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.
SESN3 has been implicated in multiple biological processes including protection against oxidative stress, regulation of glucose and lipid metabolism. However, little is known about the factors and ...mechanisms controlling its gene expression at the transcriptional level. We performed in silico phylogenetic footprinting analysis of 5 kb upstream regions of a diverse set of human SESN3 orthologs for the identification of high confidence conserved binding motifs (BMo). We further analyzed the predicted BMo by a motif comparison tool to identify the TFs likely to bind these discovered motifs. Predicted TFs were then integrated with experimentally known protein-protein interactions and experimentally validated to delineate the important transcriptional regulators of SESN3. Our study revealed high confidence set of BMos (integrated with DNase I hypersensitivity sites) in the upstream regulatory regions of SESN3 that could be bound by transcription factors from multiple families including FOXOs, SMADs, SOXs, TCFs and HNF4A. TF-TF network analysis established hubs of interaction that include SMAD3, TCF3, SMAD2, HDAC2, SOX2, TAL1 and TCF12 as well as the likely protein complexes formed between them. We show using ChIP-PCR as well as over-expression and knock out studies that FOXO3 and SOX2 transcriptionally regulate the expression of SESN3 gene. Our findings provide an important roadmap to further our understanding on the regulation of SESN3.
Protein deacetylase Sirt1 has been implicated in the regulation of hepatic gluconeogenesis; however, the mechanisms are not fully understood. To further elucidate how Sirt1 regulates gluconeogenesis, ...we took a loss-of-function approach by deleting the coding DNA sequence for the catalytic domain of the Sirt1 gene in the liver of a wild-type mouse (LKO(Sirt)¹) or a genetic diabetic mouse in which hepatic insulin receptor substrates 1 and 2 are deleted (DKO(Irs½)). Whereas LKO(Sirt)¹ mice exhibited normal levels of fasting and fed blood glucose, inactivation of Sirt1 in DKO(Irs½) mice (TKO(Irs½:Sirt)¹) reduced blood glucose levels and moderately improved systemic glucose tolerance. Pyruvate tolerance was also significantly improved in TKO(Irs½:Sirt)¹ mice, suggesting that Sirt1 promotes hepatic gluconeogenesis in this diabetic mouse model. To understand why inactivation of hepatic Sirt1 does not alter blood glucose levels in the wild-type background, we searched for a potential cause and found that expression of small heterodimer partner (SHP, encoded by the Nr0b2 gene), an orphan nuclear receptor, which has been shown to suppress the activity of forkhead transcription factor FoxO1, was decreased in the liver of LKO(Sirt)¹ mice. Furthermore, our luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the Nr0b2 gene is a target of FoxO1, which is also regulated by Sirt1. After the gene is upregulated, Nr0b2 can feed back and repress FoxO1- and Sirt1-activated G6pc and Pdk4 gene expression. Thus, our results suggest that Sirt1 can both positively and negatively regulate hepatic gluconeogenesis through FoxO1 and Nr0b2 and keep this physiological process in control.
Dysregulation of metabolic functions in the liver impacts the development of diabetes and metabolic disorders. Normal liver function can be compromised by increased inflammation via the activation of ...signaling such as nuclear factor (NF)-κB signaling. Notably, we have previously identified lysine demethylase 2A (KDM2A)-as a critical negative regulator of NF-κB. However, there are no studies demonstrating the effect of KDM2A on liver function. Here, we established a novel liver-specific
knockout mouse model to evaluate KDM2A's role in liver functions. An inducible hepatic deletion of
(
KO), was generated by crossing the
floxed mice (
) we established with commercial albumin-Cre transgenic mice (B6.Cg-Tg(Alb-cre)21Mgn/J). We show that under a normal diet,
KO mice exhibited increased serum alanine aminotransferase (ALT) activity, L-type triglycerides (TG) levels, and liver glycogen levels vs. WT (
) animals. These changes were further enhanced in
liver KO mice in high-fat diet (HFD) conditions. We also observed a significant increase in NF-κB target gene expression in
liver KO mice under HFD conditions. Similarly, the KO mice exhibited increased immune cell infiltration. Collectively, these data suggest liver-specific KDM2A deficiency may enhance inflammation in the liver, potentially through NF-κB activation, and lead to liver dysfunction. Our study also suggests that the established
mouse model may serve as a powerful tool for studying liver-related metabolic diseases.
