The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells.

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T (Treg) cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the 'master regulator' of adipocyte differentiation, as a crucial molecular orchestrator of VAT Treg cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT Treg cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of Treg cells with unique functions can be precisely targeted to therapeutic ends.

Insulin receptor activation with transmembrane domain ligands.

Complementary surfaces are buried when peptide hormones, growth factors, or cytokines bind and activate cellular receptors. Although these extended surfaces provide high affinity and specificity to the interactions, they also present great challenges to the design of small molecules that might either mimic or antagonize the process. We show that the insulin receptor (IR) and downstream signals can be activated by targeting a site outside of its ligand-binding domain. A 24-residue peptide having the IR transmembrane (TM) domain sequence activates IR, but not related growth factor receptors, through specific interactions with the receptor TM domain. Like insulin-dependent activation, IR-TM requires that IR have a competent ATP-binding site and kinase activation loop. IR-TM also activates mutated receptors from patients with severe insulin resistance, which do not respond to insulin. These results show that IR can be activated through a pathway that bypasses its canonical ligand-binding domain.

Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance.

There is increasing evidence showing that inflammation is an important pathogenic mediator of the development of obesity-induced insulin resistance. It is now generally accepted that tissue-resident immune cells play a major role in the regulation of this obesity-induced inflammation. The roles that adipose tissue (AT)-resident immune cells play have been particularly extensively studied. AT contains most types of immune cells and obesity increases their numbers and activation levels, particularly in AT macrophages (ATMs). Other pro-inflammatory cells found in AT include neutrophils, Th1 CD4 T cells, CD8 T cells, B cells, DCs, and mast cells. However, AT also contains anti-inflammatory cells that counter the pro-inflammatory immune cells that are responsible for the obesity-induced inflammation in this tissue. These anti-inflammatory cells include regulatory CD4 T cells (Tregs), Th2 CD4 T cells, and eosinophils. Hence, AT inflammation is shaped by the regulation of pro- and anti-inflammatory immune cell homeostasis, and obesity skews this balance towards a more pro-inflammatory status. Recent genetic studies revealed several molecules that participate in the development of obesity-induced inflammation and insulin resistance. In this review, the cellular and molecular players that participate in the regulation of obesity-induced inflammation and insulin resistance are discussed, with particular attention being placed on the roles of the cellular players in these pathogeneses. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Adipose tissue macrophage heterogeneity in the single-cell genomics era.

It is now well-accepted that obesity-induced inflammation plays an important role in the development of insulin resistance and type 2 diabetes. A key source of the inflammation is the murine epididymal and human visceral adipose tissue. The current paradigm is that obesity activates multiple proinflammatory immune cell types in adipose tissue, including adipose-tissue macrophages (ATMs), T Helper 1 (Th1) T cells, and natural killer (NK) cells, while concomitantly suppressing anti-inflammatory immune cells such as T Helper 2 (Th2) T cells and regulatory T cells (Tregs). A key feature of the current paradigm is that obesity induces the anti-inflammatory M2 ATMs in lean adipose tissue to polarize into proinflammatory M1 ATMs. However, recent single-cell transcriptomics studies suggest that the story is much more complex. Here we describe the single-cell genomics technologies that have been developed recently and the emerging results from studies using these technologies. While further studies are needed, it is clear that ATMs are highly heterogeneous. Moreover, while a variety of ATM clusters with quite distinct features have been found to be expanded by obesity, none truly resemble classical M1 ATMs. It is likely that single-cell transcriptomics technology will further revolutionize the field, thereby promoting our understanding of ATMs, adipose-tissue inflammation, and insulin resistance and accelerating the development of therapies for type 2 diabetes.

NK cell-activating receptor NKp46 does not participate in the development of obesity-induced inflammation and insulin resistance.

