Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated ...inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.
Insulin resistance in diet-induced obesity (DIO) is associated with a chronic systemic low-grade inflammation, and Toll-like receptor 4 (TLR4) plays an important role in the link among insulin ...resistance, inflammation, and obesity. The current study aimed to analyze the effect of exercise on TLR4 expression and activation in obese rats and its consequences on insulin sensitivity and signaling.
The effect of chronic and acute exercise was investigated on insulin sensitivity, insulin signaling, TLR4 activation, c-Jun NH(2)-terminal kinase (JNK) and IκB kinase (IKKβ) activity, and lipopolysaccharide (LPS) serum levels in tissues of DIO rats.
The results showed that chronic exercise reduced TLR4 mRNA and protein expression in liver, muscle, and adipose tissue. However, both acute and chronic exercise blunted TLR4 signaling in these tissues, including a reduction in JNK and IKKβ phosphorylation and IRS-1 serine 307 phosphorylation, and, in parallel, improved insulin-induced IR, IRS-1 tyrosine phosphorylation, and Akt serine phosphorylation, and reduced LPS serum levels.
Our results show that physical exercise in DIO rats, both acute and chronic, induces an important suppression in the TLR4 signaling pathway in the liver, muscle, and adipose tissue, reduces LPS serum levels, and improves insulin signaling and sensitivity. These data provide considerable progress in our understanding of the molecular events that link physical exercise to an improvement in inflammation and insulin resistance.
Objective
It has become clear that exercise may be a useful therapy in the insulin resistance treatment, as it has anti‐inflammatory effects and improves insulin sensitivity. However, it remains ...uncertain whether exercise affects the adipocytes or infiltrated macrophages. Thus, the aim was to investigate the effects of acute exercise on the inflammatory status and insulin signaling of the white adipose tissue (WAT) fractions (stromal‐vascular fraction SVF and adipocytes).
Design and Methods
The effect of acute swimming exercise was investigated on insulin sensitivity, insulin signaling, inflammatory pathways in the WAT fractions of high‐fat fed Wistar rats. Additionally, macrophage infiltration and polarization were analyzed in the WAT.
Results
Acute exercise can improve insulin signaling in WAT fractions, along with a phenotypic switch from M1‐ to M2‐macrophages in obese rats, as indicated by a marked increase in macrophage galactose‐type C‐type lectin 1‐positive cells in WAT was observed. Additionally, exercise promoted a reduction in circulating levels of lipopolysaccharide, and toll‐like receptor 4 activity along with TNF‐alpha, IL‐1‐beta and MCP‐1 mRNA levels in WAT fractions.
Conclusions
These data suggest that acute exercise improves insulin signaling in the WAT, at least in part by inducing macrophage polarization toward the M2‐state.
Obesity and type 2 diabetes are characterized by insulin resistance, and the common basis of these events is a chronic and systemic inflammatory process marked by the activation of the c-Jun ...N-terminal kinase (JNK) and inhibitor-κB kinase (IKKβ)/nuclear factor-κB (NFκB) pathways, up-regulated cytokine synthesis, and endoplasmic reticulum dysfunction. The aim of this study was to evaluate the effects of diacerhein administration, an antiinflammatory drug that reduces the levels of inflammatory cytokines, on insulin sensitivity and signaling in diet-induced obese (DIO) mice. Swiss mice were fed with conventional chow (control group) or a high-fat diet (DIO group). Later, DIO mice were randomly subdivided into a new subgroup (DAR) that received 20 mg/kg diacerhein for 10 d. Western blotting was used to quantify the expression and phosphorylation of insulin receptor, insulin receptor substrate 1, and Akt and of inflammatory mediators that modulate insulin signaling in a negative manner (IKKβ, JNK, and inducible nitric oxide synthase). We show here, for the first time, that the administration of diacerhein in DIO mice improved endoplasmic reticulum stress, reduced JNK and IKKβ phosphorylation, and resulted in a marked improvement in fasting glucose, a decrease in macrophage infiltration in adipose tissue, and a reduced expression and activity of proinflammatory mediators accompanied by an improvement in the insulin signaling mainly in the liver and adipose tissue. Taken together, these results indicate that diacerhein treatment improves insulin sensitivity in obesity, mediated by the reversal of subclinical inflammation, and that this drug may be an alternative therapy for insulin resistance.
