Guoxun Chen

Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1

Blood glucose levels are maintained by the balance between glucose uptake by peripheral tissues and glucose secretion by the liver. Gluconeogenesis is strongly stimulated during fasting and is aberrantly activated in diabetes mellitus. Here we show that the transcriptional coactivator PGC-1 is strongly induced in liver in fasting mice and in three mouse models of insulin action deficiency: streptozotocin-induced diabetes, ob/ob genotype and liver insulin-receptor knockout. PGC-1 is induced synergistically in primary liver cultures by cyclic AMP and glucocorticoids. Adenoviral-mediated expression of PGC-1 in hepatocytes in culture or in vivo strongly activates an entire programme of key gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, leading to increased glucose output. Full transcriptional activation of the PEPCK promoter requires coactivation of the glucocorticoid receptor and the liver-enriched transcription factor HNF-4α (hepatic nuclear factor-4α) by PGC-1. These results implicate PGC-1 as a key modulator of hepatic gluconeogenesis and as a central target of the insulin–cAMP axis in liver.

Tissue triglycerides, insulin resistance, and insulin production: implications for hyperinsulinemia of obesity

Obesity is associated with both insulin resistance and hyperinsulinemia. Initially hyperinsulinemia compensates for the insulin resistance and thereby maintains normal glucose homeostasis. Obesity is also associated with increased tissue triglyceride (TG) content. To determine whether both insulin resistance and hyperinsulinemia might be secondary to increased tissue TG, we studied correlations between TG content of skeletal muscle, liver, and pancreas and plasma insulin, plasma [insulin] x [glucose], and beta-cell function in four rat models with widely varying fat content: obese Zucker diabetic fatty rats, free-feeding lean Wistar rats, hyperleptinemic Wistar rats with profound tissue lipopenia, and rats pair fed to hyperleptinemics. Correlation coefficients >0.9 (P < 0.05) were obtained among TG of skeletal muscle, liver, and pancreas and among plasma insulin, [insulin] x [glucose] product, and beta-cell function as gauged by basal, glucose-stimulated, and arginine-stimulated insulin secretion by the isolated perfused pancreas. Although these correlations cannot prove cause and effect, they are consistent with the hypothesis that the TG content of tissues sets the level of both insulin resistance and insulin production.

β-Cell function in normal rats made chronically hyperleptinemic by adenovirus-leptin gene therapy

Leptin was overexpressed in the liver of normal Wistar rats by infusing recombinant adenovirus containing the cDNA encoding leptin. Plasma leptin levels rose to 12–24 ng/ml (vs. <2 ng/ml in control rats), and food intake and body weight fell. Visible fat disappeared within 7 days. Plasma insulin fell to <50% of normal in association with hypoglycemia, suggesting enhanced insulin sensitivity. Although β-cells appeared histologically normal, the pancreases were unresponsive to perfusion with stimulatory levels of glucose and arginine. Since islet triglyceride content was 0, compared with 14 ng/islet in pair-fed control rats, we coperfused a 2:1 oleate:palmitate mixture (0.5 mmol/l). This restored insulin responses to supranormal levels. When normal islets were cultured with 20 ng/ml of leptin, they too became triglyceride-depleted and failed to respond when perifused with glucose or arginine. Perifusion of fatty acids restored both responses. We conclude that in normal rats, hyperleptinemia for 2 weeks causes reversible β-cell dysfunction by depleting tissue lipids, thereby depriving β-cells of a lipid-derived signal required for the insulin response to other fuels.

Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors.

Leptin regulates appetite and body weight via hypothalamic targets, but it can act directly on cultured pancreatic islets to regulate their fat metabolism. To obtain in vivo evidence that leptin may act peripherally as well as centrally, we compared the effect of adenovirally induced hyperleptinemia on food intake, body weight, and islet fat content in ventromedial hypothalamic-lesioned (VMHL) rats, sham-lesioned (SL) controls, and Zucker Diabetic Fatty (ZDF) rats in which the leptin receptor is mutated. Infusion with recombinant adenovirus containing the rat leptin cDNA increased plasma leptin by approximately 20 ng/ml in VMHL and ZDF rats but had no effect on their food intake, body weight, or fat tissue weight. Caloric matching of hyperphagic VMHL rats to SL controls did not reduce their resistance to hyperleptinemia. Whereas prediabetic ZDF rats had a fourfold elevation in islet fat, in VMHL rats islet fat was normal and none of them became diabetic. Isolated islets from ZDF rats were completely resistant to the lipopenic action of leptin, while VMHL islets exhibited 50% of the normal response; caloric matching of VMHL rats to SL controls increased leptin responsiveness of their islets to 92% of controls. We conclude that leptin regulation of adipocyte fat requires an intact VMH but that islet fat content is regulated independently of the VMH.

