Endurance exercise training increases APPL1 expression and improves insulin signaling in the hepatic tissue of diet-induced obese mice, independently of weight loss

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1a), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3 beta) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1a association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3 beta phosphorylation levels and glycogen content at 24?h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss. J. Cell. Physiol. 227: 29172926, 2012. (C) 2011 Wiley Periodicals, Inc.

Altered Glucose Homeostasis and Hepatic Function in Obese Mice Deficient for Both Kinin Receptor Genes

The Kallikrein-Kinin System (KKS) has been implicated in several aspects of metabolism, including the regulation of glucose homeostasis and adiposity. Kinins and des-Arg-kinins are the major effectors of this system and promote their effects by binding to two different receptors, the kinin B2 and B1 receptors, respectively. To understand the influence of the KKS on the pathophysiology of obesity and type 2 diabetes (T2DM), we generated an animal model deficient for both kinin receptor genes and leptin (obB1B2KO). Six-month-old obB1B2KO mice showed increased blood glucose levels. Isolated islets of the transgenic animals were more responsive to glucose stimulation releasing greater amounts of insulin, mainly in 3-month-old mice, which was corroborated by elevated serum C-peptide concentrations. Furthermore, they presented hepatomegaly, pronounced steatosis, and increased levels of circulating transaminases. This mouse also demonstrated exacerbated gluconeogenesis during the pyruvate challenge test. The hepatic abnormalities were accompanied by changes in the gene expression of factors linked to glucose and lipid metabolisms in the liver. Thus, we conclude that kinin receptors are important for modulation of insulin secretion and for the preservation of normal glucose levels and hepatic functions in obese mice, suggesting a protective role of the KKS regarding complications associated with obesity and T2DM.

GLUT4 content decreases along with insulin resistance and high levels of inflammatory markers in rats with metabolic syndrome

Background: Metabolic syndrome is characterized by insulin resistance, which is closely related to GLUT4 content in insulin-sensitive tissues. Thus, we evaluated the GLUT4 expression, insulin resistance and inflammation, characteristics of the metabolic syndrome, in an experimental model. Methods: Spontaneously hypertensive neonate rats (18/group) were treated with monosodium glutamate (MetS) during 9 days, and compared with Wistar-Kyoto (C) and saline-treated SHR (H). Blood pressure (BP) and lipid levels, C-reactive protein (CRP), interleukin 6 (IL-6), TNF-alpha and adiponectin were evaluated. GLUT4 protein was analysed in the heart, white adipose tissue and gastrocnemius. Studies were performed at 3 (3-mo), 6 (6-mo) and 9 (9-mo) months of age. Results: MetS rats were more insulin resistant (p<0.001, all ages) and had higher BP (3-mo: p<0.001, 6-mo: p = 0.001, 9-mo: p = 0.015) as compared to C. At 6 months, CRP, IL-6 and TNF-alpha were higher (p<0.001, all comparisons) in MetS rats vs H, but adiponectin was lower in MetS at 9 months (MetS: 32 +/- 2, H: 42 +/- 2, C: 45 +/- 2 pg/mL; p<0.001). GLUT4 protein was reduced in MetS as compared to C rats at 3, 6 and 9-mo, respectively (Heart: 54%, 50% and 57%; Gastrocnemius: 37%, 56% and 50%; Adipose tissue: 69%, 61% and 69%). Conclusions: MSG-treated SHR presented all metabolic syndrome characteristics, as well as reduced GLUT4 content, which must play a key role in the impaired glycemic homeostasis of the metabolic syndrome.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Quercetin decreases inflammatory response and increases insulin action in skeletal muscle of ob/ob mice and in L6 myotubes

Quercetin is a potent anti-inflammatory flavonoid, but its capacity to modulate insulin sensitivity in obese insulin resistant conditions is unknown. This study investigated the effect of quercetin treatment upon insulin sensitivity of ob/ob mice and its potential molecular mechanisms. Obese ob/ob mice were treated with quercetin for 10 weeks, and L6 myotubes were treated with either palmitate or tumor necrosis factor-alpha (TNF alpha) plus quercetin. Cells and muscles were processed for analysis of glucose transporter 4 (GLUT4), TNF alpha and inducible nitric oxide synthase (iNOS) expression, and c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-kappa B (NF-kappa B) kinase (I kappa K) phosphorylation. Myotubes were assayed for glucose uptake and NF-kappa B translocation. Chromatin immunoprecipitation assessed NF-kappa B binding to GLUT4 promoter. Quercetin treatment improved whole body insulin sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, and TNF alpha and iNOS expression in skeletal muscle. Quercetin suppressed palmitate-induced upregulation of TNF alpha and iNOS and restored normal levels of GLUT4 in myotubes. In parallel, quercetin suppressed TNF alpha-induced reduction of glucose uptake in myotubes. Nuclear accumulation of NF-kappa B in myotubes and binding of NF-kappa B to GLUT4 promoter in muscles of ob/ob mice were also reduced by quercetin. We demonstrated that quercetin decreased the inflammatory status in skeletal muscle of obese mice and in L6 myotubes. This effect was followed by increased muscle GLUT4, with parallel improvement of insulin sensitivity. These results point out quercetin as a putative strategy to manage inflammatory-related insulin resistance. (C) 2012 Elsevier B.V. All rights reserved.