Abstract Background The toll-like receptor 4 (TLR4) and inducible nitric oxide synthase are proteins from the innate immune system that, when activated, can induce insulin resistance. Polymorphisms ...in these genes, TLR4 and NOS2 , respectively, could affect the immune response, as well as the prevalence of Type 2 diabetes (T2DM). Objective The aim of the present study was to investigate the contribution of four polymorphisms (two from TLR4 and two from NOS2 ) to susceptibility to T2DM in a southeast Brazilian population. Design A total of 211 patients with T2DM and 200 unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene and for the insertion (I)/deletion (D) AAAT and (CCTTT)n polymorphisms of the NOS2 promoter gene. Results With regard to the NOS2 promoter region, the data showed that the I allele of the I/D AAAT polymorphism was more prevalent in the T2DM group and that the L/L genotype of the (CCTTT)n polymorphism was also more frequent in the same group. In contrast, the 299Gly allele and the 399Ile allele from the Asp299Gly and Thr399Ile TLR4 gene polymorphisms, respectively, were associated with protection of T2DM. It is believed that the persistence of these genetic variations in human populations may be indicative of a selective advantage in the face of different environmental pressures. Conclusions Genetic variations in the NOS2 gene promoter and TLR4 coding sequence may lead to deleterious and protective effects, respectively, arising from altered function of the innate immune system in patients with T2DM.
Orientador: Mario Jose Abdalla Saad
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Previous issue date: 2009
Resumo: Atualmente, o DM2 constitui-se como um grande problema de saúde pública mundial devido à sua elevada prevalência, morbimortalidade, além do impacto que apresenta nos custos públicos com a saúde. Os mecanismos fisiopatológicos desta doença têm se tornado cada vez mais evidentes, estando relacionados com o envolvimento exclusivo de células, receptores e mediadores do sistema imune inato. Dentre estes compostos destacam-se o TLR4, um receptor de antígenos deste sistema, responsável por ativar a transcrição de citocinas pró-inflamatórias, e a iNOS, uma enzima cálcio independente, que produz elevadas concentrações de NO. Ambas as proteínas estão envolvidas no desenvolvimento de resistência à insulina. Por esta razão, este presente trabalho investigou se 2 polimorfismos no promotor do gene da iNOS (NOS2) (deleção / inserção AAAT e (CCTTT)n) e 2 polimorfismos na seqüência codificadora do gene do TLR4 (TLR4) (Asp299Gly e Thr399Ile), tanto isolados como em conjunto, possuíam alguma associação com a susceptibilidade de desenvolvimento DM2. Além disso, também foi avaliado se os polimorfismos estavam associados com fatores de risco para a resistência à insulina e características clínicas do DM2. Foram coletadas amostras de sangue de 411 indivíduos, sendo 211 portadores de DM2 e 200 controles (doadores de sangue) saudáveis acima de 50 anos, de ambos os sexos. A glicemia de jejum foi determinada e o DNA de cada sujeito foi extraído para posterior amplificações dos 4 fragmentos desejados por PCR. Para a determinação dos genótipos, os 2 polimorfismos da iNOS - AAAT e (CCTTT)n - foram submetidos à eletroforese em seqüenciador automático e os 2 polimorfismos do TLR4 - Asp299Gly e Thr299Ile - foram submetidos a reação de digestão utilizando-se enzima de restrição. Além das análises estatísticas convencionais, os resultados foram avaliados pelo método MDR (multifactor-dimensionality reduction), que visa verificar interações entre polimorfismos. As freqüências genotípicas para os polimorfismos, considerando ambos os grupos, estão em equilíbrio de Hardy-Weinberg. Para o polimorfismo de deleção / inserção AAAT nós encontramos que a presença da inserção