Aims/hypothesis
A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations ...of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration.
Methods
Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry.
Results
Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase β (IKKβ) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5′-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80
+
macrophage cells in the adipose tissue of antibiotic-treated mice.
Conclusions/interpretation
These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.
Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free ...conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Obesity and insulin resistance are the major predisposing factors to comorbidities, such as Type 2 diabetes, nonalcoholic fatty liver disease, cardiovascular and neurodegenerative diseases, and ...several types of cancer. The prevalence of obesity is still increasing worldwide and now affects a large number of individuals. Here, we review the role of the gut microbiota in the pathophysiology of insulin resistance/obesity. The human intestine is colonized by ∼100 trillion bacteria, which constitute the gut microbiota. Studies have shown that lean and overweight rodents and humans may present differences in the composition of their intestinal flora. Over the past 10 years, data from different sources have established a causal link between the intestinal microbiota and obesity/insulin resistance. It is important to emphasize that diet-induced obesity promotes insulin resistance by mechanisms independent and dependent on gut microbiota. In this review, we present several mechanisms that contribute to explaining the link between intestinal flora and insulin resistance/obesity. The LPS from intestinal flora bacteria can induce a chronic subclinical inflammatory process and obesity, leading to insulin resistance through activation of TLR4. The reduction in circulating SCFA may also have an essential role in the installation of reduced insulin sensitivity and obesity. Other mechanisms include effects of bile acids, branched-chain amino acids (BCAA), and some other lesser-known factors. In the near future, this area should open new therapeutic avenues for obesity/insulin resistance and its comorbidities.
Patients with proximal deep-vein thrombosis were assigned to undergo anticoagulation either alone or combined with pharmacomechanical thrombolysis. After 6 to 24 months, there was no significant ...between-group difference in the incidence of the post-thrombotic syndrome.
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 κB kinase β. 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 κB kinase β 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.
Loss-of-Function Mutation in Toll-Like Receptor 4 Prevents Diet-Induced Obesity and Insulin Resistance
Daniela M.L. Tsukumo 1 ,
Marco A. Carvalho-Filho 1 ,
José B.C. Carvalheira 1 ,
Patrícia O. Prada ...1 ,
Sandro M. Hirabara 2 ,
André A. Schenka 3 ,
Eliana P. Araújo 1 ,
José Vassallo 3 ,
Rui Curi 2 ,
Lício A. Velloso 1 and
Mario J.A. Saad 1
1 Department of Internal Medicine, State University of Campinas, Campinas, São Paulo, Brazil
2 Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
3 Department of Pathology, State University of Campinas, Campinas, São Paulo, Brazil
Address correspondence and reprint requests to Mario J.A. Saad, MD, Departamento de Clínica Médica, FCM-UNICAMP, Cidade Universitária
Zeferino Vaz, Campinas, SP, Brazil, 13081-970. E-mail: msaad{at}fcm.unicamp.br
Abstract
Obesity is associated with insulin resistance and a state of abnormal inflammatory response. The Toll-like receptor (TLR)4
has an important role in inflammation and immunity, and its expression has been reported in most tissues of the body, including
the insulin-sensitive ones. Because it is activated by lipopolysaccharide and saturated fatty acids, which are inducers of
insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals.
Here, we show that C3H/HeJ mice, which have a loss-of-function mutation in TLR4, are protected against the development of
diet-induced obesity. In addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory
exchange ratio, improved insulin sensitivity, and enhanced insulin-signaling capacity in adipose tissue, muscle, and liver
compared with control mice during high-fat feeding. Moreover, in these tissues, control mice fed a high-fat diet show an increase
in IκB kinase complex and c-Jun NH 2 -terminal kinase activity, which is prevented in C3H/HeJ mice. In isolated muscles from C3H/HeJ mice, protection from saturated
fatty acid–induced insulin resistance is observed. Thus, TLR4 appears to be an important mediator of obesity and insulin resistance
and a potential target for the therapy of these highly prevalent medical conditions.
CLS, crown-like structure
ELISA, enzyme-linked immunosorbent assay
FFA, free fatty acid
HFD, high-fat diet
IGTT, intraperitoneal glucose tolerance test
IκBα, inhibitor of nuclear factor-κB
IKKβ, IκB kinase complex
IL, interleukin
IR, insulin receptor
IRS-1, insulin receptor substrate-1
JNK, c-Jun NH2-terminal kinase
LPS, lipopolysaccharide
NK-κB, nuclear factor-κB
RER, respiratory exchange ratio
TNF, tumor necrosis factor
TLR, Toll-like receptor
WAT, white adipose tissue
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 22 May 2007. DOI: 10.2337/db06-1595.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted May 18, 2007.
Received November 14, 2006.
DIABETES
Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin ...of rats.
The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway.
We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing.
Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1α in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes.
ClinicalTrials.gov NCT01295177.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The development of obesity and insulin resistance has been extensively studied in the last decades, but the mechanisms underlying these alterations are still not completely understood. The gut ...microbiota has been identified as a potential contributor to metabolic diseases. It has been shown that obese individuals present different proportions of bacterial phyla compared with lean individuals, with an increase in Firmicutes and Actinobacteria and a decrease in Bacteroidetes. This alteration seems to interfere with intestinal permeability, increasing the absorption of lipopolysaccharide (LPS), which reaches circulation and initiates activation of Toll-like receptor (TLR) 4 and 2 and LPS receptor CD14, leading to increased activation of inflammatory pathways. With these activations, an impairment of the insulin signaling is observed, with decreased phosphorylation of the insulin receptor, insulin receptor substrate (IRS) and Akt, as well as increased inhibitory serine phosphorylation of IRS-1. Altered proportions of bacterial phyla have also been demonstrated to interfere with host's biochemical pathways, increasing energy extraction and depot in adipose tissue. Therefore, understanding the mechanisms by which the alteration in the gut microbiota produces different signaling activations and phenotype changes may offer an interesting opportunity for the treatment of obesity and type 2 diabetes.
The identification of brown/beige adipose tissue in adult humans has motivated the search for methods aimed at increasing its thermogenic activity as an approach to treat obesity. In rodents, the ...brown adipose tissue is under the control of sympathetic signals originating in the hypothalamus. However, the putative connection between the depots of brown/beige adipocytes and the hypothalamus in humans has never been explored. The objective of this study was to evaluate the response of the hypothalamus and brown/beige adipose tissue to cold stimulus in obese subjects undergoing body mass reduction following gastric bypass.
We evaluated twelve obese, non-diabetic subjects undergoing Roux-in-Y gastric bypass and 12 lean controls. Obese subjects were evaluated before and approximately 8 months after gastric bypass. Lean subjects were evaluated only at admission. Subjects were evaluated for hypothalamic activity in response to cold by functional magnetic resonance, whereas brown/beige adipose tissue activity was evaluated using a (F 18) fluorodeoxyglucose positron emisson tomography/computed tomography scan and real-time PCR measurement of signature genes.
Body mass reduction resulted in a significant increase in brown/beige adipose tissue activity in response to cold; however, no change in cold-induced hypothalamic activity was observed after body mass reduction. No correlation was found between brown/beige adipose tissue activation and hypothalamus activity in obese subjects or in lean controls.
In humans, the increase in brown/beige adipose tissue activity related to body mass reduction occurs independently of changes in hypothalamic activity as determined by functional magnetic resonance.
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