Kaiwen Ma

Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice.

Adipose tissue inflammation and reduced pancreatic β-cell function are key issues in the development of cardiovascular disease and progressive metabolic dysfunction in type 2 diabetes mellitus. The aim of this study was to determine the effect of the DPP IV inhibitor sitagliptin on adipose tissue and pancreatic islet inflammation in a diet-induced obesity model. C57Bl/6J mice were placed on a high-fat (60% kcal fat) diet for 12 wk, with or without sitagliptin (4 g/kg) as a food admix. Sitagliptin significantly reduced fasting blood glucose by 21% as well as insulin by ∼25%. Sitagliptin treatment reduced body weight without changes in overall body mass index or in the epididymal and retroperitoneal fat mass. However, sitagliptin treatment led to triple the number of small adipocytes despite reducing the number of the very large adipocytes. Sitagliptin significantly reduced inflammation in the adipose tissue and pancreatic islet. Macrophage infiltration in adipose tissue evaluated by immunostaining for Mac2 was reduced by sitagliptin (P < 0.01), as was the percentage of CD11b+/F4/80+ cells in the stromal vascular fraction (P < 0.02). Sitagliptin also reduced adipocyte mRNA expression of inflammatory genes, including IL-6, TNFα, IL-12(p35), and IL-12(p40), 2.5- to fivefold as well as 12-lipoxygenase protein expression. Pancreatic islets were isolated from animals after treatments. Sitagliptin significantly reduced mRNA expression of the following inflammatory cytokines: MCP-1 (3.3-fold), IL-6 (2-fold), IL-12(p40) (2.2-fold), IL-12(p35) (5-fold, P < 0.01), and IP-10 (2-fold). Collectively, the results indicate that sitagliptin has anti-inflammatory effects in adipose tissue and in pancreatic islets that accompany the insulinotropic effect.

12-Lipoxygenase Products Reduce Insulin Secretion and β-Cell Viability in Human Islets

CONTEXT Inflammation is increasingly recognized as an important contributing factor in diabetes mellitus. Lipoxygenases (LOs) produce active lipids that promote inflammatory damage by catalyzing the oxidation of linoleic and arachidonic acid, and LO is expressed in rodent and human islets. Little is known about the differential effect of the various hydroxyeicosatetraenoic acids (HETEs) that result from LO activity in human islets. OBJECTIVE We compared the effects of 12-LO products on human islet viability and function. DESIGN Human islets were treated with stable compounds derived from LOs: 12(S)-HETE, 15HETE, 12HPETE, and 12RHETE and then examined for insulin secretion and islet viability. The p38-MAPK (p38) and JNK stress-activated pathways were investigated as mechanisms of 12-LO-mediated islet inhibition in rodent and human islets. RESULTS Insulin secretion was consistently reduced by 12(S)-HETE and 12HPETE. 12(S)-HETE at 1 nm reduced viability activity by 32% measured by MTT assay and increased cell death by 50% at 100 nm in human islets. These effects were partially reversed with lisofylline, a small-molecule antiinflammatory compound that protects mitochondrial function. 12(S)-HETE increased phosphorylated p38-MAPK (pp38) protein activity in human islets. Injecting 12-LO siRNA into C57BL/6 mice reduced 12-LO and pp38-MAPK protein levels in mouse islets. The addition of proinflammatory cytokines increased pp38 levels in normal mouse islets but not in siRNA-treated islets. CONCLUSIONS These data suggest that 12(S)-HETE reduces insulin secretion and increases cell death in human islets. The 12-LO pathway is present in human islets, and expression is up-regulated by inflammatory cytokines. Reduction of 12-LO activity could thus provide a new therapeutic approach to protect human beta-cells from inflammatory injury.

Nonobese Diabetic (NOD) Mice Congenic for a Targeted Deletion of 12/15-Lipoxygenase Are Protected From Autoimmune Diabetes

OBJECTIVE— 12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic β-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of β-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in β-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS— We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15null) using a “speed congenic” protocol, and the mice were monitored for development of insulitis and diabetes. RESULTS— NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. >60% in females by 30 weeks). Nondiabetic female NOD-Alox15null mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved β-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15null mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4+ T cells and increased Foxp3+ cells. CONCLUSIONS— These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation.

