Sestrin 3 protein enhances hepatic insulin sensitivity by direct activation of the mTORC2-Akt signaling

Sestrin proteins have been implicated in multiple biological processes including resistance to oxidative and genotoxic stresses, protection against aging-related pathologies, and promotion of metabolic homeostasis; however, the underlying mechanisms are incompletely understood. Some evidence suggests that sestrins may inhibit mTORC1 (mechanistic target of rapamycin complex 1) through inhibition of RagA/B GTPases or activation of AMPK; however, whether sestrins are also involved in mTORC2 regulation and function is unclear. To investigate the functions and mechanisms of Sestrin 3 (Sesn3), we generated Sesn3 liver-specific transgenic and knockout mice. Our data show that Sesn3 liver-specific knockout mice exhibit insulin resistance and glucose intolerance, and Sesn3 transgenic mice were protected against insulin resistance induced by a high-fat diet. Using AMPK liver-specific knockout mice, we demonstrate that the Sesn3 insulin-sensitizing effect is largely independent of AMPK. Biochemical analysis reveals that Sesn3 interacts with and activates mTORC2 and subsequently stimulates Akt phosphorylation at Ser473. These findings suggest that Sesn3 can activate Akt via mTORC2 to regulate hepatic insulin sensitivity and glucose metabolism

Genetic Inactivation of Pyruvate Dehydrogenase Kinases Improves Hepatic Insulin Resistance Induced Diabetes

Pyruvate dehydrogenase kinases (PDK1-4) play a critical role in the inhibition of the mitochondrial pyruvate dehydrogenase complex especially when blood glucose levels are low and pyruvate can be conserved for gluconeogenesis. Under diabetic conditions, the Pdk genes, particularly Pdk4, are often induced, and the elevation of the Pdk4 gene expression has been implicated in the increased gluconeogenesis in the liver and the decreased glucose utilization in the peripheral tissues. However, there is no direct evidence yet to show to what extent that the dysregulation of hepatic Pdk genes attributes to hyperglycemia and insulin resistance in vivo. To address this question, we crossed Pdk2 or Pdk4 null mice with a diabetic model that is deficient in hepatic insulin receptor substrates 1 and 2 (Irs1/2). Metabolic analyses reveal that deletion of the Pdk4 gene had better improvement in hyperglycemia and glucose tolerance than knockout of the Pdk2 gene whereas the Pdk2 gene deletion showed better insulin tolerance as compared to the Pdk4 gene inactivation on the Irs1/2 knockout genetic background. To examine the specific hepatic effects of Pdks on diabetes, we also knocked down the Pdk2 or Pdk4 gene using specific shRNAs. The data also indicate that the Pdk4 gene knockdown led to better glucose tolerance than the Pdk2 gene knockdown. In conclusion, our data suggest that hepatic Pdk4 may be critically involved in the pathogenesis of diabetes

The inhibitory effect of ethanol on Sestrin3 in the pathogenesis of ethanol-induced liver injury

Sestrins (Sesns) are a family of stress-sensitive genes that have been suggested to regulate lipid metabolism. Chronic ethanol feeding is known to cause lipid accumulation in hepatocytes. This study was designed to investigate the role of Sesn3 in the pathogenesis of alcohol-induced hepatic steatosis. We demonstrated that ethanol inhibited the expression of Sesn3 in VL-17A cells. Overexpression of Sesn3 ameliorated triglyceride accumulation; downregulation using short hairpin RNA significantly deteriorated triglyceride accumulation in these cells. The expression of Sesn3 was also reduced in mice fed with ethanol for 4 wk. Overexpression of Sesn3 prevented hepatic steatosis, whereas knockdown of Sesn3 worsened hepatic steatosis in ethanol-fed mice. Overexpression of Sesn3 significantly reduced the expression of genes encoding for lipid synthesis through AMPK pathway. Overexpression of Sesn3 augmented the effect of ethanol on phospho-p70 S6 kinase. The levels of hepatic light chain 3, a marker for autophagy, expression were significantly decreased in ethanol-fed mice after Sesn3 gene was knocked down. Our findings suggest that inhibitory effect of ethanol on Sesn3 may play an important role in the development of ethanol-induced fatty liver