Recent evidence establishes a pivotal role for obesity-induced inflammation in precipitating insulin resistance and type-2 diabetes. Central to this process is the proinflammatory M1 adipose-tissue macrophages (ATMs) in epididymal white adipose tissue (eWAT). Notably, natural killer (NK) cells are a crucial regulator of ATMs since their cytokines induce ATM recruitment and M1 polarization. The importance of NK cells is shown by the strong increase in NK-cell numbers in eWAT, and by studies showing that removing and expanding NK cells respectively improve and worsen obesity-induced insulin resistance. It has been suggested that NK cells are activated by unknown ligands on obesity-stressed adipocytes that bind to NKp46 (encoded by Ncr1), which is an activating NK-cell receptor. This was supported by a study showing that NKp46-knockout mice have improved obesity-induced inflammation/insulin resistance. We therefore planned to use the NKp46-knockout mice to further elucidate the molecular mechanism by which NKp46 mediates eWAT NK-cell activation in obesity. We confirmed that obesity increased eWAT NKp46+ NK-cell numbers and NKp46 expression in wild-type mice and that NKp46-knockout ablated these responses. Unexpectedly, however, NKp46-knockout mice demonstrated insulin resistance similar to wild-type mice, as shown by fasting blood glucose/insulin levels and glucose/insulin tolerance tests. Obesity-induced increases in eWAT ATM numbers and proinflammatory gene expression were also similar. Thus, contrary to previously published results, NKp46 does not regulate obesity-induced insulin resistance. It is therefore unclear whether NKp46 participates in the development of obesity-induced inflammation and insulin resistance. This should be considered when elucidating the obesity-mediated molecular mechanisms that activate NK cells.

Metabolic syndrome, insulin resistance, and roles of inflammation--mechanisms and therapeutic targets.

Obesity and its comorbidities, including type 2 diabetes mellitus and cardiovascular disease, are associated with a state of chronic low-grade inflammation that can be detected both systemically and within specific tissues. Areas of active investigation focus on the molecular bases of metabolic inflammation and potential pathogenic roles in insulin resistance, diabetes, and cardiovascular disease. An increased accumulation of macrophages occurring in obese adipose tissue has emerged as a key process in metabolic inflammation. Recent studies have also begun to unravel the heterogeneity of adipose tissue macrophages, and their physical and functional interactions with adipocytes, endothelial cells, and other immune cells within the adipose tissue microenvironment. Translating the information gathered from experimental models of insulin resistance and diabetes into meaningful therapeutic interventions is a tantalizing goal with long-term global health implications. In this context, ongoing clinical studies are testing the effects of targeting inflammation systemically on metabolic and cardiovascular outcomes.

Inflammation and adipose tissue macrophages in lipodystrophic mice.

Lipodystrophy and obesity are opposites in terms of a deficiency versus excess of adipose tissue mass, yet these conditions are accompanied by similar metabolic consequences, including insulin resistance, dyslipidemia, hepatic steatosis, and increased risk for diabetes and atherosclerosis. Hepatic and myocellular steatosis likely contribute to metabolic dysregulation in both states. Inflammation and macrophage infiltration into adipose tissue also appear to participate in the pathogenesis of obesity-induced insulin resistance, but their contributions to lipodystrophy-induced insulin resistance have not been evaluated. We used aP2-nSREBP-1c transgenic (Tg) mice, an established model of lipodystrophy, to ask this question. Circulating cytokine elevations suggested systemic inflammation but even more dramatic was the number of infiltrating macrophages in all white and brown adipose tissue depots of the Tg mice; in contrast, there was no evidence of inflammatory infiltrates or responses in any other tissue including liver. Despite there being overt evidence of adipose tissue inflammation, antiinflammatory strategies including salicylate treatment and genetic suppression of myeloid NF-kappaB signaling that correct insulin resistance in obesity were ineffective in the lipodystrophic mice. We further showed that adipose tissue macrophages (ATMs) in lipodystrophy and obesity are very different in terms of activation state, gene expression patterns, and response to lipopolysaccharide. Although ATMs are even more abundant in lipodystrophy than in obesity, they have distinct phenotypes and likely roles in tissue remodeling, but do not appear to be involved in the pathogenesis of insulin resistance.

Self-organized insulin-producing ß-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential.