Differential expression and localization of 12/15 lipoxygenases in adipose tissue in human obese subjects.

Adipose tissue inflammation in obesity is a major factor leading to cardiovascular disease and type 2 diabetes.12/15 lipoxygenases (ALOX) play an important role in the generation of inflammatory mediators, insulin resistance and downstream immune activation in animal models of obesity. However, the expression and roles of 12/15ALOX isoforms, and their cellular sources in human subcutaneous (sc) and omental (om) fat in obesity is unknown. The objective of this study was to examine the gene expression and localization of ALOX isoforms and relevant downstream cytokines in subcutaneous (sc) and omental (om) adipose tissue in obese humans. Paired biopsies of sc and om fat were obtained during bariatric surgeries from 24 morbidly obese patients. Gene and protein expression for ALOX15a, ALOX15b and ALOX 12 were measured by real-time PCR and western blotting in adipocytes and stromal vascular fractions (SVF) from om and sc adipose tissue along with the mRNA expression of the downstream cytokines IL-12a, IL-12b, IL-6, IFNγ and the chemokine CXCL10. In a paired analysis, all ALOX isoforms, IL-6, IL-12a and CXCL10 were significantly higher in om vs. sc fat. ALOX15a mRNA and protein expression was found exclusively in om fat. All of the ALOX isoforms were expressed solely in the SVF. Further fractionation of the SVF in CD34+ and CD34- cells indicated that ALOX15a is predominantly expressed in the CD34+ fraction including vascular and progenitor cells, while ALOX15B is mostly expressed in the CD34- cells containing various leucocytes and myeloid cells. This result was confirmed by immunohistochemistry showing exclusive localization of ALOX15a in the om fat and predominantly in the vasculature and non-adipocyte cells. Our finding is identifying selective expression of ALOX15a in human om but not sc fat. This is a study showing a major inflammatory gene exclusively expressed in visceral fat in humans.

Deletion of 12/15-Lipoxygenase Alters Macrophage and Islet Function in NOD-Alox15null Mice, Leading to Protection against Type 1 Diabetes Development

Aims Type 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D. Methods 12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15null macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass. Results 12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15null mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass. Conclusions These results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.

STAT4 Deficiency Reduces Obesity-Induced Insulin Resistance and Adipose Tissue Inflammation

Signal transducer and activator of transcription (STAT) 4 is one of the seven members of the STAT family. STAT4 has a prominent role in mediating interleukin-12–induced T-helper cell type 1 lineage differentiation. T cells are key players in the maintenance of adipose tissue (AT) inflammation. The role of STAT4 in obesity and AT inflammation is unknown. We sought to determine the role of STAT4 in AT inflammation in obesity-induced insulin resistance. We studied STAT4-null mice on the C57Bl6/J background. We have found that STAT4−/−C57Bl6/J mice develop high-fat diet–induced obesity (DIO) similar to wild-type controls, but that they have significantly improved insulin sensitivity and better glucose tolerance. Using flow cytometry and real-time PCR, we show that STAT4−/− mice with DIO produce significantly reduced numbers of inflammatory cytokines and chemokines in adipocytes, have reduced numbers of CD8+ cells, and display increased alternative (M2) macrophage polarization. CD8+ cells, but not CD4+ cells, from STAT4−/− mice displayed reduced in vitro migration. Also, we found that adipocyte inflammation is reduced and insulin signaling is improved in STAT4−/− mice with DIO. We have identified STAT4 as a key contributor to insulin resistance and AT inflammation in DIO. Targeting STAT4 activation could be a novel approach to reducing AT inflammation and insulin resistance in obesity.

12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets

Context The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1β, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting In vitro study. Participants Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention Glucose stimulation. Main Outcome Measures Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.