RESUMO
BACKGROUND: Obesity and particularly the metabolic syndrome, which is often associated with obesity, combine a major risk for type 2 diabetes and cardiovascular disease. Emerging evidence indicate obesity-associated subclinical inflammation primarily originating from adipose tissue as a common cause for type 2 diabetes and cardiovascular disease. However, a suitable and well-characterized mouse model to simultaneously study obesity-associated metabolic disorders and atherosclerosis is not available yet. Here we established and characterized a murine model combining diet-induced obesity and associated adipose tissue inflammation and metabolic deteriorations as well as atherosclerosis, hence reflecting the human situation of cardio-metabolic disease. METHODS: We compared a common high-fat diet with 0.15% cholesterol (HFC), and a high-fat, high-sucrose diet with 0.15% cholesterol (HFSC) fed to LDL receptor-deficient (LDLR-/-) mice. Insulin resistance, glucose tolerance, atherosclerotic lesion formation, hepatic lipid accumulation, and inflammatory gene expression in adipose tissue and liver were assessed. RESULTS: After 12-16 weeks, LDLR-/- mice fed HFSC or HFC developed significant diet-induced obesity, adipose tissue inflammation, insulin resistance, and impaired glucose tolerance compared to lean controls. Notably, HFSC-fed mice developed significantly higher adipose tissue inflammation in parallel with significantly elevated atherosclerotic lesion area compared to those on HFC. Moreover, LDLR-/- mice on HFSC showed increased insulin resistance and impaired glucose tolerance relative to those on HFC. After prolonged feeding (20 weeks), however, no significant differences in inflammatory and metabolic parameters as well as atherosclerotic lesion formation were detectable any more between LDLR-/- mice fed HFSC or HFC. CONCLUSION: The use of high sucrose rather than more complex carbohydrates in high-fat diets significantly accelerates development of obesity-driven metabolic complications and atherosclerotic plaque formation parallel to obesity-induced adipose tissue inflammation in LDLR-/- mice. Hence LDLR-/- mice fed high-fat high-sucrose cholesterol-enriched diet appear to be a suitable and time-saving animal model for cardio-metabolic disease. Moreover our results support the suggested interrelation between adipose tissue inflammation and atherosclerotic plaque formation.
Assuntos
Tecido Adiposo/metabolismo , Aterosclerose/sangue , Gorduras na Dieta/efeitos adversos , Sacarose Alimentar/efeitos adversos , Modelos Animais de Doenças , Receptores de LDL/deficiência , Tecido Adiposo/patologia , Animais , Aterosclerose/etiologia , Gorduras na Dieta/administração & dosagem , Sacarose Alimentar/administração & dosagem , Inflamação/sangue , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
T cells are crucial players in obesity-mediated adipose tissue inflammation. We hypothesized that osteopontin (OPN), an inflammatory protein with enhanced activity when proteolytically cleaved, affects the number of viable T cells in adipose tissue and assessed inhibition of the interaction between T cells and thrombin and matrix metalloproteinases-cleaved OPN using antibodies and postimmune sera. Gene expression of T cell markers in adipose tissue from wild-type (wt) and Spp1-/- (OPN deficient) mice was analyzed after 16 weeks of high fat diet (HFD) or low fat diet (LFD) feeding. CD3, CD8 and OPN gene expression in omental adipose tissue from individuals with obesity was measured. OPN-T cell interactions were assessed with a fluorescence-based adhesion assay and blocked with antibodies targeting OPN. Comparison of T cell gene expression in adipose tissue from wt and Spp1-/- mice showed that OPN affected the number of T cells while in humans, levels of OPN correlated with T cell markers in omental adipose tissue. The interaction between T cells and cleaved OPN was blocked by postimmune sera following OPN peptide vaccinations and with monoclonal antibodies. In conclusion, levels of OPN affected the number of T cells in obesity and antibodies against cleaved OPN antagonize OPN-T cell interactions.
Assuntos
Tecido Adiposo/imunologia , Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Obesidade/imunologia , Osteopontina/imunologia , Paniculite/imunologia , Proteólise , Linfócitos T/imunologia , Tecido Adiposo/patologia , Animais , Anticorpos/genética , Antígenos CD5/genética , Antígenos CD5/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Osteopontina/genética , Paniculite/genética , Paniculite/patologia , Linfócitos T/patologiaRESUMO
Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.
