MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

No abstract

Obesity-induced insulin resistance is a major factor in the etiology of type 2 diabetes, and Jun kinases (JNKs) are key negative regulators of insulin sensitivity in the obese state. Activation of JNKs (mainly JNK1) in insulin target cells results in phosphorylation of insulin receptor substrates (IRSs) at serine and threonine residues that inhibit insulin signaling. JNK1 activation is also required for accumulation of visceral fat. Here we used reciprocal adoptive transfer experiments to determine whether JNK1 in myeloid cells, such as macrophages, also contributes to insulin resistance and central adiposity. Our results show that deletion of Jnk1 in the nonhematopoietic compartment protects mice from high-fat diet (HFD)-induced insulin resistance, in part through decreased adiposity. By contrast, Jnk1 removal from hematopoietic cells has no effect on adiposity but confers protection against HFD-induced insulin resistance by decreasing obesity-induced inflammation.

Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-(PKC-) in rat adipocytes. Concomitantly, insulin provoked increases in 32 P labeling of PKC-both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-enzyme activity. Also, PI-3,4-(PO 4 ) 2 , PI-3,4,5-(PO 4 ) 3 , and PI-4,5-(PO 4 ) 2 directly stimulated enzyme activity and autophosphoralytion in control PKC-immunoprecipitates to levels observed in insulin-treated PKC-immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-enzyme activity in vitro by both the PKC-pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigentagged GLUT4, co-transfection of wild-type or constitutive PKC-stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-partially inhibited it. Our findings suggest that insulin activates PKC-through PI 3-kinase, and PKC-may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.The atypical protein kinase C (PKC), 1 PKC-, is ubiquitously expressed, but little is known about its activation or actions. This ignorance partly derives from the fact that PKC-is not activated by membrane-associated diacylglycerol (DAG) or phorbol esters, generally does not translocate appreciably from cytosol to membrane when activated, and is not depleted by prolonged phorbol ester treatment. Consequently, methods used to evaluate DAG-sensitive conventional (␣, ␤, and ␥) and novel (␦, ⑀, , and ) PKCs are not relevant to PKC-and other DAG-insensitive, atypical PKCs. Although not activated by DAG, PKC-is activated in vitro by phosphatidylserine and polyphosphoinositides, including D3-PO 4 derivatives of phosphatidylinositol (PI) (1, 2). Because of its activation by polyphosphoinositides, PKC-has been suspected to operate downstream of PI 3-kinase; however, direct experimental evidence for this suspicion is lacking, particularly in intact cells. Since insulin increases total polyphosphoinositide levels (3-6), probably largely through PI 3-kinase activation (7), we examined the possibility that insulin activates PKC-by a PI 3-kinasedependent mechanism. To this end, we assayed immunoprecipitable PKC-(a) following treatment of intact adipocytes with insulin in the presence and absence of PI 3-kinase inhibitors; (b) following PI 3-kinase activation in vitro by a pYXXMcontaining peptide; and (c) in response to polyphosphoinositides added directly to the assay of PKC-in vitro. Also...

Thiazolidinediones (TZDs) are insulin-sensitizing drugs and are potent agonists of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Although muscle is the major organ responsible for insulin-stimulated glucose disposal, PPAR-gamma is more highly expressed in adipose tissue than in muscle. To address this issue, we used the Cre-loxP system to knock out Pparg, the gene encoding PPAR-gamma, in mouse skeletal muscle. As early as 4 months of age, mice with targeted disruption of PPAR-gamma in muscle showed glucose intolerance and progressive insulin resistance. Using the hyperinsulinemic-euglycemic clamp technique, the in vivo insulin-stimulated glucose disposal rate (IS-GDR) was reduced by approximately 80% and was unchanged by 3 weeks of TZD treatment. These effects reveal a crucial role for muscle PPAR-gamma in the maintenance of skeletal muscle insulin action, the etiology of insulin resistance and the action of TZDs.

Summary Chronic, low-grade inflammation, particularly in adipose tissue, is an important modulator of obesity-induced insulin resistance and the toll-like receptor 4 (Tlr4) is a key initiator of inflammatory responses in macrophages. We performed bone marrow transplantation (BMT) of Tlr4lps-del or control C57Bl/10J bone marrow cells into irradiated wild type C57Bl6 recipient mice to generate hematopoietic cell specific Tlr4 deletion mutant (BMT-Tlr4-/-) and control (BMT-wt) mice. When mice were fed a high-fat diet (HFD) for 16 weeks, BMT-wt mice developed obesity, hyperinsulinemia, glucose intolerance and insulin resistance. In contrast, BMT-Tlr4-/- mice became obese, but did not develop fasting hyperinsulinemia, and had improved hepatic and skeletal muscle insulin sensitivity during euglycemic clamp studies compared to HFD BMT-wt mice. The HFD BMT-Tlr4-/- mice showed markedly reduced adipose tissue inflammatory markers and macrophage content compared to HFD BMT-wt mice. In summary, our results indicate that Tlr4 signaling in hematopoietic-derived cells is important for the development of hepatic and adipose tissue insulin resistance in obese mice.

Summary Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2, encoded by the Sesn2 locus whose expression is induced upon hypernutrition, maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.

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).