Adipose tissue macrophages (ATMs) infiltrate adipose tissue during obesity and contribute to insulin resistance. We hypothesized that macrophages migrating to adipose tissue upon high-fat feeding may differ from those that reside there under normal diet conditions. To this end, we found a novel F4/80 + CD11c + population of ATMs in adipose tissue of obese mice that was not seen in lean mice. ATMs from lean mice expressed many genes characteristic of M2 or "alternatively activated" macrophages, including Ym1, arginase 1, and Il10. Dietinduced obesity decreased expression of these genes in ATMs while increasing expression of genes such as those encoding TNF-α and iNOS that are characteristic of M1 or "classically activated" macrophages. Interestingly, ATMs from obese C-C motif chemokine receptor 2-KO (Ccr2-KO) mice express M2 markers at levels similar to those from lean mice. The antiinflammatory cytokine IL-10, which was overexpressed in ATMs from lean mice, protected adipocytes from TNF-α-induced insulin resistance. Thus, diet-induced obesity leads to a shift in the activation state of ATMs from an M2-polarized state in lean animals that may protect adipocytes from inflammation to an M1 proinflammatory state that contributes to insulin resistance.

The epidemic of type 2 diabetes and impaired glucose tolerance is one of the main causes of morbidity and mortality worldwide. In both disorders, tissues such as muscle, fat and liver become less responsive or resistant to insulin. This state is also linked to other common health problems, such as obesity, polycystic ovarian disease, hyperlipidaemia, hypertension and atherosclerosis. The pathophysiology of insulin resistance involves a complex network of signalling pathways, activated by the insulin receptor, which regulates intermediary metabolism and its organization in cells. But recent studies have shown that numerous other hormones and signalling events attenuate insulin action, and are important in type 2 diabetes.

Stimulation of cells with growth factors and cytokines, or exposure to cellular stresses, activates several signal transduction pathways that have specific physiological roles. These include at least three in which a mitogen-activated protein kinase (MAPK) 1 homologue is involved. In one pathway, cell stimulation leads to the sequential activation of p21 ras and the protein kinases c-Raf, MAP kinase kinase-1 and -2 (MAPKK1, MAPKK2), and p42 and p44 MAP kinases (p42 MAPK , p44 MAPK ). These MAPKs phosphorylate a variety of proteins in vivo including MAP kinase-activated protein (MAPKAP) kinases 1␣ and 1␤ (also known as Rsk-1 and Rsk-2 (1)). The sustained activation of p42/p44MAPK is not only required, but it is sufficient to induce the proliferation or differentiation of several cells (2).In order to dissect MAPK pathways and to elucidate their physiological roles, one approach has been to generate dominant negative mutants and overexpress them in cells. For example, dominant negative forms of p21 ras , c-Raf, and MAPKK1 all inhibit the activation of p42/p44 MAPK and the growth factor-induced proliferation or differentiation of several cells (3). However, although dominant-negative mutants are useful, the generation of cell lines that stably express them is time consuming, and their expression may lead to erroneous conclusions. For example, overexpression of an inactive form of MAPKK1 that can be phosphorylated by Raf may not only prevent the activation of endogenous wild-type MAPKK1, but also the activation of other cellular substrates of Raf that might lie in distinct signaling pathways. The need to express a dominant negative mutant for many hours may also result in unwanted secondary effects. Similarly, the use of dominant negative mutants of Raf may affect Ras-dependent processes that are independent of Raf.An alternative strategy is to identify small cell-permeant molecules that are specific inhibitors of particular protein kinases. An advantage of this approach is that the effects of these inhibitors can be investigated in any cell in vivo. Moreover, these inhibitors may have therapeutic potential as anti-cancer, or anti-inflammatory agents, or as immunosuppressants. Several such inhibitors have recently been described, including an inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase (4), which may be useful for treating human tumors that overexpress this receptor, and a specific inhibitor of the MAP kinase homologue termed reactivating kinase (RK) or p38 (5). The latter inhibitor prevents the synthesis of interleukin-1

