Relationship between blood glucose levels and hepatic Fto mRNA expression in mice

Common variants in the fat mass and obesity associated (FTO) gene are associated with obesity and type 2 diabetes. Fto-deficient mice develop hepatic insulin resistance, leading to the hypothesis that hepatic Fto plays a role in the regulation of glucose metabolism and that hepatic Fto expression is regulated by metabolic states. We found that hepatic Fto mRNA levels were increased by fasting in mice. Intraperitoneal glucose injection reduced hepatic Fto mRNA levels without significant changes in body weight in fasted mice. The inverse correlation between Fto mRNA and glucose remained significant after adjusting for body weight. There were positive correlations between hepatic Fto mRNA expression and gluconeogenic gene expression. These data support the hypothesis that hepatic Fto expression changes in response to metabolic states and glucose reduces hepatic Fto mRNA expression independently of body weight. Hepatic Fto may participate in the feedback regulation of glucose metabolism via gluconeogenesis.     

Vitamin A status and its metabolism contribute to the regulation of hepatic genes during the cycle of fasting and refeeding in rats

Vitamin A (VA) status and its metabolism affect hepatic metabolic homeostasis. We investigated if VA status and metabolism contribute to energy metabolism and expression of hepatic genes in the cycle of fasting and refeeding. Zucker lean rats with VA sufficient (VAS) or VA deficient (VAD) status were respectively grouped as: ad libitum (VAS-AD or VAD-AD), 48-h fasted (VAS-Fasted or VAD-Fasted), 48-h fasted and refed a VAS diet (VAS-Refed-VAS or VAD-Refed-VAS), or refed a VAD diet (VAS-Refed-VAD or VAD-Refed-VAD) for 6 h. Respiratory exchange ratio (RER) of rats fed the VAS or VAD diet was monitored for 6 weeks. From week four, rats fed the VAS diet had higher RER than those fed the VAD diet. VAS-Refed rats had higher plasma levels of glucose, triglyceride, insulin and leptin than VAD-Refed rats. The mRNA and protein levels of hepatic genes for fuel metabolism in the fasting and refeeding cycle were determined using real-time polymerase chain reaction and immunoblot, respectively. The mRNA levels of glucokinase (Gck), sterol regulatory element-binding protein 1c (Srebp-1c), and fatty acid synthase (Fas) were lowered in VAS-Fasted and VAD-Fasted rats, and increased in VAS-Refed-VAS, VAS-Refed-VAD and VAD-Refed-VAS, but not VAD-Refed-VAD, rats. The ACL and FAS protein levels only dropped in VAS-Fasted rats and increased in VAS-Refed-VAS rats. The GK protein level decreased only in VAS-Fasted rats, and increased in VAS-Refed-VAS, VAS-Refed-VAD and VAD-Refed-VAS (but not VAD-Refed-VAD) rats. We conclude that VA status and its metabolism in the fasting and refeeding cycle contribute to the regulation of hepatic gene expression in rats.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

Alteration of De Novo Glucose Production Contributes to Fasting Hypoglycaemia in Fyn Deficient Mice

Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of ¹³C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

A hormone-dependent module regulating energy balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.     

Forkhead Box O1 (FOXO1) Protein,but Not p53, Contributes to Robust Induction of p21 Expression in Fasted Mice

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.