Retinoids synergize with insulin to induce hepatic Gck expression

Hepatic GK (glucokinase) plays a key role in maintaining glucose homoeostasis. Many stimuli regulate GK activity by controlling its gene transcription. We hypothesized that endogenous lipophilic molecules modulate hepatic Gck expression. Lipophilic molecules were extracted from rat livers, saponified and re-constituted as an LE (lipophilic extract). LE synergized with insulin to induce primary hepatocyte, but not beta-cell, Gck expression in an SREBP-1c (sterol-regulatory-element-binding protein-1c)-independent manner. The dramatic induction of Gck mRNA resulted in a significant increase in GK activity. Subsequently, the active molecules were identified as retinol and retinal by MS after the purification of the active LE fractions. Retinoids synergized with insulin to induce Gck expression by the activation of both RAR [RA (retinoic acid) receptor] and RXR (retinoid X receptor). Inhibition of RAR activation completely abolished the effect of retinal. The hepatic GK specific activity and Gck mRNA levels of Zucker lean rats fed with a VAD [VA (vitamin A)-deficient] diet were significantly lower than those of rats fed with VAS (VA-sufficient) diet. Additionally, the hepatic Gck mRNA expression of Sprague-Dawley rats fed with a VAD diet was lower than that of rats fed with VA-marginal, -adequate or -supplemented diets. The reduced expression of Gck mRNA was increased after an intraperitoneal dose of RA in VAD rats. Furthermore, an intravenous injection of RA rapidly raised hepatic Gck expression in rats fed with a VAS control diet. Understanding the underlying mechanism that mediates the synergy may be helpful for developing a treatment strategy for patients with diabetes.

Retinoids synergized with insulin to induce Srebp-1c expression and activated its promoter via the two liver X receptor binding sites that mediate insulin action

We have reported that the rat liver lipophilic extract (LE) synergized with insulin to induce Gck and Srebp-1c in primary rat hepatocytes. After identification of retinol and retinal in LE, only their effects in the absence or presence of insulin on Gck, but not that on Srebp-1c, were investigated subsequently. The retinoid effects on the Srebp-1c expression and the activation of its promoter were examined with real-time PCR and reporter gene assays, respectively. In primary hepatocytes, retinal and retinoic acid (RA) synergized with insulin to induce Srebp-1c expression. This induction was followed by the elevation of its target gene, fatty acid synthase. Activation of retinoid X receptor, but not retinoic acid receptor, was responsible for the induction of Srebp-1c expression. RA, but not retinal, also induced Srebp-1c expression in a dose dependent manner in INS-1 cells. The RA responsive elements in Srebp-1c promoter were determined as previously identified two liver X receptor elements responsible for mediating insulin action. We conclude that retinoids regulate hepatic Srebp-1c expression through activation of retinoid X receptor. The RA- and insulin-induced Srebp-1c expression converged at the same sites in its promoter, indicating the roles of vitamin A in regulation of hepatic gene expression.

Expression of the Transcription Factor STAT-1α in Insulinoma Cells Protects against Cytotoxic Effects of Multiple Cytokines

Destruction of pancreatic islet β-cells in type 1 diabetes appears to result from direct contact with infiltrating T-cells and macrophages and exposure to inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor TNF-α that such cells produce. We recently reported on a method for selection of insulinoma cells that are resistant to the cytotoxic effects of inflammatory cytokines (INS-1res), involving their growth in progressively increasing concentrations of IL-1β plus IFN-γ, and selection of surviving cells. In the current study, we have investigated the molecular mechanism of cytokine resistance in INS-1rescells. By focusing on the known components of the IFN-γ receptor signaling pathway, we have discovered that expression levels of signal transducer and activator of transcription (STAT)-1α are closely correlated with the cytokine-resistant and -sensitive phenotypes. That STAT-1α is directly involved in development of cytokine resistance is demonstrated by an increase of viability from 10 ± 2% in control cells to 50 ± 6% in cells with adenovirus-mediated overexpression of STAT-1α (p < 0.001) after culture of both cell groups in the presence of 100 units/ml IFN-γ plus 10 ng/ml IL-1β for 48 h. The resistance to IL-1β plus IFN-γ in STAT-1α-expressing cells is due in part to interference with IL-1β-mediated stimulation of inducible nitric-oxide synthase expression and nitric oxide production. Furthermore, overexpression of STAT-1α does not impair robust glucose-stimulated insulin secretion in the INS-1-derived cell line 832/13. We conclude that expression of STAT-1α may be a means of protecting insulin-producing cell lines from cytokine damage, which, in conjunction with appropriate cell-impermeant macroencapsulation devices, may allow such cells to be used for insulin replacement in type 1 diabetes.