Many diverse extracellular stimuli-including growth factors, hormones, osmolar shock, stress, and elevated temperatureresult in activation of phosphorylation cascades utilizing mitogen-activated protein kinases (MAPKs) (1-8). MAPKs (sometimes called extracellular signal-regulated kinases, or ERKs) comprise a family of related protein kinases that are themselves activated by phosphorylation on threonine and tyrosine residues. The MAPK-activating enzymes (MAPK/ ERK kinases, or MEKs) are unusual in their ability to catalyze phosphorylation on both threonine and tyrosine residues (9, 10). MEKs are in turn activated by phosphorylation on serine residues by upstream kinases. These MEK kinases, which appear to require activation by the ras protooncogene product (11, 12), include members of the Raf family (13-15), a mammalian homologue of the yeast STEll gene product (16), the tpll2 prc_ oncogene product (17), and a growth-factor sensitive enz--:e derived from PC12 rat pheochromocytoma cells (18). However, the precise specificity of these kinases in vivo is unclear, since some of them may participate in cascades leading to activation of the related stress-activated protein kinases (19,20).While the MAPK pathway is activated under many circumstances in tissue culture cells, the exact role of this pathway in vivo remains undefined. Approaches using dominant negative interfering mutant constructs of MEK have indicated that this pathway is required for nerve growth factor-dependent differentiation of PC12 cells. Furthermore, expression of constitutively activated mutants has resulted in transformation (21,22). We sought a more widely applicable method to determine the physiological role of this pathway by identifying selective inhibitors of specific components of the MAPK cascade. MATERIALS AND METHODSIn Vitro Kinase Assay. Incorporation of 32p into myelin basic protein (MBP) was assayed in the presence of glutathione S-transferase (GST) fusion proteins containing the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1). For direct evaluation of MEK activity, 10 ,tg of GST-MEK1 was incubated with 5 ,ug of a GST fusion protein containing 44-kDa MAPKwith a lysine-to-alanine mutation at position 71 (GST-MAPK-KA). This mutation eliminates kinase activity of MAPK, so that only kinase activity attributed to the added MEK remains. Similar incubations were performed with 5 ,ug of a fusion protein containing artificially partially activated MEK with serine-to-glutamate mutations at positions 218 and 222 (GST-MEK-2E). These assays utilized the same buffer and incubation conditions as described above. Phosphorylated MAPK-KA was resolved by SDS/10% PAGE and detected by autoradiography.Immunoprecipitation and Imminoblot Analysis. Tyrosinephosphorylated MAPK-KA was determined by using the same incubation protocol as for phosphorylation, but without radiolabeled, ATP. After electrophoresis, proteins on the gel were transferred to a nitrocellulose membrane, and nonspecific binding sites on the membrane were blocked by incubation with 1% ova...

The obesity epidemic has forced us to evaluate the role of inflammation in the health complications of obesity. This has led to a convergence of the fields of immunology and nutrient physiology and the understanding that they are inextricably linked. The reframing of obesity as an inflammatory condition has had a wide impact on our conceptualization of obesity-associated diseases. In this Review, we highlight the cellular and molecular mechanisms at play in the generation of obesity-induced inflammation. We also emphasize how defining the immune regulation in metabolic tissues has broadened the understanding of the diversity of inflammatory responses. IntroductionThe burden of obesity on health extends across multiple organ systems and diseases. While its impact on tissues involved in nutrient regulation is manifest in the development of insulin resistance and type 2 diabetes, there are also unexpected connections between obesity and the risk of cancer and pulmonary diseases. Over the past decade, the search for a potential unifying mechanism behind the pathogenesis of obesity-associated diseases has revealed a close relationship between nutrient excess and derangements in the cellular and molecular mediators of immunity and inflammation. This has given birth to the concept of "metainflammation" (1) to describe the chronic low-grade inflammatory response to obesity. We present here a broad overview of the links between obesity and immune responses with a focus on metabolic disease and argue that the intersection between the pathways that control nutrient metabolism and inflammatory responses may be broadly applicable to our understanding of inflammation and the immune system.

