Obesity is a significant factor in the development of type 2 diabetes (T2D). Treatment of obesity is pivotal in the prevention and management of T2D, and the development of new pharmacological therapies are studied for improving insulin resistance and glucose intolerance. Oleanolic acid-derived triterpenoids, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acids (CDDOs), are studied to elucidate the mechanisms by which they protect against obesity. However, fundamental knowledge gaps remain regarding the physiological and molecular mechanisms by which CDDOs protect against obesity. Our recently published studies showed that CDDO-ethyl amide (CDDO-EA) prevents skeletal muscle inflammation by inhibiting activation of nuclear factor-kappa B (NF-κB) signaling. Moreover, CDDO-EA induced translocation of glucose transporter 4, GLUT4, in skeletal muscle cells. We hypothesized that CDDO-EA protects from obesity-induced hyperglycemia in mice fed a high-fat diet (HFD). Our results show that CDDO-EA protects from HFD-induced obesity but has no effect on body weight in mice fed a low-fat diet (LFD). Our data show that CDDO-EA inhibition of weight gain is associated with reduced caloric intake and glucose and insulin levels in mice fed an HFD. This highlights the potential of CDDO-EA as a therapeutic agent for obesity treatment and the protection against the development of T2D.

IntroductionHigh density lipoproteins (HDL) exert cardiovascular protection in part through their antioxidant capacity and cholesterol efflux function. Effects of exercise training on HDL function are yet to be well established, while impact on triacylglycerol (TG)-lowering has been often reported. We previously showed that a short-term high-intensity interval training (HIIT) program improves insulin sensitivity but does not inhibit inflammatory pathways in immune cells in insulin-resistant subjects. The purpose of this study is to evaluate HDL function along with changes of lipoproteins after the short-term HIIT program in lean, obese nondiabetic, and obese type 2 diabetic (T2DM) subjects.MethodsAll individuals underwent a supervised 15-day program of alternative HIIT for 40 minutes per day. VO2peak was determined before and after this training program. A pre-training fasting blood sample was collected, and the post-training fasting blood sample collection was performed 36 hours after the last exercise session.ResultsBlood lipid profile and HDL function were analyzed before and after the HIIT program. Along with improved blood lipid profiles in obese and T2DM subjects, the HIIT program affected circulating apolipoprotein amounts differently. The HIIT program increased HDL-cholesterol levels and improved the cholesterol efflux capacity only in lean subjects. Furthermore, the HIIT program improved the antioxidant capacity of HDL in all subjects. Data from multiple logistic regression analysis showed that changes in HDL antioxidant capacity were inversely associated with changes in atherogenic lipids and changes in HDL-TG content.DiscussionWe show that a short-term HIIT program improves aspects of HDL function depending on metabolic contexts, which correlates with improvements in blood lipid profile. Our results demonstrate that TG content in HDL particles may play a negative role in the anti-atherogenic function of HDL.

Mexican Americans living in the Rio Grande Valley (RGV) have a high prevalence of type 2 diabetes (T2D). The US-Mexico border frontier has a unique blended culture of American lifestyle and Mexican traditions. Some examples of the cultural traditions are the food and the use of herbal medicine, but these traditions are in danger of disappearing after a very short number of generations living in the United States. This article describes the use of animal models under experimental conditions to solve practical questions (etiology or treatment). We performed studies with murine (ie, mouse and rat) models to elucidate the characteristics of medicinal plants that modulate glucose metabolism and inflammation and protect from bone loss, complications related to T2D. The University of Texas Rio Grande Valley researchers also have collaborated with the University of Texas Health Science Center at San Antonio researchers in performing studies in nonhuman primates (NHP) (ie, baboon) to understand the effect of T2D and diets on organs and tissues. With the new knowledge gained from the use of animal models (murine and NHP), new therapies are discovered for the prevention and treatment of T2D and its related complications, such as bone loss and nonalcoholic fatty liver disease, all of which the Mexican American and other human populations are at high risk of developing.

