Physiologically important cell-signalling networks are complex, and contain several points of regulation, signal divergence and crosstalk with other signalling cascades. Here, we use the concept of 'critical nodes' to define the important junctions in these pathways and illustrate their unique role using insulin signalling as a model system.

Sirt3 is a member of the sirtuin family of protein deacetylases that is localized in mitochondria and regulates mitochondrial function. Sirt3 expression in skeletal muscle is decreased in models of type 1 and type 2 diabetes and regulated by feeding, fasting, and caloric restriction. Sirt3 knockout mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling. This effect is mimicked by knockdown of Sirt3 in cultured myoblasts, which exhibit reduced mitochondrial oxidation, increased reactive oxygen species, activation of JNK, increased serine and decreased tyrosine phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 plays an important role in diabetes through regulation of mitochondrial oxidation, reactive oxygen species production, and insulin resistance in skeletal muscle.mitochondrial metabolism | protein acetylation I nsulin resistance in skeletal muscle is a major and early feature in the pathogenesis of type 2 diabetes (1, 2). This pathological condition has been shown to involve decreased activity of the insulin signaling network with reduced tyrosine phosphorylation of the insulin receptor and its substrates, decreased activation of phosphatidylinositol 3-kinase (PI 3-kinase), and decreased activation of Akt/PKB (protein kinase B), leading to reduced glucose uptake and other metabolic abnormalities (3-5). Another early feature of type 2 diabetes is altered mitochondrial function in muscle. Reduced expression of multiple nuclear-encoded genes involved in mitochondrial oxidative phosphorylation and alterations in mitochondrial morphology have been observed in skeletal muscle of both rodent models of diabetes and humans with type 2 diabetes (6-8). Impaired mitochondrial lipid oxidation and glycolytic capacity have also been observed in individuals with diabetes and obesity, whereas enhanced mitochondrial lipid oxidation capacity has been associated with improved insulin resistance (9, 10). This reduced expression and/or activity of mitochondrial proteins has been closely associated with altered skeletal muscle physiology and metabolism. Some, but not all, studies have found similar alterations in skeletal muscle of individuals with a family history positive for type 2 diabetes (8, 11). Reduced oxidative capacity and reduced ATP synthesis rates have also been shown in individuals with type 2 diabetes and in some nondiabetic individuals with a family history for diabetes (12).In addition to its role in substrate metabolism, the mitochondrion is the major production site of reactive oxygen species (ROS). When ROS level is excessive or there is impaired ROS clearance, the oxidative stress response can activate serine/ threonine kinases such as protein kinase C, S6 kinase, and Jun N-terminal kinase (JNK), which can phosphorylate the insulin receptor (IR) and/or insulin receptor substrate (IRS) proteins (13-15), leading to a decrease in their tyrosine phosphorylation, decreased activation of PI 3-kinase and A...

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr 960 of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-␣ (TNF-␣), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-␣ induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-␣ target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.

The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.