An insulin-independent mechanism for transcriptional regulation of Foxo1 in type 2 diabetic mice.
ABSTRACT: Hepatic gluconeogenesis is the major contributor to the hyperglycemia observed in both patients and animals with type 2 diabetes. The transcription factor FOXO1 plays a dominant role in stimulating hepatic gluconeogenesis. FOXO1 is mainly regulated by insulin under physiological conditions, but liver-specific disruption of Foxo1 transcription restores normal gluconeogenesis in mice in which insulin signaling has been blocked, suggesting that additional regulatory mechanisms exist. Understanding the transcriptional regulation of Foxo1 may be conducive to the development of insulin-independent strategies for the control of hepatic gluconeogenesis. Here, we found that elevated plasma levels of adenine nucleotide in type 2 diabetes are the major regulators of Foxo1 transcription. We treated lean mice with 5'-AMP and examined their transcriptional profiles using RNA-seq. KEGG analysis revealed that the 5'-AMP treatment led to shifted profiles that were similar to db/db mice. Many of the upregulated genes were in pathways associated with the pathology of type 2 diabetes including Foxo1 signaling. As observed in diabetic db/db mice, lean mice treated with 5'-AMP displayed enhanced Foxo1 transcription, involving an increase in cellular adenosine levels and a decrease in the S-adenosylmethionine to S-adenosylhomocysteine ratio. This reduced methylation potential resulted in declining histone H3K9 methylation in the promoters of Foxo1, G6Pc, and Pepck. In mouse livers and cultured cells, 5'-AMP induced expression of more FOXO1 protein, which was found to be localized in the nucleus, where it could promote gluconeogenesis. Our results revealed that adenine nucleotide-driven Foxo1 transcription is crucial for excessive glucose production in type 2 diabetic mice.
Project description:SREBP1c is a key lipogenic transcription factor activated by insulin in the postprandial state. Although SREBP1c appears to be involved in suppression of hepatic gluconeogenesis, the molecular mechanism is not thoroughly understood. Here we show that CRY1 is activated by insulin-induced SREBP1c and decreases hepatic gluconeogenesis through FOXO1 degradation, at least, at specific circadian time points. SREBP1c(-/-) and CRY1(-/-) mice show higher blood glucose than wild-type (WT) mice in pyruvate tolerance tests, accompanied with enhanced expression of PEPCK and G6Pase genes. CRY1 promotes degradation of nuclear FOXO1 by promoting its binding to the ubiquitin E3 ligase MDM2. Although SREBP1c fails to upregulate CRY1 expression in db/db mice, overexpression of CRY1 attenuates hyperglycaemia through reduction of hepatic FOXO1 protein and gluconeogenic gene expression. These data suggest that insulin-activated SREBP1c downregulates gluconeogenesis through CRY1-mediated FOXO1 degradation and that dysregulation of hepatic SREBP1c-CRY1 signalling may contribute to hyperglycaemia in diabetic animals.
Project description:Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM.
Project description:Liver plays a key role in maintaining glucose homeostasis and impaired hepatic glucose metabolism is associated with type 2 diabetes. In the present study, we used RNA sequencing to profile the transcriptome of the livers of diabetic db/db mice as compared to the normal db/+?mice and identified 218 differentially expressed genes. Amongst these, there were 3 lncRNAs that were significantly downregulated and H19 was the most altered lncRNA in the livers of db/db mice. H19 expression significantly correlated with the expression of genes of the glycolysis and gluconeogenesis pathways, which suggest that altered hepatic H19 levels can directly or indirectly modulate their expression. Inhibition of H19 using specific siRNA in HepG2 cells and primary mouse hepatocytes significantly increased the levels of gluconeogenic genes. This was subsequently accompanied by increased hepatic glucose output. Further,H19 depletion in HepG2 cells impaired insulin signaling and increased nuclear localization of FoxO1, an important transcriptional regulator of gluconeogenic gene expression. Our results reveal a novel link between decreased H19 levels and impaired gluconeogenesis via regulation of FoxO1 nuclear levels. These put forth interesting observations on the regulatory role of H19 in altering hepatic physiology during diabetes.
