SIRT4, a member of the sirtuin family, has been implicated in the regulation of insulin secretion by modulation of glutamate dehydrogenase. However, the role of this enzyme in the regulation of metabolism in other tissues is unknown. In this study we investigated whether depletion of SIRT4 would enhance liver and muscle metabolic functions. To do this SIRT4 was knocked down using an adenoviral shRNA in mouse primary hepatocytes and myotubes. We observed a significant increase in gene expression of mitochondrial and fatty acid metabolism enzymes in hepatocytes with reduced SIRT4 levels. SIRT4 knockdown also increased SIRT1 mRNA and protein levels both in vitro and in vivo. In agreement with the increased fatty acid oxidation (FAO) gene expression, we showed a significant increase in FAO in SIRT4 knockdown primary hepatocytes compared with control, and this effect was dependent on SIRT1. In primary myotubes, knockdown of SIRT4 resulted in increased FAO, cellular respiration, and pAMPK levels. When SIRT4 was knocked down in vivo by tail vein injection of a shRNA adenovirus, we observed a significant increase in hepatic mitochondrial and FAO gene expression consistent with the findings in primary hepatocytes.Taken together these findings demonstrate that SIRT4 inhibition increases fat oxidative capacity in liver and mitochondrial function in muscle, which might provide therapeutic benefits for diseases associated with ectopic lipid storage such as type 2 diabetes. Sirtuins (SIRTs)2 are an evolutionarily conserved family of NAD-dependent family of deacetylases that regulate diverse set of biological processes including stress response, genome maintenance, and energy metabolism (1, 2). The sirtuin family consists of seven family members (SIRT1-7), each containing a conserved catalytic core domain (3). SIRT1, SIRT2, SIRT3, and SIRT5 catalyze the NAD-dependent deacetylation, whereas SIRT4 and SIRT6 have been proposed to mediate ADP-ribosylation of protein substrates. The sirtuins are located in different organelles (4), with SIRT4 mainly localized in the mitochondria (5). Recent studies suggested that SIRT4 uses NAD to ADPribosylate and inactivate glutamate dehydrogenase, an enzyme that converts glutamate to ␣-ketoglutarate in mitochondria and thereby suppresses insulin secretion from pancreatic beta cells (5, 6). In addition to pancreatic beta cells, SIRT4 is highly expressed in brain, liver, kidney, and heart with moderate expression in skeletal muscle (5). The function of SIRT4 in these other tissues is unknown. Based on its mitochondrial localization, we hypothesized that SIRT4 is involved in mitochondrial oxidative metabolism. To test our hypothesis, we knocked down SIRT4 expression in primary hepatocytes and myotubes with an adenovirus expressing a SIRT4 shRNA and assessed fatty acid oxidation and oxygen consumption. In addition, we delivered an shRNA adenovirus in vivo to knock down SIRT4 specifically in liver in mice. EXPERIMENTAL PROCEDURESCell Culture and Adenoviral Transduction-The sequence of Ad-shRNA-SIRT4 ...

In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C. elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction

SIRT1 is a NAD-dependent deacetylase that regulates a variety of pathways including the stress protection pathway. SIRT1 deacetylates a number of protein substrates, including histones, FOXOs, PGC-1α, and p53, leading to cellular protection. We identified a functional interaction between cJUN N-terminal kinase (JNK1) and SIRT1 by coimmunoprecipitation of endogenous proteins. The interaction between JNK1 and SIRT1 was identified under conditions of oxidative stress and required activation of JNK1 via phosphorylation. Modulation of SIRT1 activity or protein levels using nicotinamide or RNAi did not modify JNK1 activity as measured by its ability to phosphorylate cJUN. In contrast, human SIRT1 was phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increased its nuclear localization and enzymatic activity. Surprisingly, JNK1 phosphorylation of SIRT1 showed substrate specificity resulting in deacetylation of histone H3, but not p53. These findings identify a mechanism for regulation of SIRT1 enzymatic activity in response to oxidative stress and shed new light on its role in the stress protection pathway.

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulinresponsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1⅐IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⅐IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300͞CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A͞SRC interaction domains of p300͞CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⅐IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300͞CBP͞SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.I nsulin signaling via the phosphatidylinositol 3-kinase (PI 3-kinase)͞protein kinase B (PKB) pathway has diverse effects on cellular metabolism and apoptosis (1, 2). A major role of insulin is to act in opposition to the catabolic effects of cAMP and glucocorticoids, agents that stimulate liver gluconeogenesis. The rate-limiting step in gluconeogenesis is catalyzed by the phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32) gene. The insulin-like growth factor binding protein 1 (IGFBP-1) gene indirectly promotes gluconeogenesis by binding insulin-like growth factor (IGF)-I and -II and inhibiting their insulin-like effects. Expression of the PEPCK and IGFBP-1 genes is controlled at the transcriptional level by a complex regulatory mechanism in which glucocorticoids activate and insulin inhibits gene expression (3-6).In the case of the PEPCK gene, the response to both glucocorticoids and insulin is mediated by the accessory factor II (AFII) site, located upstream of the glucocorticoid-response element (GRE); this site is also referred to as the PEPCK insulin-response sequence, IRS-1 (7). Similarly the response of the IGFBP-1 promoter to glucocorticoids and insulin is mediated by one site, the insulin-response element (IRE) site located upstream of its GRE (5, 8). Biochemical evidence first showed that the forkhead (FK...