resultou em um aumento de risco para a doença. A inserção também se associou com a história familiar de diabetes em homens diabéticos e fatores de risco para a resistência à insulina. Para o polimorfismo (CCTTT)n notamos que os genótipos que continham apenas as repetições longas (12-16 repetições) do microssatélite estavam associados com uma chance aumentada de desenvolver DM2, além destas mesmas repetições estarem relacionadas com colesterol total e nefropatia diabética. As freqüências dos polimorfismos Asp299Gly e Thr399Ile do TLR4 também foram avaliadas. Para ambas as mutações, os alelos menos prevalentes foram significativamente maior nos controles, mostrando um papel protetor para os alelos que codificam os aminoácidos 299Gly e 399Ile, respectivamente. Pelo método MDR também foi observado que existe uma forte interação entre os quatro polimorfismos estudados e que a combinação dos genótipos D/I do polimorfismo de deleção / inserção AAAT no gene NOS2, C/L (formas curtas / formas longas) do polimorfismo (CCTTT)n no gene NOS2 e Asp/Asp ou Thr/Thr dos polimorfismos Asp299Gly ou Thr299Ile, respectivamente, no gene TLR4 apresenta alto risco para o desenvolvimento do DM2. Sendo assim, as variações genéticas no promotor do gene NOS2 e na seqüência codificadora do TLR4, consideradas tanto isoladas como em conjunto, podem levar a efeitos deletérios ou protetores, respectivamente, que são oriundos de funções alteradas do sistema imune inato em pacientes com DM2.
Abstract: Background: The toll-like receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS) are proteins from the innate immune system that, when activated, can induce insulin resistance. Polymorphisms in these genes could affect the immune response, as well as the prevalence of type 2 diabetes (T2DM). Objective: The aims of the present study were: to investigate the contribution, isolated or together, of four polymorphisms (Asp299Gly and Thr399Ile from TLR4; deletion / insertion AAAT and (CCTTT)n from NOS2) to susceptibility to T2DM in a southeast Brazilian population; and to verify if these polymorphisms are associated with risk factors for insulin resistance syndrome and clinical characteristics for T2DM. Design: A total of 211 patients with T2DM and 200 unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene and for the deletion / insertion AAAT and (CCTTT)n polymorphisms of the NOS2 promoter gene. Besides the conventional statistics analysis, the data was also analyzed for gene-to-gene interactions among the four polymorphic loci using the multifactor-dimensionality reduction (MDR) method. Results: With regard to the NOS2 promoter region, the data showed that the I allele of the deletion / insertion AAAT polymorphism was more prevalent in the T2DM group and also was related with some risk factors for insulin resistance syndrome (body mass index, waist circumference). Similarly, the L/L genotype of (CCTTT)n polymorphism were more frequent in the T2DM group and the L allele was associated with total cholesterol and diabetic nephropathy. In contrast, the 299Gly allele and the 399Ile allele from the Asp299Gly and Thr399Ile TLR4 gene polymorphisms, respectively, were associated with protection of T2DM. The MDR analysis showed a significant gene-to-gene interaction between the four polymorphisms studied. Moreover, the combination of the NOS2 deletion / insertion AAAT heterozygote, the NOS2 (CCTTT)n (stratified in short and long forms) heterozygote and the TLR4 Asp299Gly Asp/Asp or Thr399Ile Thr/Thr, homozygotes was associated with a increased risk of T2DM. Conclusions: Genetic variations in the NOS2 gene promoter and TLR4 coding sequence, when analyzed together or isolated, may lead to deleterious and protective effects, respectively, arising from altered function of the innate immune system in patients with T2DM.