Assuntos
Epitopos/imunologia , Gelatinases/imunologia , Osteopontina/imunologia , Proteólise , Trombina/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Adesão Celular/genética , Adesão Celular/imunologia , Epitopos/genética , Gelatinases/genética , Células HEK293 , Humanos , Camundongos , Osteopontina/genética , Trombina/genéticaRESUMO
Obesity-induced chronic low-grade inflammation originates from adipose tissue and is crucial for obesity-driven metabolic deterioration, including insulin resistance and type 2 diabetes. Chronic inflammation may be a consequence of a failure to actively resolve inflammation and could result from a lack of local specialized proresolving lipid mediators (SPMs), such as resolvins and protectins, which derive from the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We assessed obesity-induced changes of n-3-derived SPMs in adipose tissue and the effects of dietary EPA/DHA thereon. Moreover, we treated obese mice with SPM precursors and investigated the effects on inflammation and metabolic dysregulation. Obesity significantly decreased DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA, resolvin D1 precursor) and protectin D1 (PD1) levels in murine adipose tissue. Dietary EPA/DHA treatment restored endogenous biosynthesis of n-3-derived lipid mediators in obesity while attenuating adipose tissue inflammation and improving insulin sensitivity. Notably, 17-HDHA treatment reduced adipose tissue expression of inflammatory cytokines, increased adiponectin expression, and improved glucose tolerance parallel to insulin sensitivity in obese mice. These findings indicate that impaired biosynthesis of certain SPM and SPM precursors, including 17-HDHA and PD1, contributes to adipose tissue inflammation in obesity and suggest 17-HDHA as a novel treatment option for obesity-associated complications.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/imunologia , Obesidade/tratamento farmacológico , Obesidade/imunologia , Tecido Adiposo/metabolismo , Animais , Western Blotting , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/uso terapêutico , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Chronic adipose tissue inflammation is a hallmark of obesity, triggering the development of associated pathologies, particularly type 2 diabetes. Long-chain n-3 PUFAs reduce cardiovascular events and exert well-established antiinflammatory effects, but their effects on human adipose tissue inflammation are unknown. OBJECTIVE: We investigated whether n-3 PUFAs reduce adipose tissue inflammation in severely obese nondiabetic patients. DESIGN: We treated 55 severely obese nondiabetic patients, scheduled to undergo elective bariatric surgery, with 3.36 g long-chain n-3 PUFAs/d (EPA, DHA) or an equivalent amount of butterfat as control, for 8 wk, in a randomized open-label controlled clinical trial. The primary efficacy measure was inflammatory gene expression in visceral and subcutaneous adipose tissue samples (subcutaneous adipose tissue and visceral adipose tissue), collected during surgery after the intervention. Secondary efficacy variables were adipose tissue production of antiinflammatory n-3 PUFA-derived eicosanoids, plasma concentrations of inflammatory markers, metabolic control, and the effect of the Pro12Ala PPARG polymorphism on the treatment response. RESULTS: Treatment with n-3 PUFAs, which was well tolerated, decreased the gene expression of most analyzed inflammatory genes in subcutaneous adipose tissue (P < 0.05) and increased production of antiinflammatory eicosanoids in visceral adipose tissue and subcutaneous adipose tissue (P < 0.05). In comparison with control subjects who received butterfat, circulating interleukin-6 and triglyceride concentrations decreased significantly in the n-3 PUFA group (P = 0.04 and P = 0.03, respectively). The Pro12Ala polymorphism affected the serum cholesterol response to n-3 PUFA treatment. CONCLUSIONS: Treatment with long-chain n-3 PUFAs favorably modulated adipose tissue and systemic inflammation in severely obese nondiabetic patients and improved lipid metabolism. These effects may be beneficial in the long-term treatment of obesity. This trial was registered at clinicaltrials.gov as NCT00760760.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Ácidos Graxos Ômega-3/administração & dosagem , Inflamação/tratamento farmacológico , Obesidade/tratamento farmacológico , Tecido Adiposo/fisiologia , Adulto , Idoso , DNA/química , DNA/genética , Ácidos Graxos/sangue , Feminino , Humanos , Imuno-Histoquímica , Inflamação/sangue , Inflamação/genética , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/genética , PPAR gama/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Triglicerídeos/sangue , Adulto JovemRESUMO
