John M. Stafford

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.

SRC-1 and GRIP1 Coactivate Transcription with Hepatocyte Nuclear Factor 4

Hepatocyte nuclear factor-4 (HNF4), a member of the nuclear receptor superfamily, plays an important role in tissue-specific gene expression, including genes involved in hepatic glucose metabolism. In this study, we show that SRC-1 and GRIP1, which act as coactivators for various nuclear receptors, associate with HNF4in vivo and enhance its transactivation potential. The AF-2 domain of HNF4 is required for this interaction and for the potentiation of transcriptional activity by these coactivators. p300 can also serve as a coactivator with HNF4, and it synergizes with SRC-1 to further augment the activity of HNF4. HNF4 is also a key regulator of the expression of hepatocyte nuclear factor-1 (HNF1). The overexpression of SRC-1 or GRIP1 enhances expression from a HNF1 gene promoter-reporter in HepG2 hepatoma cells, and this requires an intact HNF4-binding site in the HNF1 gene promoter. Type 1 maturity onset diabetes of young (MODY), which is characterized by abnormal glucose-mediated insulin secretion, is caused by mutations of the HNF4 gene. A mutation of the HNF4-binding site in the HNF1 gene promoter has also been associated with MODY. Thus, HNF4 is involved in the regulation of glucose homeostasis at several levels and along with the SRC-1, GRIP1, and p300 may play an important role in the pathophysiology of non-insulin-dependent diabetes mellitus.

Estrogen Treatment After Ovariectomy Protects Against Fatty Liver and May Improve Pathway-Selective Insulin Resistance

Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

The Repression of Hormone-activated PEPCK Gene Expression by Glucose Is Insulin-independent but Requires Glucose Metabolism*

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-controlling enzyme in hepatic gluconeogenesis, and it therefore plays a central role in glucose homeostasis. The rate of transcription of the PEPCK gene is increased by glucagon (via cAMP) and glucocorticoids and is inhibited by insulin. Under certain circumstances glucose also decreases PEPCK gene expression, but the mechanism of this effect is poorly understood. The glucose-mediated stimulation of a number of glycolytic and lipogenic genes requires the expression of glucokinase (GK) and increased glucose metabolism. HL1C rat hepatoma cells are a stably transfected line of H4IIE rat hepatoma cells that express a PEPCK promoter-chloramphenicol acetyltransferase fusion gene that is regulated in the same manner as the endogenous PEPCK gene. These cells do not express GK and do not normally exhibit a response of either the endogenous PEPCK gene, or of thetrans-gene, to glucose. A recombinant adenovirus that directs the expression of glucokinase (AdCMV-GK) was used to increase glucose metabolism in HL1C cells to test whether increased glucose flux is also required for the repression of PEPCK gene expression. In AdCMV-GK-treated cells glucose strongly inhibits hormone-activated transcription of the endogenous PEPCK gene and of the expressed fusion gene. The glucose effect on PEPCK gene promoter activity is blocked by 5 mm mannoheptulose, a specific inhibitor of GK activity. The glucose analog, 2-deoxyglucose mimics the glucose response, but this effect does not require GK expression. 3-O-methylglucose is ineffective. Glucose exerts its effect on the PEPCK gene within 4 h, at physiologic concentrations, and with an EC50 of 6.5 mm, which approximates theK m of glucokinase. The effects of glucose and insulin on PEPCK gene expression are additive, but only at suboptimal concentrations of both agents. The results of these studies demonstrate that, by inhibiting PEPCK gene transcription, glucose participates in a feedback control loop that governs its production from gluconeogenesis.

Central Nervous System Neuropeptide Y Signaling Modulates VLDL Triglyceride Secretion