Mestrado
Medicina Experimental
Mestre em Fisiopatologia Médica
Orientador: Mario Jose Abdalla Saad
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Previous issue date: 2013
Resumo: A obesidade é caracterizada por processo inflamatório crônico e resistência à insulina (RI), os quais são responsáveis por grande parte de suas doenças associadas. Sabe-se que diversas moléculas do sistema imune inato estão associadas à RI e obesidade, destacando-se o receptor toll-like-receptor 4 (TLR4). Sua via de sinalização está ativada na obesidade, devido à presença aumentada na circulação de seu principal ligante, lipopolissacarídeo (LPS). Acredita-se que esta endotoxemia metabólica seja causada por alterações na microbiota e na permeabilidade intestinais, o que torna o intestino e as bactérias que o habitam, grandes alvos para o tratamento da obesidade. O objetivo deste presente trabalho foi avaliar os efeitos dos probióticos (PB) na sensibilidade à insulina e na sinalização de TLR4 em tecidos insulino-sensíveis, além de verificar suas possíveis ações na microbiota intestinal. Dessa forma, camundongos Swiss foram divididos em 4 grupos: controles (C), controles tratados com PB (C+PB), obesos (DIO) e obesos tratados com PB (DIO+PB). O tratamento teve a duração de 5 semanas. O uso de PB em animais obesos proporcionou grandes melhoras nos parâmetros fisiológicos e moleculares de RI, além de atenuar a ativação da via de sinalização do TLR4, provavelmente pela redução dos níveis circulantes de LPS. O grupo DIO+PB ainda mostrou mudanças positivas na distribuição dos filos de bactérias intestinais e menor permeabilidade intestinal, quando comparado com o grupo DIO. Analisando o hipotálamo, observou-se que o uso de PB nesse modelo de obesidade regulou de forma favorável os mecanismos centrais de controle da fome, bem como alguns parâmetros de inflamação. Assim, conclui-se que a regulação da microbiota intestinal promovida pela administração de probióticos pode trazer benefícios no controle da obesidade, por reduzir ou atenuar mecanismos moleculares de resistência à insulina
Abstract: Obesity is the main risk factor to the development of insulin resistance and type 2 diabetes. The common basis among these events is an inflammatory process characterized by the activation of toll-like receptor 4 (TLR4) by its main ligand lipopolysaccharide, LPS. Its concentration is higher in obese people and it is believed that changes in composition of the gut microbiota and epithelial functions may play a role in the inflammation associated with obesity. The aim of the study was to evaluate the effects of probiotic on the insulin sensitivity, TLR4 signaling, intestinal permeability and microbiota composition in diet-induced obese mice. Male adult Swiss mice composed randomly 2 groups: chow diet (CTL) and high-fat diet by 5 consecutive weeks (DIO). During these 5 weeks, some mice of the DIO and CTL groups received daily a pool of probiotics. The DIO animals that received probiotic presented an expressive improvement in their glucose tolerance test, fasting glucose and in parallel a significant increase in the phosphorylation levels of insulin induced IR, IRS1 and Akt in muscle, liver and adipose tissue. There was a relevant reduction in the TLR4-Myd88 interaction, IKK? and JNK phosphorylation and iNOS expression in DIO mice treated with probiotic. This treatment also improved the expression of ileal tight-junctions proteins (ZO-1, Occludin), decreased LPS portal levels and the concentrations of bacteria of the phylum Firmicutes (associated with obesity) in feces. Analyzing the hypothalamus, it was observed that the use of probiotics in this model of obesity favorably regulated central mechanisms of food intake, as well as some inflammation parameters. In conclusion, our results show that probiotics, through their effects on intestinal permeability and microbiota composition, can improve insulin sensitivity and signaling of DIO mice, reducing their inflammation and suggesting potential beneficial effects in the treatment of insulin resistance and type 2 diabetes
Doutorado
Medicina Experimental
Doutora em Ciências
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