Summary In yeast, worms and flies, an extra copy of the gene encoding the Sirtuin Sir2 increases metabolic efficiency, as does administration of polyphenols like resveratrol, thought to act through Sirtuins. But evidence that Sirtuin gain-of-function results in increased metabolic efficiency in mammals is limited. We generated transgenic mice with moderate overexpression of SirT1, designed to mimic the Sirtuin gain-of-function that improves metabolism in C.elegans. These mice exhibit normal insulin sensitivity, but decreased food intake and locomotor activity, resulting in decreased energy expenditure. However, in various models of insulin resistance and diabetes, SirT1 transgenics display improved glucose tolerance due to decreased hepatic glucose production and increased adiponectin levels, without changes in body weight or composition. We conclude that SirT1 gain-of-function primes the organism for metabolic adaptation to insulin resistance, increasing hepatic insulin sensitivity and decreasing whole-body energy requirements. These findings have important implications for Sirtuin-based therapies in humans.

The hallmark of type 2 diabetes is excessive hepatic glucose production. Several transcription factors and coactivators regulate this process in cultured cells. But gene ablation experiments have yielded few clues as to the physiologic mediators of this process in vivo. We show that inactivation of the gene encoding forkhead protein Foxo1 in mouse liver results in 40% reduction of glucose levels at birth and 30% reduction in adult mice after a 48 hr fast. Gene expression and glucose clamp studies demonstrate that Foxo1 ablation impairs fasting- and cAMP-induced glycogenolysis and gluconeogenesis. Pgc1alpha is unable to induce gluconeogenesis in Foxo1-deficient hepatocytes, while the cAMP response is significantly blunted. Conversely, Foxo1 deletion in liver curtails excessive glucose production caused by generalized ablation of insulin receptors and prevents neonatal diabetes and hepatosteatosis in insulin receptor knockout mice. The data provide a unifying mechanism for regulation of hepatic glucose production by cAMP and insulin.

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKC and PKC) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKC (KD or ⌬NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone-or hyperosmolarity-induced glucose uptake, were inhibited by KD or ⌬NKD in a dosedependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC was ϳ50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC mutant that lacks the pseudosubstrate domain (⌬PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of ⌬PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKC. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKC pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46,48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21,33,41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (...

Hepatic insulin resistance affects both carbohydrate and lipid metabolism. It has been proposed that insulin controls these 2 metabolic branches through distinct signaling pathways. FoxO transcription factors are considered effectors of the pathway regulating hepatic glucose production. Here we show that adenoviral delivery of constitutively nuclear forkhead box O1 (FoxO1) to mouse liver results in steatosis arising from increased triglyceride accumulation and decreased fatty acid oxidation. FoxO1 gain of function paradoxically increased insulin sensitivity by promoting Akt phosphorylation, while FoxO1 inhibition via siRNA decreased it. We show that FoxO1 regulation of Akt phosphorylation does not require DNA binding and is associated with repression of the pseudokinase tribble 3 (Trb3), a modulator of Akt activity. This unexpected dual role of FoxO1 in promoting insulin sensitivity and lipid synthesis in addition to glucose production has the potential to explain the peculiar admixture of insulin resistance and sensitivity that is commonly observed in the metabolic syndrome.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr 308 and Ser 473 ) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ϳ75% in CHO cells and ϳ30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4,14,25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11,23). These observations indicate that Akt is a downstream effector of PI 3-kinase.PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6,16,31,39,61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase ...

STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.

Ghrelin was identified in the stomach as an endogenous ligand specific for the growth hormone secretagogue receptor (GHS-R). GHS-R is found in various tissues, but its function is unknown. Here we show that GHS-R is found in hepatoma cells. Exposure of these cells to ghrelin caused up-regulation of several insulininduced activities including tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, mitogen-activated protein kinase activity, and cell proliferation. Unlike insulin, ghrelin inhibited Akt kinase activity as well as up-regulated gluconeogenesis. These findings raise the possibility that ghrelin modulates insulin activities in humans.