Project description:Sodium meta-arsenite (SA) is implicated in the regulation of hepatic gluconeogenesis-related genes in vitro; however, the effects in vivo have not been studied. We investigated whether SA has antidiabetic effects in a type 2 diabetic mouse model. Diabetic db/db mice were orally intubated with SA (10?mg?kg(-1) body weight/day) for 8 weeks. We examined hemoglobin A1c (HbA1c), blood glucose levels, food intake, and body weight. We performed glucose, insulin, and pyruvate tolerance tests and analyzed glucose production and the expression of gluconeogenesis-related genes in hepatocytes. We analyzed energy metabolism using a comprehensive animal metabolic monitoring system. SA-treated diabetic db/db mice had reduced concentrations of HbA1c and blood glucose levels. Exogenous glucose was quickly cleared in glucose tolerance tests. The mRNA expressions of genes for gluconeogenesis-related enzymes, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly reduced in the liver of SA-treated diabetic db/db mice. In primary hepatocytes, SA treatment decreased glucose production and the expression of G6Pase, PEPCK, and hepatocyte nuclear factor 4 alpha (HNF-4?) mRNA. Small heterodimer partner (SHP) mRNA expression was increased in hepatocytes dependent upon the SA concentration. The expression of Sirt1 mRNA and protein was reduced, and acetylated forkhead box protein O1 (FoxO1) was induced by SA treatment in hepatocytes. In addition, SA-treated diabetic db/db mice showed reduced energy expenditure. Oral intubation of SA ameliorates hyperglycemia in db/db mice by reducing hepatic gluconeogenesis through the decrease of Sirt1 expression and increase in acetylated FoxO1.
Project description:Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Here, through the generation and analysis of a liver-specific Med23-knockout mouse, we found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila. Collectively, our data revealed Mediator MED23 as a novel regulator for energy homeostasis, suggesting potential therapeutic strategies against metabolic diseases.
Project description:Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of <sup>13</sup>C-alanine into newly synthesized glucose by hepatocytes. In vivo Gpt2 knockdown or knockout in liver has no effect on glucose concentrations in lean mice, but Gpt2 suppression alleviates hyperglycemia in db/db mice. These data suggest that ALT2 plays a significant role in hepatic gluconeogenesis from amino acids in diabetes.
Project description:<h4>Background</h4>Protease-activated protein-2 (PAR2) has been reported to regulate hepatic insulin resistance condition in type 2 diabetes mice. However, the mechanism of lipid metabolism through PAR2 in obesity mice have not yet been examined. In liver, Forkhead box O1 (FoxO1) activity induces peroxisome proliferator-activated receptor ? (PPAR?), leading to accumulation of lipids and hyperlipidemia. Hyperlipidemia significantly influence hepatic steatoses, but the mechanisms underlying PAR2 signaling are complex and have not yet been elucidated.<h4>Methods</h4>To examine the modulatory action of FoxO1 and its altered interaction with PPAR?, we utilized db/db mice and PAR2-knockout (KO) mice administered with high-fat diet (HFD).<h4>Results</h4>Here, we demonstrated that PAR2 was overexpressed and regulated downstream gene expressions in db/db but not in db+ mice. The interaction between PAR2/?-arrestin and Akt was also greater in db/db mice. The Akt inhibition increased FoxO1 activity and subsequently PPAR? gene in the livers that led to hepatic lipid accumulation. Our data showed that FoxO1 was negatively controlled by Akt signaling and consequently, the activity of a major lipogenesis-associated transcription factors such as PPAR? increased, leading to hepatic lipid accumulation through the PAR2 pathway under hyperglycemic conditions in mice. Furthermore, the association between PPAR? and FoxO1 was increased in hepatic steatosis condition in db/db mice. However, HFD-fed PAR2-KO mice showed suppressed FoxO1-induced hepatic lipid accumulation compared with HFD-fed control groups.<h4>Conclusion</h4>Collectively, our results provide evidence that the interaction of FoxO1 with PPAR? promotes hepatic steatosis in mice. This might be due to defects in PAR2/?-arrestin-mediated Akt signaling in diabetic and HFD-fed mice.