We are in the midst of a worldwide obesity epidemic. Over 1.9 billion people are now obese or overweight, including more than 50 million children under age 5, and the worldwide prevalence of obesity has doubled since 1980 (1). The complications of obesity are taking a major public health toll, which will only worsen in future years. Among the most devastating of these complications is type 2 diabetes (T2D). Most patients with T2D are obese or overweight, and there are numerous longitudinal studies linking obesity with insulin resistance, a frequent forerunner of diabetes.Over the past two decades, the search for a mechanism linking the pathogenesis of obesity with insulin resistance and diabetes has revealed a close relationship between nutrient excess and activation of the innate immune system in most organs pertinent to energy homeostasis (2-4). Numerous studies indicate that inflammation occurs as a consequence of obesity, and recent insights suggest that it may play a causative role in generating insulin resistance, defective insulin secretion, and disruption of other aspects of energy homeostasis. The nature of obesityinduced inflammation differs from other inflammatory paradigms in that it involves tonic activation of the innate immune system that impacts metabolic homeostasis, in some cases over a lifetime. Inflammation also leads to maladaptive responses such as fibrosis and necrosis that can cause significant tissue damage. Moreover, obesity-induced inflammation is unique in that it involves multiple organs, including adipose, pancreas, liver, skeletal muscle, heart, and brain. These features of obesity-induced inflammation present a challenge to understanding the underlying mechanisms and the manner by which they impact metabolic systems. In this issue of the JCI, a group of experts have provided reviews that dive deeply into the mechanisms by which obesity-induced inflammation influences metabolic homeostasis, surveying the modes and mechanisms of inflammation in different tissues and how they might contribute to disease. These Reviews also discuss how inflammation can lead to other aspects of metabolic syndrome, including hyperphagia, reduced energy expenditure, β cell dysfunction, and liver disease, and summarize the status of therapeutic interventions that target inflammation in the context of metabolic syndrome. Activation of the innate immune system in obesityMuch of what has been learned about the immune response to obesity has come from studies in adipose tissue, although it is clear that inflammation occurs in other organs as well. Adipose depots normally contain multiple immune cells that together surveil and maintain the integrity and hormonal sensitivity of adipocytes. In lean animals, these immune cells operate in an overall Th2 or type 2 state that coordinately controls tissue integrity and metabolism by controlling the activity of subsets of T lymphocytes. These cells release a cascade of cytokines that coordinately regulate other immune cells, including eosinophils, mast cells, and othe...

Although recent studies show that adipose tissue macrophages (ATMs) participate in the inflammatory changes in obesity and contribute to insulin resistance, the properties of these cells are not well understood. We hypothesized that ATMs recruited to adipose tissue during a high-fat diet have unique inflammatory properties compared with resident tissue ATMs. Using a dye (PKH26) to pulse label ATMs in vivo, we purified macrophages recruited to white adipose tissue during a high-fat diet. Comparison of gene expression in recruited and resident ATMs using real-time RT-PCR and cDNA microarrays showed that recruited ATMs overexpress genes important in macrophage migration and phagocytosis, including interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and C-C chemokine receptor 2 (CCR2). Many of these genes were not induced in ATMs from high-fat diet-fed CCR2 knockout mice, supporting the importance of CCR2 in regulating recruitment of inflammatory ATMs during obesity. Additionally, expression of Apoe was decreased, whereas genes important in lipid metabolism, such as Pparg, Adfp, Srepf1, and Apob48r, were increased in the recruited macrophages. In agreement with this, ATMs from obese mice had increased lipid content compared with those from lean mice. These studies demonstrate that recruited ATMs in obese animals represent a subclass of macrophages with unique properties. Diabetes 56:16 -23, 2007

Adipose tissue not only has an important role in the storage of excess nutrients but also senses nutrient status and regulates energy mobilization. An overall positive energy balance is associated with overnutrition and leads to excessive accumulation of fat in adipocytes. These cells respond by initiating an inflammatory response that, although maladaptive in the long run, might initially be a physiological response to the stresses obesity places on adipose tissue. In this Review, we characterize adipose tissue inflammation and review the current knowledge of what triggers obesity-associated inflammation in adipose tissue. We examine the connection between adipose tissue inflammation and the development of insulin resistance and catecholamine resistance and discuss the ensuing state of metabolic inflexibility. Finally, we review the current and potential new anti-inflammatory treatments for obesity-associated metabolic disease.