OBJECTIVE—Elevated triglyceride (TG) is the major plasma lipid abnormality in obese and diabetic patients and contributes to cardiovascular morbidity in these disorders. We sought to identify novel mechanisms leading to hypertriglyceridemia. Resistance to negative feedback signals from adipose tissue in key central nervous system (CNS) energy homeostatic circuits contributes to the development of obesity. Because triglycerides both represent the largest energy depot in the body and are elevated in both the plasma and adipose in obesity and diabetes, we hypothesized that the same neural circuits that regulate energy balance also regulate the secretion of TGs into plasma. RESEARCH DESIGN AND METHODS—In normal fasting rats, the TG secretion rate was estimated by serial blood sampling after intravascular tyloxapol pretreatment. Neuropeptide Y (NPY) signaling in the CNS was modulated by intracerebroventricular injection of NPY, receptor antagonist, and receptor agonist. RESULTS—A single intracerebroventricular injection of NPY increased TG secretion by 2.5-fold in the absence of food intake, and this was determined to be VLDL by fast performance liquid chromatography (FPLC). This effect was recapitulated by activating NPY signaling in downstream neurons with an NPY-Y5 receptor agonist. An NPY-Y1 receptor antagonist decreased the elevated TGs in the form of VLDL secretion rate by 50% compared with vehicle. Increased TG secretion was due to increased secretion of VLDL particles, rather than secretion of larger particles, because apolipoprotein B100 was elevated in FPLC fractions corresponding to VLDL. CONCLUSIONS—We find that a key neuropeptide system involved in energy homeostasis in the CNS exerts control over VLDL-TG secretion into the bloodstream.

Pathway-selective Insulin Resistance and Metabolic Disease: The Importance of Nutrient Flux

Hepatic glucose and lipid metabolism are altered in metabolic disease (e.g. obesity, metabolic syndrome, and Type 2 diabetes). Insulin-dependent regulation of glucose metabolism is impaired. In contrast, lipogenesis, hypertriglyceridemia, and hepatic steatosis are increased. Because insulin promotes lipogenesis and liver fat accumulation, to explain the elevation in plasma and tissue lipids, investigators have suggested the presence of pathway-selective insulin resistance. In this model, insulin signaling to glucose metabolism is impaired, but insulin signaling to lipid metabolism is intact. We discuss the evidence for the differential regulation of hepatic lipid and glucose metabolism. We suggest that the primary phenotypic driver is altered substrate delivery to the liver, as well as the repartitioning of hepatic nutrient handling. Specific alterations in insulin signaling serve to amplify the alterations in hepatic substrate metabolism. Thus, hyperinsulinemia and its resultant increased signaling may facilitate lipogenesis, but are not the major drivers of the phenotype of pathway-selective insulin resistance.

Estrogen signaling prevents diet-induced hepatic insulin resistance in male mice with obesity

The development of insulin resistance in the liver is a key event that drives dyslipidemia and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor-α (ERα). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ERα signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ERα-knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared with their wild-type floxed (fl/fl) littermates during hyperinsulinemic euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of Akt-Ser(473) and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin-stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ERα in the liver helps prevent whole body and hepatic insulin resistance associated with HFD feeding in males. Augmenting hepatic estrogen signaling through ERα may lessen the impact of obesity on diabetes and cardiovascular risk in males.

Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.

Treatment update: thiazolidinediones in combination with metformin for the treatment of type 2 diabetes

Type 2 diabetes mellitus (DM2) is characterized by excessive hepatic gluconeogenesis, increased insulin resistance and a progressive inability of pancreatic beta cells to produce sufficient insulin. DM2 evolves as a progression from normal glucose tolerance, to impaired glucose tolerance (IGT) to frank diabetes mellitus, reflecting the establishment of insulin resistance and beta cell dysfunction. Insulin resistance not only contributes to impaired glycemic control in DM2, but to the development of hypertension, dyslipidemia and endothelial dysfunction. Cardiovascular disease is the primary morbidity for patients with DM2. The onset of insulin resistance and cardiovascular insult likely occurs well before the onset of IGT is detected clinically. Biguanides and thiazolidinediones (TZDs) are two classes of oral agents for the management of DM2 that improve insulin resistance, and thus have potential cardiovascular benefits beyond glycemic control alone. Metformin additionally inhibits hepatic gluconeogenesis. The combined use of two of these agents targets key pathophysiologic defects in DM2. Single pill combinations of rosiglitazone/metformin and pioglitazone/metformin have recently been approved for use in the US and Europe. This article reviews the clinical data behind the use of metformin in combination with TZDs for the management of diabetes, its impact on vascular health, side effects and potential mechanisms of action for combined use.

Stress-impaired transcription factor expression and insulin secretion in transplanted human islets

Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.