Chronic inflammation is a major contributor to the development of obesity-induced insulin resistance, which then can lead to the development of type 2 diabetes (T2D). Skeletal muscle plays a pivotal role in insulin-stimulated whole-body glucose disposal. Therefore, dysregulation of glucose metabolism by inflammation in skeletal muscle can adversely affect skeletal muscle insulin sensitivity and contribute to the pathogenesis of T2D. The mechanism underlying insulin resistance is not well known; however, macrophages are important initiators in the development of the chronic inflammatory state leading to insulin resistance. Skeletal muscle consists of resident macrophages which can be activated by lipopolysaccharide (LPS). These activated macrophages affect myocytes via a paracrine action of pro-inflammatory mediators resulting in secretion of myokines that contribute to inflammation and ultimately skeletal muscle insulin resistance. Therefore, knowing that synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acids (CDDOs) can attenuate macrophage pro-inflammatory responses in chronic disorders, such as cancer and obesity, and that macrophage pro-inflammatory responses can modulate skeletal muscle inflammation, we first examined whether CDDO-ethyl amide (CDDO-EA) inhibited chemokine and cytokine production in macrophages since this had not been reported for CDDO-EA. CDDO-EA blocked LPS-induced tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukine-1beta (IL-1β), and interleukine-6 (IL-6) production in RAW 264.7 mouse and THP-1 human macrophages. Although many studies show that CDDOs have anti-inflammatory properties in several tissues and cells, little is known about the anti-inflammatory effects of CDDOs on skeletal muscle. We hypothesized that CDDO-EA protects skeletal muscle from LPS-induced inflammation by blocking nuclear factor kappa B (NF-κB) signaling. Our studies demonstrate that CDDO-EA prevented LPS-induced TNF-α and MCP-1 gene expression by inhibiting the NF-κB signaling pathway in L6-GLUT4myc rat myotubes. Our findings suggest that CDDO-EA suppresses LPS-induced inflammation in macrophages and myocytes and that CDDO-EA is a promising compound as a therapeutic agent for protecting skeletal muscle from inflammation.

Insulin resistance is associated with circulating levels of lipopolysaccharide (LPS) released from the gut. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. Binding of LPS to Toll like receptor 4 (TLR4) leads to the internalization and trafficking of TLR4, inducing activation of downstream signaling pathways. TLR4 internalization in macrophages leads to activation of pro-inflammatory signaling pathways and production of factors linked to the development of insulin resistance. The extracellular signal-regulated kinase 1 and 2 (ERK1 and 2) are activated downstream of TLR4 and this activation is associated with insulin resistance. We hypothesized that ERK1 and ERK2 regulate TLR4 internalization in macrophages that are exposed to LPS. We examined whether inhibition of ERK activity blocks LPS-mediated internalization of TLR4 in bone marrow derived macrophages (BMDM) .We used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr) . Loss of cell surface TLR4 expression was measured by flow cytometry as a readout for TLR4 internalization. LPS decreased TLR4 surface expression by 31.3%, but knockdown of ERK1, ERK2, or both prevented LPS-induced decrease of TLR4 surface expression (5.6%, no decrease, 0.9%, respectively) . In addition, knockdown of either ERK1, ERK2, or both in RAW264.7 cells prevented LPS-induced activity of Rab5, the early endosomal protein associated with TLR4 translocation. In summary, ERK regulates TLR4 endocytosis and trafficking in macrophages. We propose that ERK positively regulates LPS-mediated TLR4 internalization and inhibition of ERK signaling will protect against insulin resistance. Disclosure P. F. Chang: n/a. S. M. Reyna: n/a. Funding National Institutes of Health (SC2GM127272)

