RESUMO
A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene (NPC1) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice (Npc1+/- ) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared with homozygous normal mice (Npc1+/+ ) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/- mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1+/- mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/- mice had significantly decreased amounts of phosphorylated hormone-sensitive lipase with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/- fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Interação Gene-Ambiente , Redes e Vias Metabólicas/genética , Proteínas/fisiologia , Aumento de Peso/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína C1 de Niemann-Pick , Proteínas/genéticaRESUMO
ABCA1 exports excess cholesterol from cells to apoA-I and is essential for HDL synthesis. Genetic studies have shown that ABCA1 protects against cardiovascular disease. We have previously shown that the interaction of apoA-I with ABCA1 activates signaling molecule Janus kinase 2 (JAK2), which optimizes the cholesterol efflux activity of ABCA1. ABCA1-mediated activation of JAK2 also activates signal transducer and activator of transcription 3 (STAT3), which significantly attenuates proinflammatory cytokine expression in macrophages. To determine the mechanisms of the anti-inflammatory effects of apoA-I/ABCA1 interaction, we identified two special ABCA1 mutants, one with normal STAT3-activating capacity but lacking cholesterol efflux ability and the other with normal cholesterol efflux ability but lacking STAT3-activating capacity. We showed that activation of STAT3 by the interaction of apoA-I/ABCA1 without cholesterol efflux could significantly decrease proinflammatory cytokine expression in macrophages. Mechanistic studies showed that the anti-inflammatory effect of the apoA-I/ABCA1/STAT3 pathway is suppressor of cytokine signaling 3 dependent. Moreover, we showed that apoA-I/ABCA1-mediated cholesterol efflux without STAT3 activation can also reduce proinflammatory cytokine expression in macrophages. These findings suggest that the interaction of apoA-I/ABCA1 activates cholesterol efflux and STAT3 branch pathways to synergistically suppress inflammation in macrophages.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Macrófagos Peritoneais/metabolismo , Fator de Transcrição STAT3/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Macrófagos Peritoneais/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
Low-grade chronic inflammation plays an important role in the pathogenesis of obesity-induced insulin resistance. ABCA1 is essential for reverse cholesterol transport and HDL synthesis, and protects against macrophage inflammation. In the present study, the effects of ABCA1 deficiency in hematopoietic cells on diet-induced inflammation and insulin resistance were tested in vivo using bone marrow transplanted (BMT)-WT and BMT-ABCA1(-/-) mice. When challenged with a high-fat high-carbohydrate diabetogenic diet with added cholesterol (HFHSC), BMT-ABCA1(-/-) mice displayed enhanced insulin resistance and impaired glucose tolerance as compared with BMT-WT mice. The worsened insulin resistance and impaired glucose tolerance in BMT-ABCA1(-/-) mice were accompanied by increased macrophage accumulation and inflammation in adipose tissue and liver. Moreover, BMT-ABCA1(-/-) mice had significantly higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis when challenged with the HFHSC diet. In vitro studies indicated that macrophages from ABCA1(-/-) mice showed significantly increased inflammatory responses induced by saturated fatty acids. Taken together, these studies point to an important role for hematopoietic ABCA1 in modulating a feed-forward mechanism in obesity such that inflamed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of inflammation and associated disease processes.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Resistência à Insulina/fisiologia , Transportador 1 de Cassete de Ligação de ATP/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Tecido Adiposo/metabolismo , Animais , Colesterol/metabolismo , Colesterol/farmacologia , Dieta Hiperlipídica , Intolerância à Glucose/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Fígado/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Monócitos/metabolismo , Monócitos/patologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Obesidade/patologia , Receptores de LDL/metabolismoRESUMO
In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1(-/-) cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Restrição Calórica , Colesterol/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Lipoproteínas/genética , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genéticaRESUMO
Inflammatory pathways are central mechanisms in diabetic kidney disease (DKD). Serum amyloid A (SAA) is increased by chronic inflammation, but SAA has not been previously evaluated as a potential DKD mediator. The aims of this study were to determine whether SAA is increased in human DKD and corresponding mouse models and to assess effects of SAA on podocyte inflammatory responses. SAA was increased in the plasma of people with DKD characterized by overt proteinuria and inversely correlated with estimated glomerular filtration rate (creatinine-based CKD-EPI). SAA was also elevated in plasma of diabetic mouse models including type 1 diabetes (streptozotocin/C57BL/6) and type 2 diabetes (BTBR-ob/ob). SAA mRNA (Nephromine) was increased in human DKD compared with non-diabetic and/or glomerular disease controls (glomerular fold change 1.5, P=0.017; tubulointerstitium fold change 1.4, P=0.021). The kidneys of both diabetic mouse models also demonstrated increased SAA mRNA (quantitative real-time PCR) expression compared with non-diabetic controls (type 1 diabetes fold change 2.9; type 2 diabetes fold change 42.5, P=0.009; interaction by model P=0.57). Humans with DKD and the diabetic mouse models exhibited extensive SAA protein deposition in the glomeruli and tubulointerstitium in similar patterns by immunohistochemistry. SAA localized within podocytes of diabetic mice. Podocytes exposed to advanced glycation end products, metabolic mediators of inflammation in diabetes, increased expression of SAA mRNA (fold change 15.3, P=0.004) and protein (fold change 38.4, P=0.014). Podocytes exposed to exogenous SAA increased NF-κB activity, and pathway array analysis revealed upregulation of mRNA for NF-κB-dependent targets comprising numerous inflammatory mediators, including SAA itself (fold change 17.0, P=0.006). Inhibition of NF-κB reduced these pro-inflammatory responses. In conclusion, SAA is increased in the blood and produced in the kidneys of people with DKD and corresponding diabetic mouse models. Podocytes are likely to be key responder cells to SAA-induced inflammation in the diabetic kidney. SAA is a compelling candidate for DKD therapeutic and biomarker discovery.
