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.
The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if close enough to the 5 9 methyl Gc ap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5 9 -untranslated region. We devised at wo-color fluorescence assay for translation efficiency in single live cells and compared aw ide range of hairpins with predicted thermal stabilities ranging from ÿ 10 to ÿ 50 kcal/mol and 5 9 Gcap-to-hairpin distances of 1-46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from D G = ÿ 25 to ÿ 35 kcal/mol. Shifting ahairpin as little as nine bases relative to the 5 9 cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5 9 -untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5 9 UTRs.
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).
Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKK, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-␣, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-␣ signaling with neutralizing antibodies to TNF-␣ or its receptors or with a dominant negative TNF-␣ peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-␣ signaling, whereas the FFA-induced increase in TNF-␣ secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-␣-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-␣ is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.
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...
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