Low-grade chronic inflammation may be a factor in the development of insulin resistance. Studies suggest that chronic inflammation may be due in part to changes in composition and function of gut microflora, which provide an intestinal barrier preventing bacterial lipopolysaccharide (LPS) release. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. We hypothesized that insulin resistance could be produced by inflammatory factors secreted by macrophages when exposed to gut-released LPS. We examined the extracellular signal-regulated kinase (ERK) signaling pathway, which may be responsible for the macrophage inflammatory response. We examined whether inhibition of ERK activity blocked LPS-mediated responses in bone marrow derived macrophages (BMDM). To determine which ERK isoform is involved in the regulation of inflammatory factor production, we used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr). LPS induced TNF-α, RANTES, MCP-1, and IFN-β production of 106371±18250 pg/ml, 11827 ± 1168 pg/ml, 851 ± 73 pg/ml, and 454 ± 46 pg/ml, respectively. Knockdown of ERK1 decreased the release of TNF-α and RANTES to 26534 ± 1471 pg/ml and 5938 ± 806 pg/ml, respectively. Knockdown of ERK2 decreased the release of TNF-α and RANTES to 11600±3610 pg/ml and 7289 ± 265 pg/ml, respectively. Knockdown of either ERK1 or ERK2 did not decrease the release of MCP-1 and IFN-β. However, double knockdown of ERK1 and ERK2 had the greatest inhibition of TNF-α, RANTES, MCP-1, and IFN-β release (not detected, 1540 ± 240 pg/ml, 549 ± 32 pg/ml, and 31 ± 1 pg/ml, respectively). In summary, knockdown of both ERK isoforms is necessary to completely abrogate the LPS effect in macrophages. We propose that ERK positively regulates LPS-mediated inflammatory responses and inhibition of ERK signaling may protect against development of insulin resistance. Disclosure P.F. Chang: None. S.M. Reyna: None. Funding National Institutes of Health; University of Texas Rio Grande Valley

Insulin resistance precedes and contributes to the development of type 2 diabetes mellitus, and it is now believed that chronic inflammation is a major contributor to insulin resistance. Both cellular and secreted factors participate in the pathological and physiological changes that occur to promote inflammation. However, the molecular signaling pathways that drive these processes remain elusive. The mitogen activated protein kinase, extracellular signal-regulated kinase 1 and 2 module (ERK1/2), and the transcription factor, interferon regulatory factor-3 (IRF3), are both activated downstream of Toll-like receptor 4 and associated with the development of insulin resistance. We examined whether inhibition of ERK activity blocked IRF3-mediated immune responses. ERK1 and ERK2 isoforms can have different cellular functions; thus, to determine which ERK isoform is involved in the regulation of IRF3 activity, we performed siRNA to knockdown either ERK1, ERK2, or both in RAW 264.7 macrophages and saw 70% knockdown of each ERK1 and ERK2. Knockdown of ERK1 or ERK2 or both blocked IRF3 translocation to the nucleus with LPS treatment (100 ng/ml, 1 hour), as determined by immunofluorescence using an antibody specific for nuclear IRF3. Because phosphorylated and dimerized IRF3 translocates to the nucleus to regulate the transcription of interferon (IFN)-beta expression, we investigated whether ERK1 or ERK2 inhibited IFN-beta release. Macrophages were treated with LPS (100 ng/ml, 6 hour). LPS induced an IFN-beta production of 791±18.0 pg/ml, but knockdown of either ERK1 or ERK2 decreased the release of IFN-beta to 199±12.3 and 282±32.7 pg/ml, respectively. However, double knockdown of ERK1 and ERK 2 had the greatest inhibition of IFN-beta release (121±15.2 pg/ml). In summary, both ERK1 and ERK2 regulate IRF3 nuclear translocation and signaling in macrophages. We propose that ERK positively regulates IRF3-mediated immune responses and inhibition of ERK signaling will protect against insulin resistance. Disclosure P.F. Chang: None. D. Acevedo: None. S.M. Reyna: None.

