipogenesis enzymes and parallel up-regulation of carnitine palmitoyltransferase-1, a key enzyme in fatty acid β-oxidation. In vitro studies demonstrated that these effects were directly induced by GLP-1. Mechanistically, GLP-1 increased cAMP in hepatocytes, resulting in the phosphorylation of cAMP-activated protein kinase (AMPK), a suppressor of lipogenesis. Indeed, hepatocytes expressing a dominant negative Ad-DN-AMPK displayed attenuated GLP-1 effects on AMPK phosphorylation and its downstream lipogenic targets. Importantly, normoglycemic DPP4-rats did not display improved hepatic insulin sensitivity in vivo, suggesting a direct effect of GLP-1 on fat metabolism. Finally, DPP4-rats expressed lower levels of hepatic proinflammatory and profibrotic cytokines in response to nutrient stimuli. CONCLUSIONS GLP-1 suppresses hepatic lipogenesis via activation of the AMPK pathway. GLP-1 inhibitory effects on hepatic fat accumulation and nutrient-induced hepatic proinflammatory response suggest GLP-1 analogs as novel therapies for non-alcoholic fatty liver diseases.

Anoikis: Roadblock to Cell Transplantation?

Cell therapy, in particular liver cell transplantation, holds great therapeutic potential and is partially hindered by the high rate of apoptosis during cell isolation, cryopreservation, and engraftment. Apoptosis occurring due to cell detachment from the extracellular matrix is a phenomenon termed “anoikis. ” The purpose of this review is to describe signaling mechanisms pertinent to anoikis in both immortalized cell lines, but particularly in primary normal epithelial cells. The mechanisms described include integrin signaling and survival molecules, caspase activation, and the role of mitochondrial proteins in anoikis. Strategies to prevent anoikis during isolation and cryopreservation of hepatocytes are discussed.

Gliotoxin Ameliorates Development of Fibrosis and Cirrhosis in a Thioacetamide Rat Model

Activation of hepatic stellate cells causes most of the pathological changes in cirrhosis. The fungal metabolite gliotoxin was shown to induce apoptosis of hepatic stellate cells in vitro. We examined whether gliotoxin may prevent or reverse liver fibrosis in a rat model of thioacetamide-inducedcirrhosis, and whether gliotoxin administration in vivo causes apoptosis of activated stellate cells. Gliotoxin treatment resulted in a significant decrease in liver fibrosis in rats, but did not improve liver functions. We observed a significant reduction in the numbers of activated hepatic stellate cells in the gliotoxin-treated rats. Gliotoxin administration also resulted in parenchymal apoptosis of hepatocytes and hepatic stellate cells. In conclusion, gliotoxin reduces hepatic fibrosis, an effect accompanied by reduction of the numbers of activated hepatic stellate cells in the liver.

Stromal extracellular matrix reduces chemotherapy-induced apoptosis in colon cancer cell lines

Several studies have shown that extracellular matrix reduces chemotherapeutic drugs-induced apoptosis in small cell lung cancer cells, myelomas and gliomas. We have investigated the protective effect of defined extracellular matrix components and of extracellular matrix from different cell types (fibroblasts, hepatocytes and intestinal epithelial cells) on the toxicity of three types of chemotherapeutic drugs on colon cancer cells. Human colon cancer cell lines LS174T and LiM6 were plated on plastic, on hepatocyte-derived ECM or on stromal ECM and in the presence of the antimetabolite 5-fluorouracil (5-FU), the topoisomerase I inhibitor camptothecin and the topoisomerase II inhibitor etoposide. We determined IC50 for the drugs for each of these culture conditions. We also determined the expression of the anti-apoptotic proteins bcl-2 and bcl-x (L) under these culture conditions. We found that stromal ECM protected LiM6 cells from the toxicity of etoposide and LS174T, but not LiM6 cells, from the toxicity of camptothecin. Collagen I, fibronectin and fibroblast-derived ECM rendered LiM6 cells, but not LS174T, more sensitive to the harmful effect of 5-FU. Both colon cell lines had increased expression of anti-apoptotic proteins bcl-2 and bcl-x(L) when cultured on the various ECMs and with the drugs, but there was no correlation between a protective ECM effect and expression of the anti-apoptotic proteins. Stromal-derived ECM may protect colon cancer cells from etoposide and camptothecin-induced apotosis, through a mechanism that is not bcl-2 or bcl-x(L) dependant.

