SIRT1 is a prominent member of a family of NAD؉ -dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.

padhyay G, Olefsky JM. SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity. Am J Physiol Endocrinol Metab 298: E419 -E428, 2010. First published December 8, 2009; doi:10.1152/ajpendo.00417.2009.-Chronic inflammation is an important etiology underlying obesity-related disorders such as insulin resistance and type 2 diabetes, and recent findings indicate that the macrophage can be the initiating cell type responsible for this chronic inflammatory state. The mammalian silent information regulator 2 homolog SIRT1 modulates several physiological processes important for life span, and a potential role of SIRT1 in the regulation of insulin sensitivity has been shown. However, with respect to inflammation, the role of SIRT1 in regulating the proinflammatory pathway within macrophages is poorly understood. Here, we show that knockdown of SIRT1 in the mouse macrophage RAW264.7 cell line and in intraperitoneal macrophages broadly activates the JNK and IKK inflammatory pathways and increases LPS-stimulated TNF␣ secretion. Moreover, gene expression profiles reveal that SIRT1 knockdown leads to an increase in inflammatory gene expression. We also demonstrate that SIRT1 activators inhibit LPS-stimulated inflammatory pathways, as well as secretion of TNF␣, in a SIRT1-dependent manner in RAW264.7 cells and in primary intraperitoneal macrophages. Treatment of Zucker fatty rats with a SIRT1 activator leads to greatly improved glucose tolerance, reduced hyperinsulinemia, and enhanced systemic insulin sensitivity during glucose clamp studies. These in vivo insulin-sensitizing effects were accompanied by a reduction in tissue inflammation markers and a decrease in the adipose tissue macrophage proinflammatory state, fully consistent with the in vitro effects of SIRT1 in macrophages. In conclusion, these results define a novel role for SIRT1 as an important regulator of macrophage inflammatory responses in the context of insulin resistance and raise the possibility that targeting of SIRT1 might be a useful strategy for treating the inflammatory component of metabolic diseases. macrophage; insulin resistance FOR MANY YEARS, IT HAS BEEN KNOWN that caloric restriction extends life span over a wide range of species, including mammals (27). Silent information regulator 2 (Sir2) is a NADdependent deacetylase that is one of the components connecting the metabolic effects of caloric restriction to longevity in yeast, worms, and flies (7). Mammals express 7 homologs of yeast Sir2, identified as the SIRTUIN family, SIRT1-7 (7). SIRT1 has the closest homology to Sir2, and recent data suggest that activation of SIRT1 may be, at least partially, responsible for the extension of life span in mammals (4, 5, 7).

GLP-1 is secreted in a nutrient-dependent manner and potentiates glucose-dependent insulin secretion in pancreatic ␤ cells and inhibits glucagon secretion from ␣ cells. Chronic administration of GLP-1 also promotes insulin synthesis, ␤ cell proliferation, and neogenesis (1-3). Recent drug discovery has focused on GLP-1 action because of its therapeutic utility in the treatment of type 2 diabetes. GLP-1 analogues and small molecule compounds that inhibit GLP-1 degrading enzyme DPP-IV are all effective at improving glycemic profiles and ␤ cell performance (4, 5). Thus, a thorough understanding of GLP-1's cellular actions assumes greater importance.The GLP-1 receptor (GLP-1 R) is a member of the seventransmembrane family of G protein-coupled receptors (7TMRs) (6). A large body of literature exists on many aspects of 7TMR signaling and function, and it is now recognized that ␤-arrestin-1 is an important adaptor protein for several 7TMRs and functions in the process of transmitting receptor-mediated downstream signals, receptor internalization, and receptor desensitization (7,8). ␤-Arrestin-1 can also function as an adaptor/signaling protein in other receptor systems, including the IGF-1 receptor (9, 10), the TNF-␣ receptor (11), and others (7,8,(12)(13)(14)(15)(16). Given the widespread functions of ␤-arrestin-1, particularly in relationship to 7TMRs, we hypothesized that ␤-arrestin-1 could play a role in GLP-1 R action. In the current work, we tested this proposition in a variety of ways and found that ␤-arrestin-1 coassociates with the GLP-1 R and plays a role in GLP-1 signaling events that stimulate cAMP production, phosphoprotein generation, and insulin secretion in the pancreatic ␤ cell line INS-1 cells. These results establish a role for ␤-arrestin-1 and provide further insight into the cellular mechanisms of GLP-1 action. Results ␤-Arrestin-1Associates with the GLP-1 Receptor. The GLP-1 receptor (GLP-1 R) is a member of the 7TMR family (1, 2, 6), and ␤-arrestin-1 has diverse functions as an adaptor molecule for several classes of receptor types (7,8,10). To determine whether there is an association between ␤-arrestin-1 and the GLP-1 R in a ␤ cell model, we conducted coimmunoprecipitation experiments in FLAG-tagged ␤-arrestin-1-expressing INS-1 cells. As shown in Fig. 1A, an interaction between the GLP-1 R and ␤-arrestin-1 was strongly enhanced in an agonist (GLP-1)-dependent manner.It is known that ␤-arrestin-1 is degraded after ligand stimulation in several receptor signaling systems (14,(17)(18)(19), and, to determine whether this was the case in our model, we measured ␤-arrestin-1 protein content after treatment of INS-1 cells with 100 nM GLP-1. As shown in Fig. 1B, GLP-1 had a timedependent effect to decrease cellular ␤-arrestin-1 levels, and ␤-arrestin-1 was decreased by Ϸ30% after 7 h of GLP-1 treatment.Effect of ␤-Arrestin-1 Knockdown on GLP-1 Signaling. The data in Fig. 1 strongly suggest that the GLP-1 signaling system directly couples into ␤-arrestin-1. Therefore, we used INS-1 cells to focus on the...

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3␤, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C (PKC ) activity was also augmented in small-t-antigenexpressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC expression or PKC siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.Protein phosphorylation plays a key role in many cellular processes, including insulin signal transduction (24), and the phosphorylation state of a target protein is regulated by opposing kinase and phosphatase activities (24). Thus, the balance of enzyme activity between kinases and phosphatases is critical for the mediation of insulin's effects and, in turn, for the pathogenesis of insulin-resistant states.Tyrosine phosphorylation is essential for insulin action, and several lines of evidence have demonstrated that protein tyrosine phosphatases can play a role in insulin-resistant states (3, 4). For example, protein tyrosine phosphatase 1B (PTP1B) directly interacts with the activated insulin receptor and exhibits high specific activity for IRS-1 (22, 49). It has been reported previously that hyperglycemia can impair insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, at least in part because of the increased expression and activity of PTP1B (37,41), and that overexpression of PTP1B inhibits insulin-stimulated glucose metabolism in 3T3-L1 adipocytes and L6 myocytes (12,18,51).Serine/thre...

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