Type 2 diabetes is a global epidemic. Increasing evidence shows that estrogen has protective effects on insulin sensitivity. Decreased estrogen levels affect adipocytes response to insulin leading to increased circulating glucose levels and insulin resistance. AMP‐activated protein kinase (AMPK) regulates cellular energy balance due to its role in glucose, lipid, and protein metabolism. Activation of AMPK results in translocation of glucose transporter 4 (GLUT4) to the plasma membrane which mobilizes glucose into the cell. The seed extract of Nigella sativa (NS) reduces elevated glucose levels. However, the molecular mechanisms involved in regulating glucose transport by NS remain elusive. We used an estrogen deficient animal model to examine whether NS and its active ingredient thymoquinone (TQ) modulate proteins involved in regulating glucose transport: AMPK, Akt, phosphorylated AMPK and Akt, and GLUT4.Sprague Dawley rats were either sham‐operated or ovariectomized and divided into control, NS and TQ fed groups. Rats were fed the respective diets for 4 months and sacrificed. The peritoneal adipose tissue was collected and proteins were analyzed. NS fed sham‐operated rats showed significant increase in AMPK phosphorylation, compared to control sham‐operated rats. Similarly, ovariectomized rats fed TQ showed an increase in AMPK phosphorylation, compared to TQ sham‐operated rats. Moreover, rats fed NS or TQ showed an increase in GLUT4 protein content, compared to the respective control groups. However, NS and TQ did not activate Akt phosphorylation.

1 Medical Research Division, Regional Academic Health Center, 1214 W. Schunior Street, Edinburg, TX 78541, USA 2Diabetes Division, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA 3 Texas Diabetes Institute, 701 S. Zarzamora, San Antonio, TX 78207, USA 4Geriatric, Research, Education, and Clinical Center, Audie L. Murphy VA Hospital, 7400 Merton Minter Boulevard, San Antonio, TX 78229, USA

Background. Exercise has an anti-inflammatory effect against, and immune cells play critical roles in the development, of insulin resistance and atherosclerotic vascular disease (AVD). Thus, the goal of this study was to determine whether exercise improves insulin sensitivity in insulin-resistant subjects by downregulating proinflammatory signaling in immune cells. Methods. Seventeen lean, 8 obese nondiabetic, and 11 obese type 2 diabetic individuals underwent an aerobic exercise program for 15 days and an insulin clamp before and after exercise. Peripheral mononuclear cells (PMNC) were obtained for determination of Toll-like receptor (TLR) 2 and 4 protein content and mitogen-activated protein kinase phosphorylation. Results. Compared with that in lean individuals, TLR4 protein content was increased by 4.2-fold in diabetic subjects. This increase in TLR4 content was accompanied by a 3.0-fold increase in extracellular signal-regulated kinase (ERK) phosphorylation. Exercise improved insulin sensitivity in the lean, obese, and type 2 diabetes groups. However, exercise did not affect TLR content or ERK phosphorylation. Conclusions. TLR4 content and ERK phosphorylation are increased in PMNC of type 2 diabetic individuals. While exercise improves insulin sensitivity, this effect is not related to changes in TLR2/TLR4 content or ERK phosphorylation in PMNC of type 2 diabetic individuals.

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased β-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow–fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924 with a known anti-inflammatory agent (dexamethasone), on certain proinflammatory cytokines (TNF-α and IL-6) and the NF-κB signaling pathway in LPS-stimulated macrophages. We also used siRNA to block neddylation to assess the role of this molecular process during LPS-induced cytokine responsiveness. Our results demonstrate that blocking neddylation, either pharmacologically or using siRNA, abrogates the increase in certain proinflammatory cytokines secreted from macrophages in response to LPS. In addition, we have shown that MLN4924 and dexamethasone inhibit LPS-induced cytokine up-regulation at the transcriptional level, albeit through different molecular mechanisms. Thus, neddylation represents a novel molecular process in macrophages that can be targeted to prevent and/or treat the LPS-induced up-regulation of proinflammatory cytokines and the disease processes associated with their up-regulation.