Project description:Long non-coding RNA Gomafu is involved in diabetes-related diseases. However, its role in insulin resistance (IR) remains unclear. Our objective is to explore the role of Gomafu in hepatic IR and glucose intolerance. Gomafu expression was determined in livers of ob/ob mice and high-fat diet (HFD) mice. The binding activity of NF-?B on the Gomafu promoter was measured by chromatin immunoprecipitation and quantitative real-time PCR assays. Increased Gomafu expression was observed in the livers of obese mice. Besides, the binding of NF-?B on the Gomafu promoter was also observed in hepatocytes from ob/ob mice. Further study showed that knockdown of NF-?B p65 alleviated the increase in hepatic Gomafu expression in vivo and in vitro. Knockdown of hepatic Gomafu inhibited hepatic glucose production (HGP) and improved insulin sensitivity in obese mice, whereas, overexpression of hepatic Gomafu resulted in an increase in random and fasting blood glucose levels in lean mice. In addition, we demonstrated that Gomafu functioned as miR-139 sponge and led to the de-repression of its target gene Foxo1, which played an important role in gluconeogenesis and HGP in hepatocytes. Finally, silenced Foxo1 expression abolished the effect of Gomafu overexpression on gluconeogenesis and glucose production in hepatocytes. Taken together, our data suggested that the increase in Gomafu expression contributed to hepatic IR in obese mice.
Project description:Renewed interest in alternative medicine among diabetic individuals prompted us to investigate anti-diabetic effects of Morinda citrifolia (noni) in high-fat diet (HFD)-fed mice. Type 2 diabetes is associated with increased glucose production due to the inability of insulin to suppress hepatic gluconeogenesis and promote glycolysis. Insulin inhibits gluconeogenesis by modulating transcription factors such as forkhead box O (FoxO1). Based on microarray analysis data, we tested the hypothesis that fermented noni fruit juice (fNJ) improves glucose metabolism via FoxO1 phosphorylation. C57BL/6 male mice were fed a HFD and fNJ for 12 weeks. Body weights and food intake were monitored daily. FoxO1 expression was analysed by real-time PCR and Western blotting. Specificity of fNJ-associated FoxO1 regulation of gluconeogenesis was confirmed by small interfering RNA (siRNA) studies using human hepatoma cells, HepG2. Supplementation with fNJ inhibited weight gain and improved glucose and insulin tolerance and fasting glucose in HFD-fed mice. Hypoglycaemic properties of fNJ were associated with the inhibition of hepatic FoxO1 mRNA expression, with a concomitant increase in FoxO1 phosphorylation and nuclear expulsion of the proteins. Gluconeogenic genes, phosphoenolpyruvate C kinase (PEPCK) and glucose-6-phosphatase (G6P), were significantly inhibited in mice fed a HFD+fNJ. HepG2 cells demonstrated more than 80 % inhibition of PEPCK and G6P mRNA expression in cells treated with FoxO1 siRNA and fNJ. These data suggest that fNJ improves glucose metabolism via FoxO1 regulation in HFD-fed mice.
Project description:Peroxisome proliferator-activated receptor ? (PPAR?) coactivator-1? (PGC-1?) promotes hepatic gluconeogenesis by activating HNF4? and FoxO1. PGC-1? expression in the liver is highly elevated in obese and diabetic conditions, leading to increased hepatic glucose production. We previously showed that the spliced form of X-box binding protein 1 (XBP1s) suppresses FoxO1 activity and hepatic gluconeogenesis. The shared role of PGC-1? and XBP1s in regulating FoxO1 activity and gluconeogenesis led us to investigate the probable interaction between PGC-1? and XBP1s and its role in glucose metabolism.We investigated the biochemical interaction between PGC-1? and XBP1s and examined the role of their interaction in glucose homeostasis using animal models.We show that PGC-1? interacts with XBP1s, which plays an anti-gluconeogenic role in the liver by suppressing FoxO1 activity. The physical interaction between PGC-1? and XBP1s leads to suppression of XBP1s activity rather than its activation. Upregulating PGC-1? expression in the liver of lean mice lessens XBP1s protein levels, and reducing PGC-1? levels in obese and diabetic mouse liver restores XBP1s protein induction.Our findings reveal a novel function of PGC-1? as a suppressor of XBP1s function, suggesting that hepatic PGC-1? promotes gluconeogenesis through multiple pathways as a co-activator for HNF4? and FoxO1 and also as a suppressor for anti-gluconeogenic transcription factor XBP1s.