OBJECTIVE: Obesity is associated with a state of chronic low-grade inflammation mediated by immune cells that are primarily located to adipose tissue and liver. The chronic inflammatory response appears to underlie obesity-induced metabolic deterioration including insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine, the expression of which is strongly upregulated in adipose tissue and liver upon obesity. Here, we studied OPN effects in obesity-induced inflammation and insulin resistance by targeting OPN action in vivo. RESEARCH DESIGN AND METHODS: C57BL/6J mice were fed a high-fat diet to induce obesity and were then intravenously treated with an OPN-neutralizing or control antibody. Insulin sensitivity and inflammatory alterations in adipose tissue and liver were assessed. RESULTS: Interference with OPN action by a neutralizing antibody for 5 days significantly improved insulin sensitivity in diet-induced obese mice. Anti-OPN treatment attenuated liver and adipose tissue macrophage infiltration and inflammatory gene expression by increasing macrophage apoptosis and significantly reducing c-Jun NH(2)-terminal kinase activation. Moreover, we report OPN as a novel negative regulator for the activation of hepatic signal transducer and activator of transcription 3 (STAT3), which is essential for glucose homeostasis and insulin sensitivity. Consequently, OPN neutralization decreased expression of hepatic gluconeogenic markers, which are targets of STAT3-mediated downregulation. CONCLUSIONS: These findings demonstrate that antibody-mediated neutralization of OPN action significantly reduces insulin resistance in obesity. OPN neutralization partially decreases obesity-associated inflammation in adipose tissue and liver and reverses signal transduction related to insulin resistance and glucose homeostasis. Hence, targeting OPN could provide a novel approach for the treatment of obesity-related metabolic disorders.
Assuntos
Inflamação/prevenção & controle , Resistência à Insulina/fisiologia , Obesidade/fisiopatologia , Osteopontina/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Especificidade de Anticorpos , Glicemia/metabolismo , Gorduras na Dieta/farmacologia , Citometria de Fluxo , Imunoglobulina G/farmacologia , Marcação In Situ das Extremidades Cortadas , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Insulina/fisiologia , Lipídeos/sangue , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/imunologia , Obesidade/patologia , Triglicerídeos/metabolismoRESUMO
Recently, we showed that holo HDL particle uptake and resecretion occur in physiologically relevant cell lines and that HDL uptake is mediated by scavenger receptor class B type I (SR-BI). Furthermore, we established that HDL resecretion is accompanied by [(3)H]cholesterol efflux. This study shows that HDL uptake and resecretion occur even when LDL uptake and cholesterol trafficking are disturbed. First, we used a set of inhibitors that block cholesterol transport out of the lysosome: chloroquine, imipramine, U18666A, and monensin. In all cases, HDL retroendocytosis occurred and HDL resecretion mediated [(3)H]cholesterol efflux, although to a lesser extent. Second, cell lines carrying somatic mutations in intracellular cholesterol transport were used: CHO 2-2 and CHO 3-6 cells accumulated LDL-derived lipid in the lysosome but showed all components of HDL retroendocytosis. SR-BI overexpression increased HDL uptake and resecretion and [(3)H]cholesterol efflux in these mutant cells. Finally, we used Niemann-Pick type C (NPC) patient fibroblast cells, which carry a defect in cholesterol transfer out of the lysosome. NPC fibroblast cells accumulate cholesterol in the lysosome as a result of a mutation in the NPC1 gene. Despite disturbed intracellular cholesterol transfer, NPC fibroblast cells exhibited HDL retroendocytosis and [(3)H]cholesterol efflux via HDL resecretion, although to a lesser extent. Thus, [(3)H]cholesterol efflux via HDL resecretion is independent of the cholesterol uptake pathway via the LDL receptor and may be an alternative way to remove excess cholesterol.
Assuntos
Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Androstenos/farmacologia , Animais , Transporte Biológico , Células CHO , Proteínas de Transporte/metabolismo , Cloroquina/farmacologia , Cricetinae , Cricetulus , Endocitose , Fibroblastos/metabolismo , Humanos , Imipramina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Monensin/farmacologia , Proteína C1 de Niemann-Pick , Receptores Depuradores Classe B/metabolismoRESUMO
The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.