To investigate the role of insulin signaling pathways in migration, proliferation, and inflammation of vascular smooth muscle cells (VSMCs), we examined the expression of active components of the phosphatidyl inositol 3 (PI-3) kinase (p-Akt) and mitogen-activated protein kinase (MAPK) (p-Erk) in primary cultures of VSMCs from human coronary arteries. VSMCs were treated in a dose-response manner with insulin (0, 1, 10, and 100 nM) for 20 min, and Akt and Erk phosphorylation were measured by Western blot analysis. In separate experiments, we evaluated the effect of 200 μM palmitate, in the presence and absence of 8 μM pioglitazone, on insulin-stimulated (100 nM for 20 min) Akt and Erk phosphorylation. The phosphorylation of Akt and Erk in VSMCs exhibited a dose dependency with a three- to fourfold increase, respectively, at the highest dose (100 nM). In the presence of palmitate, insulin-induced Akt phosphorylation was completely abolished, and there was a threefold increase in p-Erk. With addition of pioglitazone, the phosphorylation of Akt by insulin remained unchanged, whereas insulin-stimulated Erk phosphorylation was reduced by pioglitazone. These data in VSMCs indicate that high palmitate decreases insulin-stimulated Akt phosphorylation and stimulates MAPK, whereas preexposure peroxisome proliferator-activated receptor-γ agonist pioglitazone preserves Akt phosphorylation and simultaneously attenuates MAPK signaling. Our results suggest that metabolic and mitogenic insulin signals have different sensitivity, are independently regulated, and may play a role in arterial smooth muscle cells migration, proliferation, and inflammation in conditions of acute hyperinsulinemia.

Background To test potential differences between the actions of antidiabetic medications, we examined the effects of oral hypoglycaemic agents versus glargine-apidra insulin therapy in T2DM.Methods T2DM subjects were randomized to either oral hypoglycaemic agents (pioglitazone, metformin and glipizide, n = 9) or insulin therapy (n = 12) for 6 months. Carotid intimal media thickness, vascular reactivity (flow-mediated vasodilatation; percent change in brachial artery basal diameter post-ischaemia) and sublingual nitrate were measured with ultrasonography. Euglycemic hyperinsulinemic (80 mU/m(2)) clamp with [3]-3H-glucose and muscle biopsies were performed.Results Fasting plasma glucose (similar to 257 to similar to 124 mg/dL, oral hypoglycaemic agents and similar to 256 to similar to 142 mg/dL, IT) and HbA(1c) (similar to 10.3 to similar to 6.4%, OHA and similar to 10.7 to similar to 7.1%, IT) improved comparably. Endogenous glucose production (similar to 2.1 to similar to 1.7 mg/kg/min, oral hypoglycaemic agents and similar to 2.3 to similar to 2.0 mg/kg/min, insulin therapy) and endogenous glucose production suppression by insulin (similar to 0.4 to similar to 0.3 mg/kg min, oral hypoglycaemic agents and similar to 0.5 to similar to 0.7 mg/kg min, insulin therapy) were different. Total glucose disposal x 100 increased in the oral hypoglycaemic agents group (similar to 5.2 to similar to 8.1; p = 0.03), but not in insulin therapy (similar to 6.0 to similar to 5.4 mg/kg/min/mu U/mL x 100). OHA reduced CIMT (similar to 0.080 to similar to 0.068 cm; p < 0.05), whereas insulin therapy did not (similar to 0.075 to similar to 0.072 cm). After sublingual nitrate, brachial artery basal diameter increased in the OHA group (similar to 8.7 to similar to 18.2%), but not in insulin therapy (similar to 11.2 to similar to 15.0%; p < 0.02). Except for plasma adiponectin (similar to 7 to similar to 15, oral hypoglycaemic agents versus similar to 6 to similar to 10, IT), changes in inflammatory markers in the circulation and in muscle (I kappa B alpha, super-oxidase dismutase 2, monocyte-chemo-attractant protein 1, p-ERK and JNK) were equivalent.Conclusions Oral hypoglycaemic agents and insulin therapy treated patients achieved adequate glycemic control and the effects on circulating and muscle inflammatory biomarkers were similar, but only oral hypoglycaemic agents improved insulin sensitivity, vascular function and carotid intimal media thickness. These findings in a small sample suggest that the use of oral hypoglycaemic agents provides additional benefits to patients with T2DM. Copyright (C) 2011 John Wiley & Sons, Ltd.

TNF-α levels are increased in obesity and type 2 diabetes. The regulation of TNF-α converting enzyme (TACE) and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), in human type 2 diabetes is unknown.

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐ D ‐ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP‐activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK‐independent effects, mainly on AMP‐regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A‐769662, a nonnucleoside compound from the thienopyridone family. We also report that A‐769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A‐769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A‐769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK‐independent side effects. © 2008 IUBMB IUBMB Life, 61(1): 18–26, 2009