Sestrin 1/2/3 (Sesn1/2/3) belong to a small family of proteins that have been implicated in the regulation of metabolic homeostasis and oxidative stress. However, the underlying mechanisms remain ...incompletely understood. The aim of this work was to illustrate the collective function of Sesn1/2/3 in the protection against hepatic lipotoxicity.
We used Sesn1/2/3 triple knockout (TKO) mouse and cell models to characterize oxidative stress and signal transduction under lipotoxic conditions. Biochemical, histologic, and physiological approaches were applied to illustrate the related processes.
After feeding with a Western diet for 8 weeks, TKO mice developed remarkable metabolic associated fatty liver disease that was manifested by exacerbated hepatic steatosis, inflammation, and fibrosis compared with wild-type counterparts. Moreover, TKO mice exhibited higher levels of hepatic lipotoxicity and oxidative stress. Our biochemical data revealed a critical signaling node from sestrins to c-Jun N-terminal kinases (JNKs) in that sestrins interact with JNKs and mitogen-activated protein kinase kinase 7 and suppress the JNK phosphorylation and activity. In doing so, sestrins markedly reduced palmitate-induced lipotoxicity and oxidative stress in both mouse and human hepatocytes.
The data from this study suggest that Sesn1/2/3 play an important role in the protection against lipotoxicity-associated oxidative stress and related pathology in the liver.
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Abstract Insulin receptor substrates (Irs1, 2, 3 and Irs4) mediate the actions of insulin/IGF1 signaling. They have similar structure, but distinctly regulate development, growth, and metabolic ...homeostasis. Irs2 contributes to central metabolic sensing, partially by acting in leptin receptor (LepRb)-expressing neurons. Although Irs4 is largely restricted to the hypothalamus, its contribution to metabolic regulation is unclear because Irs4-null mice barely distinguishable from controls. We postulated that Irs2 and Irs4 synergize and complement each other in the brain. To examine this possibility, we investigated the metabolism of whole body Irs4−/y mice that lacked Irs2 in the CNS (bIrs2−/− ·Irs4−/y ) or only in LepRb-neurons ( Lepr ∆Irs2 · Irs4 − / y ). bIrs2−/− ·Irs4−/y mice developed severe obesity and decreased energy expenditure, along with hyperglycemia and insulin resistance. Unexpectedly, the body weight and fed blood glucose levels of Lepr ∆Irs2 · Irs4 − / y mice were not different from Lepr ∆Irs2 mice, suggesting that the functions of Irs2 and Irs4 converge upon neurons that are distinct from those expressing LepRb.
Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of ...oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma.
Sesn3 belongs to the three-member sestrin protein family. Sestrins have been implicated in anti-oxidative stress, AMPK and mTOR signal transduction, and metabolic homeostasis. However, the role of ...Sesn3 in the development of nonalcoholic steatohepatitis (NASH) has not been previously studied. In this work, we generated
Sesn3
whole-body knockout and liver-specific transgenic mice to investigate the hepatic function of Sesn3 in diet-induced NASH. With only 4 weeks of dietary treatment,
Sesn3
knockout mice developed severe NASH phenotype as characterized by hepatic steatosis, inflammation, and fibrosis. Strikingly, after 8-week feeding with a NASH-inducing diet,
Sesn3
transgenic mice were largely protected against NASH development. Transcriptomic analysis revealed that multiple extracellular matrix related processes were upregulated including TGFβ signaling and collagen production. Further biochemical and cell biological analyses have illustrated a critical control of the TGFβ-Smad pathway by Sesn3 at the TGFβ receptor and Smad3 levels. First, Sesn3 inhibits the TGFβ receptor through an interaction with Smad7; second, Sesn3 directly inhibits the Smad3 function through protein-protein interaction and cytosolic retention.
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