Double-Stranded RNA-Activated Protein Kinase Is a Key Modulator of Insulin Sensitivity in Physiological Conditions and in Obesity in Mice

The molecular integration of nutrient-and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of kappa B kinase beta. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of kappa B kinase beta phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity. (Endocrinology 153:5261-5274, 2012)

Oral treatment with Hev b 13 ameliorates experimental colitis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human mononuclear cells to release interleukin (IL)-10 in vitro. This immunoregulatory cytokine appears to play an important role in preventing inflammation and mucosal damage in animal models of colitis and in Crohn's disease patients. The aim of this study was to evaluate the therapeutic effect of Hev b 13 in mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. Two hours following colonic instillation of the haptenizing agent, and daily thereafter for 5 days, Hev b 13 was administered by oral gavage. In mice treated with daily doses of either 0.5 mg/kg or 5.0 mg/kg of Hev b 13, the clinical signs of diarrhoea, rectal prolapse and body weight loss and also histological damage of the distal colon, were reduced significantly, in comparison with water-treated diseased mice. These findings suggest a potent anti-inflammatory activity of Hev b 13; this activity is speculated to be related to its interaction with cells from the immune system.

Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses

Hypertension is a disorder affecting millions worldwide, and is a leading cause of death and debilitation in the United States. It is widely accepted that during hypertension and other cardiovascular diseases the vasculature exhibits endothelial dysfunction; a deficit in the relaxatory ability of the vessel, attributed to a lack of nitric oxide (NO) bioavailability. Recently, the one electron redox variant of NO, nitroxyl anion (NO-) has emerged as an endothelium-derived relaxing factor (EDRF) and a candidate for endothelium-derived hyperpolarizing factor (EDRF). NO- is thought to exist protonated (HNO) in vivo, which would make this species more resistant to scavenging. However, no studies have investigated the role of this redox species during hypertension, and whether the vasculature loses the ability to relax to HNO. Thus, we hypothesize that aorta from angiotensin II (AngII)-hypertensive mice will exhibit a preserved relaxation response to Angeli's Salt, an HNO donor. Male C57B16 mice, aged 12-14 weeks were implanted with mini-osmotic pumps containing AngII (90 ng/min, 14 days plus high salt chow) or sham surgery. Aorta were excised, cleaned and used to perform functional studies in a myograph. We found that aorta from AngII-hypertensive mice exhibited a significant endothelial dysfunction as demonstrated by a decrease in acetylcholine (ACh)-mediated relaxation. However, vessels from hypertensive mice exhibited a preserved response to Angeli's Salt (AS), the HNO donor. To confirm that relaxation responses to HNO were maintained, concentration response curves (CRCs) to ACh were performed in the presence of scavengers to both NO and HNO (carboxy-PTIO and L-cys, resp.). We found that ACh-mediated relaxation responses were significantly decreased in aorta from sham and almost completely abolished in aorta from AngII-treated mice. Vessels incubated with L-cys exhibited a modest decrease in ACh-mediated relaxations responses. These data demonstrate that aorta from AngII-treated hypertensive mice exhibit a preserved relaxation response to AS, an HNO donor, regardless of a significant endothelial dysfunction. (C) 2011 Elsevier Ltd. All rights reserved,

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Sunflower Oil Supplementation Has Proinflammatory Effects and Does Not Reverse Insulin Resistance in Obesity Induced by High-Fat Diet in C57BL/6Mice

High consumption of polyunsaturated fatty acids, such as sunflower oil has been associated to beneficial effects in plasma lipid profile, but its role on inflammation and insulin resistance is not fully elucidated yet. We evaluated the effect of sunflower oil supplementation on inflammatory state and insulin resistance condition in HFD-induced obese mice. C57BL/ 6 male mice (8 weeks) were divided in four groups: (a) control diet (CD), (b) HFD, (c) CD supplemented with n-6 (CD + n-6), and (d) HFD supplemented with n-6 (HFD + n-6). CD + n-6 and HFD + n-6 were supplemented with sunflower oil by oral gavage at 2 g/ Kg of body weight, three times per week. CD and HFD were supplemented with water instead at the same dose. HFD induced whole andmuscle-specific insulin resistance associated with increased inflammatory markers in insulin-sensitive tissues andmacrophage cells. Sunflower oil supplementation was not efficient in preventing or reducing these parameters. In addition, the supplementation increased pro-inflammatory cytokine production by macrophages and tissues. Lipid profile, on the other hand, was improved with the sunflower oil supplementation in animals fed HFD. In conclusion, sunflower oil supplementation improves lipid profile, but it does not prevent or attenuate insulin resistance and inflammation induced by HFD in C57BL/ 6 mice.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Involvement of nitric oxide on the pathogenesis of irinotecan-induced intestinal mucositis: role of cytokines on inducible nitric oxide synthase activation