The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes.

We explored the effect of extracellular matrix (ECM) produced by fetal and adult hepatocytes on tissue-specific gene expression and proliferation of fetal and adult hepatocytes. Adult hepatocytes ECM strongly induced expression of both albumin and HNF-4 in adult hepatocytes. In contrast, fibroblast ECM reduced the expression of mRNAs for albumin and alpha-fetoprotein in fetal hepatocytes. Adult hepatocytes ECM also increased the activity of liver-specific enzymes of adult hepatocytes (DPP IV and glucose-6-phosphatase) in both fetal and adult hepatocytes, while fetal hepatocyte-derived ECM increased activity of the fetal hepatocyte enzyme GGT in fetal hepatocytes. Fibroblast ECM was inhibitory for the activity of all enzymes assayed. Removal of heparin chains from the various matrices by pretreatment of the ECM with heparinase resulted in reduction of glucose-6-phosphatase and DPP IV in adult hepatocytes. Removal of chondroitin sulfate chains from fetal hepatocyte-derived ECM resulted in loss of induction of GGT in the fetal cells. Fetal hepatocytes proliferated best on adult hepatocyte-derived ECM. Adult hepatocytes showed only modest proliferation on both fetal and adult hepatocytes ECM and their growth was inhibited by fibroblast ECM. In conclusion, adult hepatocyte ECM better supports the expression of adult genes, whereas fetal hepatocyte ECM induced expression of fetal genes. Fibroblast derived-ECM was inhibitory for both proliferation and tissue-specific gene expression in fetal and adult hepatocytes. The data support a role for heparan sulfate being the active element in adult ECM, and chondroitin sulfate being the active element in fetal ECM.

Induced hypothyroidism accelerates the regression of liver fibrosis in rats

Background and Aim:  It has been shown in previous studies that hypothyroidism prevents the development of liver fibrosis in bile duct ligated rats and in rats chronically treated with thioacetamide (TAA). In recent years, regression of liver fibrosis (occurring spontaneously or during treatment) has been demonstrated in rodent models such as bile duct ligation and CCl4 administration. Therefore, in the present study, the potential therapeutic effect of hypothyroidism on liver fibrosis was investigated.

Nicotinamide induces apoptosis and reduces collagen I and pro-inflammatory cytokines expression in rat hepatic stellate cells.

OBJECTIVE Nicotinamide has been shown to inhibit proliferation and induce apoptosis in a variety of cells. Moreover, nicotinamide treatment attenuates collagen accumulation and fibrogenesis in the bleomycin model of lung fibrosis. We hypothesized that nicotinamide may be useful as an antifibrotic agent in liver fibrosis and we investigated the in vitro effect of nicotinamide on hepatic stellate cells proliferation, apoptosis and collagen I expression. MATERIAL AND METHODS Transforming growth factor beta1 (TGF-beta1) was used for activation of the rat HSC-T6 cell line. Apoptosis was determined by fluorescence activated cell sorter (FACS) analysis after propidium iodide staining and by immunohistochemistry showing presence of the active form of caspase 3. Expression of activation marker alpha-smooth muscle actin (alpha-SMA), apoptotic and cell cycle markers cyclin D1, P53 and caspase 3 was determined by Western blotting. Collagen I expression was assessed by Northern blotting. RESULTS Nicotinamide inhibits hepatic stellate cell proliferation and induces apoptosis with caspase-3 activation. There is no effect of nicotinamide on the levels of cell cycle stimulator cyclin D1. Expression of p53 is induced in the presence of nicotinamide. Nicotinamide reduces activation marker alpha-SMA and decreases both basal and TGFbetaepsilon-induced collagen I expression. Moreover, in TGFbeta-activated cells, nicotinamide reduces expression of pro-inflammatory and pro-fibrotic cytokines TGFbeta2, IL-1beta, TNFalpha and macrophage chemotactic protein-1. CONCLUSIONS The in vitro effect of nicotinamide on activation and proliferation of hepatic stellate cells suggests that nicotinamide may have a potential beneficial role in attenuation of liver fibrogenesis.