BACKGROUND: Human omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived ß-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional ß-cells remains challenging. METHODS: We aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing ß-cells via cell-matrix/cell-cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the ß-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes. RESULTS: Our findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing ß-cell progenitors, as evident from the upregulation of pancreatic ß-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated ß-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation. CONCLUSIONS: Our findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic ß-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.

Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB.

We show that NF-kappaB and transcriptional targets are activated in liver by obesity and high-fat diet (HFD). We have matched this state of chronic, subacute 'inflammation' by low-level activation of NF-kappaB in the liver of transgenic mice, designated LIKK, by selectively expressing constitutively active IKK-b in hepatocytes. These mice exhibit a type 2 diabetes phenotype, characterized by hyperglycemia, profound hepatic insulin resistance, and moderate systemic insulin resistance, including effects in muscle. The hepatic production of proinflammatory cytokines, including IL-6, IL-1beta and TNF-alpha, was increased in LIKK mice to a similar extent as induced by HFD in in wild-type mice. Parallel increases were observed in cytokine signaling in liver and mucscle of LIKK mice. Insulin resistance was improved by systemic neutralization of IL-6 or salicylate inhibition of IKK-beta. Hepatic expression of the IkappaBalpha superrepressor (LISR) reversed the phenotype of both LIKK mice and wild-type mice fed an HFD. These findings indicate that lipid accumulation in the liver leads to subacute hepatic 'inflammation' through NF-kappaB activation and downstream cytokine production. This causes insulin resistance both locally in liver and systemically.

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14.

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14+ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P3 antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP+ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP+ cells represented more significant populations in tissues of Cd14-/- than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14+ phagocytes.

Role of obesity-induced inflammation in the development of insulin resistance and type 2 diabetes: history of the research and remaining questions.

The prevalence of obesity has increased alarmingly both worldwide and in Korea. This has also dramatically increased the prevalence of chronic obesity-associated diseases, including type 2 diabetes (T2D). Extensive studies on the molecular etiology of T2D have revealed several potential mechanisms by which obesity induces the development of insulin resistance and T2D. One of these is low-grade chronic inflammation. Studies hinting at the existence of this phenomenon were first published about 30 years ago. Ten years later, several seminal papers confirmed its existence, which then led to a rapid and massive escalation of research in this field. Today, the notion that obesity-induced inflammation mediates T2D is now well-accepted. This paper will review the key developments in this field, including the discovery that obesity-induced inflammation and insulin resistance is mainly regulated by adipose tissue-resident immune cells, particularly those in visceral adipose tissue. This review further details the research areas, including (1) the obesity-related factors that induce adipose tissue macrophage (ATM) inflammation, (2) the precise effector functions by which adipose tissue immune cells promote insulin resistance, (3) whether there are early immunological events that have an outsize effect on later events and could be targeted to arrest the development of insulin resistance, (4) the roles played by nonimmunological functions of ATMs and other immune cells, and (5) whether there are noncanonical immune responses to obesity (i.e., immune responses that are unique to obesity and cannot be detected by following the discoveries in the classical immunity field).

Inflammation and insulin resistance.

Over a hundred years ago, high doses of salicylates were shown to lower glucose levels in diabetic patients. This should have been an important clue to link inflammation to the pathogenesis of type 2 diabetes (T2D), but the antihyperglycemic and antiinflammatory effects of salicylates were not connected to the pathogenesis of insulin resistance until recently. Together with the discovery of an important role for tissue macrophages, these new findings are helping to reshape thinking about how obesity increases the risk for developing T2D and the metabolic syndrome. The evolving concept of insulin resistance and T2D as having immunological components and an improving picture of how inflammation modulates metabolism provide new opportunities for using antiinflammatory strategies to correct the metabolic consequences of excess adiposity.

Pyruvate Dehydrogenase Kinase Is a Metabolic Checkpoint for Polarization of Macrophages to the M1 Phenotype.

Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Here, we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the pyruvate dehydrogenase-mediated conversion of cytosolic pyruvate to mitochondrial acetyl-CoA, functions as a metabolic checkpoint in M1 macrophages. Polarization was not prevented by PDK2 or PDK4 deletion but was fully prevented by the combined deletion of PDK2 and PDK4; this lack of polarization was correlated with improved mitochondrial respiration and rewiring of metabolic breaks that are characterized by increased glycolytic intermediates and reduced metabolites in the TCA cycle. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). Transplantation of PDK2/4-deficient bone marrow into irradiated wild-type mice to produce mice with PDK2/4-deficient myeloid cells prevented M1 polarization, reduced obesity-associated insulin resistance, and ameliorated adipose tissue inflammation. A novel, pharmacological PDK inhibitor, KPLH1130, improved high-fat diet-induced insulin resistance; this was correlated with a reduction in the levels of pro-inflammatory markers and improved mitochondrial function. These studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages, which could potentially be exploited as a novel therapeutic target for obesity-associated metabolic disorders and other inflammatory conditions.

Kinase activation through dimerization by human SH2-B.

The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.

Macrophage Lamin A/C Regulates Inflammation and the Development of Obesity-Induced Insulin Resistance.

Obesity-induced chronic low-grade inflammation, in particular in adipose tissue, contributes to the development of insulin resistance and type 2 diabetes. However, the mechanism by which obesity induces adipose tissue inflammation has not been completely elucidated. Recent studies suggest that alteration of the nuclear lamina is associated with age-associated chronic inflammation in humans and fly. These findings led us to investigate whether the nuclear lamina regulates obesity-mediated chronic inflammation. In this study, we show that lamin A/C mediates inflammation in macrophages. The gene and protein expression levels of lamin A/C are significantly increased in epididymal adipose tissues from obese rodent models and omental fat from obese human subjects compared to their lean controls. Flow cytometry and gene expression analyses reveal that the protein and gene expression levels of lamin A/C are increased in adipose tissue macrophages (ATMs) by obesity. We further show that ectopic overexpression of lamin A/C in macrophages spontaneously activates NF-κB, and increases the gene expression levels of proinflammatory genes, such as Il6, Tnf, Ccl2, and Nos2. Conversely, deletion of lamin A/C in macrophages reduces LPS-induced expression of these proinflammatory genes. Importantly, we find that myeloid cell-specific lamin A/C deficiency ameliorates obesity-induced insulin resistance and adipose tissue inflammation. Thus, our data suggest that lamin A/C mediates the activation of ATM inflammation by regulating NF-κB, thereby contributing to the development of obesity-induced insulin resistance.

The role of GSK3 in glucose homeostasis and the development of insulin resistance.

GSK3 has been implicated in the development of insulin resistance, primarily based on its role in regulation of glycogen synthesis. However, GSK3 is involved in many other important signaling cascades which may regulate glucose homeostasis and the development of insulin resistance. In addition, GSK3 is composed of two isoforms, GSK3alpha and beta, which do not completely share their physiological roles, and this raises a possibility that GSK3alpha and beta may function differently in glucose homeostasis. In this review, we will give an overview to examine potential mechanisms for the roles of GSK3 in the development of insulin resistance.

Unusual Suspects in the Development of Obesity-Induced Inflammation and Insulin Resistance: NK cells, iNKT cells, and ILCs.

The notion that obesity-induced inflammation mediates the development of insulin resistance in animal models and humans has been gaining strong support. It has also been shown that immune cells in local tissues, in particular in visceral adipose tissue, play a major role in the regulation of obesity-induced inflammation. Specifically, obesity increases the numbers and activation of proinflammatory immune cells, including M1 macrophages, neutrophils, Th1 CD4 T cells, and CD8 T cells, while simultaneously suppressing anti-inflammatory cells such as M2 macrophages, CD4 regulatory T cells, regulatory B cells, and eosinophils. Recently, however, new cell types have been shown to participate in the development of obesity-induced inflammation and insulin resistance. Some of these cell types also appear to regulate obesity. These cells are natural killer (NK) cells and innate lymphoid cells (ILCs), which are closely related, and invariant natural killer T (iNKT) cells. It should be noted that, although iNKT cells resemble NK cells in name, they are actually a completely different cell type in terms of their development and functions in immunity and metabolism. In this review, we will focus on the roles that these relatively new players in the metabolism field play in obesity-induced insulin resistance and the regulation of obesity.