FoxO3 transcription factor and Sirt6 deacetylase regulate LDL-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression

Elevated LDL-cholesterol is a risk factor for the development of cardiovascular disease. Thus, proper control of LDL-cholesterol homeostasis is critical for organismal health. Genetic analysis has identified PCSK9 (proprotein convertase subtilisin/kexin type 9) as a crucial gene in the regulation of LDL-cholesterol via control of LDL receptor degradation. Although biochemical characteristics and clinical implications of PCSK9 have been extensively investigated, epigenetic regulation of this gene is largely unknown. In this work we have discovered that Sirt6, an NAD+-dependent histone deacetylase, plays a critical role in the regulation of the Pcsk9 gene expression in mice. Hepatic Sirt6 deficiency leads to elevated Pcsk9 gene expression and LDL-cholesterol as well. Mechanistically, we have demonstrated that Sirt6 can be recruited by forkhead transcription factor FoxO3 to the proximal promoter region of the Pcsk9 gene and deacetylates histone H3 at lysines 9 and 56, thereby suppressing the gene expression. Also remarkably, overexpression of Sirt6 in high fat diet-fed mice lowers LDL-cholesterol. Overall, our data suggest that FoxO3 and Sirt6, two longevity genes, can reduce LDL-cholesterol levels through regulation of the Pcsk9 gene

BRD7 improves glucose homeostasis independent of IRS proteins

Bromodomain-containing protein 7 (BRD7) has emerged as a player in the regulation of glucose homeostasis. Hepatic BRD7 levels are decreased in obese mice, and the reinstatement of hepatic BRD7 in obese mice has been shown to establish euglycemia and improve glucose homeostasis. Of note, the upregulation of hepatic BRD7 levels activates the AKT cascade in response to insulin without enhancing the sensitivity of the insulin receptor (InsR)-insulin receptor substrate (IRS) axis. In this report, we provide evidence for the existence of an alternative insulin signaling pathway that operates independently of IRS proteins and demonstrate the involvement of BRD7 in this pathway. To investigate the involvement of BRD7 as a downstream component of InsR, we utilized liver-specific InsR knockout mice. Additionally, we employed liver-specific IRS1/2 knockout mice to examine the requirement of IRS1/2 for the action of BRD7. Our investigation of glucose metabolism parameters and insulin signaling unveiled the significance of InsR activation in mediating BRD7's effect on glucose homeostasis in the liver. Moreover, we identified an interaction between BRD7 and InsR. Notably, our findings indicate that IRS1/2 is not necessary for BRD7's regulation of glucose metabolism, particularly in the context of obesity. The upregulation of hepatic BRD7 significantly reduces blood glucose levels and restores glucose homeostasis in high-fat diet-challenged liver-specific IRS1/2 knockout mice. These findings highlight the presence of an alternative insulin signaling pathway that operates independently of IRS1/2 and offer novel insights into the mechanisms of a previously unknown insulin signaling in obesity.Nutritional Science

Inactivating hepatic follistatin alleviates hyperglycemia

Unsuppressed hepatic glucose production (HGP) contributes substantially to glucose intolerance and diabetes, which can be modeled by the genetic inactivation of hepatic insulin receptor substrate 1 (Irs1) and Irs2 (LDKO mice). We previously showed that glucose intolerance in LDKO mice is resolved by hepatic inactivation of the transcription factor FoxO1 (that is, LTKO mice)-even though the liver remains insensitive to insulin. Here, we report that insulin sensitivity in the white adipose tissue of LDKO mice is also impaired but is restored in LTKO mice in conjunction with normal suppression of HGP by insulin. To establish the mechanism by which white adipose tissue insulin signaling and HGP was regulated by hepatic FoxO1, we identified putative hepatokines-including excess follistatin (Fst)-that were dysregulated in LDKO mice but normalized in LTKO mice. Knockdown of hepatic Fst in the LDKO mouse liver restored glucose tolerance, white adipose tissue insulin signaling and the suppression of HGP by insulin; however, the expression of Fst in the liver of healthy LTKO mice had the opposite effect. Of potential clinical significance, knockdown of Fst also improved glucose tolerance in high-fat-fed obese mice, and the level of serum Fst was reduced in parallel with glycated hemoglobin in obese individuals with diabetes who underwent therapeutic gastric bypass surgery. We conclude that Fst is a pathological hepatokine that might be targeted for diabetes therapy during hepatic insulin resistance

Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic beta cells

AIMS/HYPOTHESIS: Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells. METHODS: To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy. RESULTS: Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets. CONCLUSIONS/INTERPRETATION: These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes

Sirt6 Regulates Insulin Secretion from the Pancreatic Beta Cells

poster abstractSirt6 is an NAD-dependent histone deacetylase, which is involved in multiple biological processes, including aging, DNA repair, and metabolism; however, it is unclear what its functions in pancreatic beta-cells are. The beta cells play an essential role in metabolic regulation by secreting insulin in response to an elevated glucose concentration in the circulation. To examine the role of Sirt6 in beta cells, we initially used adenovirus-mediated shRNA to knock down the Sirt6 gene expression in a mouse pancreatic beta cell line - MIN6. Knockdown of the Sirt6 gene significantly reduced glucose-stimulated insulin secretion. To further validate this phenotype in vivo, we generated pancreatic beta-cell-specific Sirt6 knockout mice (bKO) using mouse genetic approach. Indeed, the bKO mice showed remarkable impairment in both first and second phases of insulin secretion in response to a glucose load. While morphometric analyses did not reveal significant difference in islet area between wild-type and bKO mice, biochemical analysis of ATP concentrations showed a 22% decrease in bKO mouse islets relative to control wild-type islets after glucose stimulation. To assess mitochondrial function in Sirt6-deficient beta cells, we also performed Seahorse bioenergetics assays in MIN6 cells after the Sirt6 gene was knocked down. Glucose oxidation in mitochondria was decreased 20-30% in Sirt6- knockdown MIN6 cells as compared to the control cells. Since calcium signaling is critical to insulin secretion, we also measured intracellular calcium concentrations using a fluorescent imaging approach. The results showed a significant decrease in cytoplasmic calcium in the bKO islets as compared to the wild-type controls. Overall, our data demonstrate that Sirt6 plays a critical role in the regulation of pancreatic insulin secretion. This work was supported in part by the NIDDK grant R01DK091592

Deletion of hepatic FoxO1/3/4 genes in mice significantly impacts on glucose metabolism through downregulation of gluconeogenesis and upregulation of glycolysis.

Forkhead transcription factors FoxO1/3/4 have pleiotrophic functions including anti-oxidative stress and metabolism. With regard to glucose metabolism, most studies have been focused on FoxO1. To further investigate their hepatic functions, we generated liver-specific FoxO1/3/4 knockout mice (LTKO) and examined their collective impacts on glucose homeostasis under physiological and pathological conditions. As compared to wild-type mice, LTKO mice had lower blood glucose levels under both fasting and non-fasting conditions and they manifested better glucose and pyruvate tolerance on regular chow diet. After challenged by a high-fat diet, wild-type mice developed type 2 diabetes, but LTKO mice remained euglycemic and insulin-sensitive. To understand the underlying mechanisms, we examined the roles of SIRT6 (Sirtuin 6) and Gck (glucokinase) in the FoxO-mediated glucose metabolism. Interestingly, ectopic expression of SIRT6 in the liver only reduced gluconeogenesis in wild-type but not LTKO mice whereas knockdown of Gck caused glucose intolerance in both wild-type and LTKO mice. The data suggest that both decreased gluconeogenesis and increased glycolysis may contribute to the overall glucose phenotype in the LTKO mice. Collectively, FoxO1/3/4 transcription factors play important roles in hepatic glucose homeostasis