Roles of vitamin A status and retinoids in glucose and fatty acid metabolism

The rising prevalence of metabolic diseases, such as obesity and diabetes, has become a public health concern. Vitamin A (VA, retinol) is an essential micronutrient for a variety of physiological processes, such as tissue differentiation, immunity, and vision. However, its role in glucose and lipid metabolism has not been clearly defined. VA activities are mediated by the metabolite of retinol catabolism, retinoic acid, which activates the retinoic acid receptor and retinoid X receptor (RXR). Since RXR is an obligate heterodimeric partner for many nuclear receptors involved in metabolism, it is reasonable to assume that VA status and retinoids contribute to glucose and lipid homeostasis. To date, the impacts of VA and retinoids on energy metabolism in animals and humans have been demonstrated in some basic and clinical investigations. This review summarizes the effects of VA status and retinoid treatments on metabolism of the liver, adipocytes, pancreatic β-cells, and skeletal muscle. It proposes a mechanism by which the dietary and hormonal signals converge on the promoter of sterol regulatory element-binding protein 1c gene to induce its expression, and in turn, the expression of lipogenic genes in hepatocytes. Future research projects relevant to the VAs roles in metabolic diseases are also discussed.

The hepatic Raldh1 expression is elevated in Zucker fatty rats and its over-expression introduced the retinal-induced Srebp-1c expression in INS-1 cells.

The roles of vitamin A (VA) in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c) expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF) rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA) for regulating gene expression. We hypothesized that the expression of RA production enzymes contributes to the regulation of the hepatic Srebp-1c expression. Therefore, we analyzed their expression levels in Zucker lean (ZL) and ZF rats. The mRNA levels of retinaldehyde dehydrogenase family 1 gene (Raldh1) were found to be higher in the isolated and cultured primary hepatocytes from ZF rats than that from ZL rats. The RALDH1 protein level was elevated in the liver of ZF rats. Retinol and retinal dose- and time-dependently induced the expression of RA responsive Cyp26a1 gene in hepatocytes and hepatoma cells. INS-1 cells were identified as an ideal tool to study the effects of RA production on the regulation of gene expression because only RA, but not retinal, induced Srebp-1c mRNA expression in them. Recombinant adenovirus containing rat Raldh1 cDNA was made and used to infect INS-1 cells. The over-expression of RALDH1 introduced the retinal-mediated induction of Srebp-1c expression in INS-1 cells. We conclude that the expression levels of the enzymes for RA production may contribute to the regulation of RA responsive genes, and determine the responses of the cells to retinoid treatments. The elevated hepatic expression of Raldh1 in ZF rats may cause the excessive RA production from retinol, and in turn, result in higher Srebp-1c expression. This excessive RA production may be one of the factors contributing to the elevated lipogenesis in the liver of ZF rats.

Zyflamend reduces the expression of androgen receptor in a model of castrate-resistant prostate cancer.

Prostate cancer is the most commonly diagnosed solid malignancy, and tumor cells eventually transform to castrate resistance through multiple pathways including activation of the androgen receptor via insulin-like growth factor receptor (IGF-1R) signaling involving phospho-AKT (pAKT). In this study, a mixture of herbal extracts, Zyflamend®, was used as a treatment in a model of castrate-resistant prostate cancer using CWR22Rv1 cells. Zyflamend reduced androgen receptor and IGF-1R expression along with a reduction of IGF-1-mediated proliferation of CWR22Rv1 cells. IGF-1 induced downstream AKT phosphorylation; however, the induction of pAKT was not associated with androgen receptor expression. Further, constitutively active form of AKT had no effect on nuclear expression of androgen receptor, indicating that upregulation of pAKT did not promote androgen receptor expression or nuclear translocation in castrate-resistant CWR22Rv1 cells. Conversely, Zyflamend reduced androgen receptor expression following IGF-1 stimulation and in cells overexpressing pAKT. These results demonstrated that Zyflamend inhibited IGF-1-stimulated cell growth, IGF-1R expression, and androgen receptor expression and its nuclear localization, but these effects were not dependent upon phosphatidylinositol 3-kinase/pAKT signaling. In conclusion, Zyflamend decreased cell proliferation and inhibited IGF-1R and androgen receptor expression in a phosphatidylinositol 3-kinase/pAKT independent manner.