Extracellular matrix modulates expression of growth factors and growth-factor receptors in liver-colonizing colon-cancer cell lines

Site‐specific metastasis is determined by the extracellular matrix (ECM) of the colonized organ. We have shown that hepatocyte‐derived ECM stimulated proliferation of colon‐cancer cells via induction of autocrine growth factors and their receptors. The ECM component responsible was heparin proteoglycan. We therefore investigated the effect of exogenously added heparin on colon cell lines of varying liver‐colonizing potential. The cells were grown on typical liver matrix components, such as fibronectin and collagens type I and IV. We assessed the effect of these matrix components on clonal growth, proliferation and expression of autocrine growth factors and their receptors. The clonal growth of the KM12 cells was not affected by heparin, while the other cell lines were inhibited by heparin. Cell proliferation in weakly metastatic KM12, but not in strongly metastatic KM12SM, was inhibited by heparin on plastic. Weakly metastatic LS174T, but not strongly metastatic LiM6, was inhibited by heparin on fibronectin. Expression of erb‐B2 was also differently modulated by heparin in weakly metastatic vs. highly metastatic cells. In weakly metastatic cells, heparin reduced erb‐B2 levels when cells were on plastic and fibronectin, while in strongly metastatic cells, erb‐B2 was induced by heparin. In all 4 cell lines, mRNA for cripto was induced by heparin when the cells were grown on fibronectin. In KM12SM cells, amphiregulin was induced by heparin in cells on fibronectin and collagen IV. We show that soluble heparin, similar in its carbohydrate chemistry to liver heparin proteoglycan, regulates the growth of colon‐cancer cells. This effect depends on other matrix components found in the liver and is mediated in part by EGF family members. Int. J. Cancer 77:295–301, 1998.© 1998 Wiley‐Liss, Inc.

Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet-induced obesity

BACKGROUND & AIMS Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Long-Acting Glucose-Dependent Insulinotropic Polypeptide Ameliorates Obesity-Induced Adipose Tissue Inflammation

Obesity induces low-grade chronic inflammation, manifested by proinflammatory polarization of adipose tissue innate and adaptive resident and recruited immune cells that contribute to insulin resistance (IR). The glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that mediates postprandial insulin secretion and has anabolic effects on the adipose tissue. Importantly, recent evidence suggested that GIP is a potential suppressor of inflammation in several metabolic models. In this study, we aimed to investigate the immunoregulatory role of GIP in a murine model of diet-induced obesity (DIO) using the long-acting GIP analog [d-Ala2]GIP. Administration of [d-Ala2]GIP resulted in adipocytes of increased size, increased levels of adipose tissue lipid droplet proteins, indicating better lipid storage capacity, and reduced adipose tissue inflammation. Flow cytometry analysis revealed reduced numbers of inflammatory Ly6Chi monocytes and F4/80hiCD11c+ macrophages, associated with IR. In addition, [d-Ala2]GIP reduced adipose tissue infiltration of IFN-γ–producing CD8+ and CD4+ T cells. Furthermore, [d-Ala2]GIP treatment induced a favorable adipose tissue adipokine profile, manifested by a prominent reduction in key inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and chemokines (CCL2, CCL8, and CCL5) and an increase in adiponectin. Notably, [d-Ala2]GIP also reduced the numbers of circulating neutrophils and proinflammatory Ly6Chi monocytes in mice fed regular chow or a high-fat diet. Finally, the beneficial immune-associated effects were accompanied by amelioration of IR and improved insulin signaling in liver and adipose tissue. Collectively, our results describe key beneficial immunoregulatory properties for GIP in DIO and reveal that its augmentation ameliorates adipose tissue inflammation and improves IR.