Purpose Intestinal mucositis and the closely associated diarrhea are common costly side effects of irinotecan. Cytokine modulators, such as thalidomide and pentoxifylline, are found capable of attenuating intestinal mucositis progression. Nitric oxide (NO) seems to be a key mediator of the antineoplastic drug toxicity. The aim of this study was to investigate the role of NO on the pathogenesis of intestinal mucositis, as well as the participation of cytokines upon inducible nitric oxide synthase (iNOS) expression in irinotecan-induced intestinal mucositis. Methods iNOS-knockout (iNOS(-/-)) and C57BL/6 (WT, wild type) animals (n = 5-6) were given either saline or irinotecan (60 mg/kg i.p for 4 days), with or without pretreatment with aminoguanidine (50 mg/kg s.c.), thalidomide (60 mg/kg s.c), infliximab (5 mg/kg i.v.), or pentoxifylline (1.7 mg/kg s.c). On day 5, diarrhea was assessed, and following euthanasia, proximal intestinal samples were obtained for myeloperoxidase (MPO) and iNOS activity, morphometric analysis, western blot and immunohistochemistry to iNOS, cytokine dosage, and for in vitro evaluation of gut contractility. Results Irinotecan induced severe diarrhea and intestinal smooth muscle over-contractility, accompanied with histopathological changes. Additionally, increased MPO and iNOS activity and iNOS immunoexpression were found in WT animals treated with irinotecan. The rise in MPO, smooth muscle over-contractility, and diarrhea were abrogated in aminoguanidine-treated and iNOS(-/-) mice. Moreover, through western blot, we verified that infliximab and pentoxifylline significantly inhibited irinotecan-induced iNOS expression. In addition, cytokine concentration was found only partially decreased in irinotecan-treated iNOS(-/-) mice when compared with wild-type animals that were given irinotecan. Conclusions This study suggests a role of nitric oxide in the pathogenesis of irinotecan-induced intestinal mucositis and also provides evidence for the participation of cytokines on iNOS induction.

The role of Nox2-derived ROS in the development of cognitive impairment after sepsis

BACKGROUND: Sepsis- associated encephalopathy (SAE) is an early and common feature of severe infections. Oxidative stress is one of the mechanisms associated with the pathophysiology of SAE. The goal of this study was to investigate the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. METHODS: Sepsis was induced in WT and gp91phox knockout mice (gp91phox-/-) by cecal ligation and puncture (CLP) to induce fecal peritonitis. We measured oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in the hippocampus at six hours, twenty-four hours and five days post-sepsis. Mice were also treated with apocynin, a NADPH oxidase inhibitor. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance test and the Morris water maze in control and apocynin-treated WT mice. RESULTS: Acute oxidative damage to the hippocampus was identified by increased 4-HNE expression in parallel with an increase in Nox2 gene expression after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibited hippocampal oxidative stress in septic animals. Pharmacologic inhibition or the absence of Nox2 in gp91phox-/- mice prevented glial cell activation, one of the central mechanisms associated with SAE. Finally, treatment with apocynin and inhibition of hippocampal oxidative stress in the acute phase of sepsis prevented the development of long-term cognitive impairment. CONCLUSIONS: Our results demonstrate that Nox2 is the main source of reactive oxygen species (ROS) involved in the oxidative damage to the hippocampus in SAE and that Nox2-derived ROS are determining factors for cognitive impairments after sepsis. These findings highlight the importance of Nox2-derived ROS as a central mechanism in the development of neuroinflammation associated with SAE.

Intestinal permeability parameters in obese patients are correlated with metabolic syndrome risk factors

Background & aims: Altered intestinal permeability has been shown to be associated with metabolic alterations in animal models of obesity, but not in humans. The aim of this study was to assess intestinal permeability in obese women and verify if there is any association with anthropometric measurements, body composition or biochemical variables. Methods: Twenty lean and twenty obese females participated in the study. Anthropometric measurements, body composition and blood pressure were assessed and biochemical analyses were performed. Administration of lactulose and mannitol followed by their quantification in urine was used to assess the intestinal permeability of volunteers. Results: The obese group showed lower HDL (p < 0.05), higher fasting glucose, insulin, HOMA index and lactulose excretion than the lean group (p < 0.05), suggesting increased paracellular permeability. Lactulose excretion showed positive correlation (p < 0.05) with waist and abdominal circumference. Blood insulin and the HOMA index also increased with the increase in mannitol and lactulose excretion and in the L/M ratio (p < 0.05). L/M ratio presented a negative correlation with HDL concentration (p < 0.05). Conclusions: We demonstrated that intestinal permeability parameters in obese women are positively correlated with anthropometric measurements and metabolic variables. Therapeutic interventions focused on intestine health and the modulation of intestinal permeability should be explored in the context of obesity. (C) 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.