Troglitazone inhibits expression of the phosphoenolpyruvate carboxykinase gene by an insulin-independent mechanism.

Troglitazone is an oral insulin-sensitizing drug used to treat patients with type 2 diabetes. A major feature of this hyperglycemic state is the presence of increased rates of hepatic gluconeogenesis, which troglitazone is able to ameliorate. In this study, we examined the molecular basis for this property of troglitazone by exploring the effects of this compound on the expression of the two genes encoding the major regulatory enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary cultures of rat hepatocytes. Insulin is able to inhibit expression of both of these genes, which was verified in our model system. Troglitazone significantly reduced mRNA levels of PEPCK and G6Pase in rat hepatocytes isolated from normal and Zucker-diabetic rats, but to a lesser extent than that observed with insulin. Interestingly, troglitazone was unable to reduce cAMP-induced levels of PEPCK mRNA, suggesting that the molecular mechanism whereby troglitazone exerted its effects on gene expression differed from that of insulin. This was further supported by the observation that troglitazone was able to reduce PEPCK mRNA levels in the presence of the insulin signaling pathway inhibitors wortmannin, rapamycin, and PD98059. These results indicate that troglitazone can regulate the expression of specific genes in an insulin-independent manner, and that genes encoding gluconeogenic enzymes are targets for the inhibitory effects of this drug.     

Sodium arsenite induces orphan nuclear receptor SHP gene expression via AMP-activated protein kinase to inhibit gluconeogenic enzyme gene expression

Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.     

Role of resveratrol in FOXO1-mediated gluconeogenic gene expression in the liver

During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation of insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.     

Different mechanisms are used by insulin to repress three genes that contain a homologous thymine-rich insulin response element

Insulin rapidly and completely inhibits expression of the hepatic insulin-like growth factor binding protein-1 (IGFBP-1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. This inhibition is mediated through a phosphatidyl inositol 3-kinase-dependent regulation of a DNA element, termed the thymine-rich insulin response element, found within the promoters of each of these genes. This has led to the conclusion that these three promoters are regulated by insulin using the same molecular mechanism. However, we recently found that the regulation of the IGFBP1 but not the PEPCK or G6Pase genes by insulin was sensitive to rapamycin, an inhibitor of mTOR. Here, we present further evidence that different regulatory pathways mediate the insulin regulation of these promoters. Importantly, we identify a protein phosphatase activity in the pathway connecting mTOR to the IGFBP-1 promoter. These data have major implications for the development of molecular therapeutics for the treatment of insulin-resistant states such as diabetes and hypertension.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

Identification of a novel variant in the phosphoenolpyruvate carboxykinase gene promoter in Japanese patients with type 2 diabetes.

Phospho enolpyruvate carboxykinase (PEPCK) plays an important role in gluconeogenesis and hepatic glucose production. To test the hypothesis that mutations of the PEPCK gene promoter contribute to the increased hepatic glucose production that leads to diabetes, we screened for polymorphisms of the PEPCK promoter region in 252 Japanese type 2 diabetic patients and 188 non-diabetic control subjects. A novel variant at position - 232 (C to G) was found at a similar frequency in type 2 diabetes patients (32 %) and control subjects (35 %) (p = 0.26). However, patients with the - 232 G/G genotype had an earlier age of onset than those with the - 232 C/C or - 232 C/G genotypes (p = 0.028). As the variant might well otherwise influence hormonal action, we transfected PEPCK-luciferase fusion gene constructs with the variant into human hepatoma cells and examined the response to dexamethasone, insulin, and cAMP. The reporter assay showed no significant difference in hormonal responses with the fusion gene containing the variant. Accordingly, the single-base variant at position - 232 of the PEPCK gene promoter is most probably not a major contributor to the pathogenesis of type 2 diabetes. However, this variation may be useful as a genetic marker for other metabolic disorders, especially in Japanese.     

Evidence for the expression of both the hydrolase and translocase components of hepatic glucose-6-phosphatase in murine pancreatic islets

Glucose-6-phosphatase (G6Pase) is a multicomponent enzyme system which regulates the catalysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6Pase can antagonize glucose phosphorylation, a step prerequisite in the regulation of insulin secretion from pancreatic beta cells, and G6Pase activity is increased in islets isolated from animal models of type II diabetes. Using RT-PCR with hepatic G6Pase catalytic subunit primers, we demonstrate that the sizes of amplified products from ob/ob mouse islets are identical to those from liver cDNA. This was confirmed by PCR-based cloning and sequencing of the hepatic G6Pase catalytic subunit open reading frame from islet cDNA. The expression in islets of the G6P transporter, G6PT1, was also demonstrated, suggesting that all of the identified hepatic G6Pase system genes are expressed in pancreatic islets. Finally, the expression of islet-specific G6Pase-related protein (IGRP) in pancreatic islets was confirmed and its expression in liver was also observed.     

Liver glyconeogenesis: a pathway to cope with postprandial amino acid excess in high-protein fed rats?

This paper provides molecular evidence for a liver glyconeogenic pathway, that is, a concomitant activation of hepatic gluconeogenesis and glycogenesis, which could participate in the mechanisms that cope with amino acid excess in high-protein (HP) fed rats. This evidence is based on the concomitant upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, downregulation of glucose 6-phosphatase catalytic subunit (G6PC1) gene expression, an absence of glucose release from isolated hepatocytes and restored hepatic glycogen stores in the fed state in HP fed rats. These effects are mainly due to the ability of high physiological concentrations of portal blood amino acids to counteract glucagon-induced liver G6PC1 but not PEPCK gene expression. These results agree with the idea that the metabolic pathway involved in glycogen synthesis is dependent upon the pattern of nutrient availability. This nonoxidative glyconeogenic disposal pathway of gluconeogenic substrates copes with amino excess and participates in adjusting both amino acid and glucose homeostasis. In addition, the pattern of PEPCK and G6PC1 gene expression provides evidence that neither the kidney nor the small intestine participated in gluconeogenic glucose production under our experimental conditions. Moreover, the main glucose-6-phosphatase (G6Pase) isoform expressed in the small intestine is the ubiquitous isoform of G6Pase (G6PC3) rather than the G6PC1 isoform expressed in gluconeogenic organs.