Assuntos
Nefropatias Diabéticas/sangue , Inflamação/sangue , Podócitos/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Antioxidantes/farmacologia , Células Cultivadas , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/sangue , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Podócitos/efeitos dos fármacos , Pirrolidinas/farmacologia , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/farmacologia , Tiocarbamatos/farmacologiaRESUMO
Diabetic nephropathy (DN) is a major complication of diabetes and the leading cause of end-stage renal disease. DN is characterized by changes in kidney structure and function but the underlying genetic and molecular factors are poorly understood. We used a mouse diversity panel to explore the genetic basis of DN traits in mice carrying the Ins2 Akita mutation. Twenty-eight Akita strains were generated by breeding this panel to DBA/2.Akita mice. Male F1 diabetic and nondiabetic littermates were evaluated for DN-related traits. Urine albumin-to-creatinine ratios (ACRs), volume and cystatin C as well as blood urea nitrogen and lipoprotein levels varied significantly among the diabetic strains. For most Akita strains, ACR values increased 2- to 6-fold over euglycemic control values. However, six strains exhibited changes in ACR exceeding 10-fold with two strains (NOD/ShiLt and CBA) showing 50- to 83- fold increases. These increases are larger than previously reported among available DN mouse models establishing these strains as useful for additional studies of renal function. ACRs correlated with cystatin C (P = 0.0286), a measure of hyperfiltration and an interstitial tubular marker associated with DN onset in humans suggesting that tubule damage as well as podocyte-stress contributed to reduced kidney function assessed by ACR. Although large changes were seen for ACRs, severe nephropathology was absent. However, glomerular hypertrophy and collagen IV content were found to vary significantly among strains suggesting a genetic basis for early onset features of DN. Our results define the range of DN phenotypes that occur among common inbred strains of mice.
RESUMO
Metabolic diseases such as obesity and atherosclerosis result from complex interactions between environmental factors and genetic variants. A panel of chromosome substitution strains (CSSs) was developed to characterize genetic and dietary factors contributing to metabolic diseases and other biological traits and biomedical conditions. Our goal here was to identify quantitative trait loci (QTLs) contributing to obesity, energy expenditure, and atherosclerosis. Parental strains C57BL/6 and A/J together with a panel of 21 CSSs derived from these progenitors were subjected to chronic feeding of rodent chow and atherosclerotic (females) or diabetogenic (males) test diets, and evaluated for a variety of metabolic phenotypes including several traits unique to this report, namely fat pad weights, energy balance, and atherosclerosis. A total of 297 QTLs across 35 traits were discovered, two of which provided significant protection from atherosclerosis, and several dozen QTLs modulated body weight, body composition, and circulating lipid levels in females and males. While several QTLs confirmed previous reports, most QTLs were novel. Finally, we applied the CSS quantitative genetic approach to energy balance, and identified three novel QTLs controlling energy expenditure and one QTL modulating food intake. Overall, we identified many new QTLs and phenotyped several novel traits in this mouse model of diet-induced metabolic diseases.
Assuntos
Aterosclerose/genética , Metabolismo Energético/genética , Obesidade/genética , Animais , Composição Corporal , Peso Corporal , Cromossomos de Mamíferos/genética , Dieta Hiperlipídica/efeitos adversos , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Locos de Características QuantitativasRESUMO
Macrophage metalloelastase, a matrix metallopeptidase (MMP12) predominantly expressed by mature tissue macrophages, is implicated in pathological processes. However, physiological functions for MMP12 have not been described. Because mRNA levels for the enzyme increase markedly in adipose tissue of obese mice, we investigated the role of MMP12 in adipose tissue expansion and insulin resistance. In humans, MMP12 expression correlated positively and significantly with insulin resistance, TNF-α expression, and the number of CD14(+)CD206(+) macrophages in adipose tissue. MMP12 was the most abundant matrix metallopeptidase detected by proteomic analysis of conditioned medium of M2 macrophages and dendritic cells. In contrast, it was detected only at low levels in bone marrow derived macrophages and M1 macrophages. When mice received a high-fat diet, adipose tissue mass increased and CD11b(+)F4/80(+)CD11c(-) macrophages accumulated to a greater extent in MMP12-deficient (Mmp12(-/-)) mice than in wild-type mice (Mmp12(+/+)). Despite being markedly more obese, fat-fed Mmp12(-/-) mice were more insulin sensitive than fat-fed Mmp12(+/+) mice. Expression of inducible nitric oxide synthase (Nos2) by Mmp12(-/-) macrophages was significantly impaired both in vivo and in vitro, suggesting that MMP12 might mediate nitric oxide production during inflammation. We propose that MMP12 acts as a double-edged sword by promoting insulin resistance while combatting adipose tissue expansion.
Assuntos
Tecido Adiposo/enzimologia , Insulina/metabolismo , Macrófagos/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Obesidade/enzimologia , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Adulto , Animais , Feminino , Humanos , Técnicas In Vitro , Resistência à Insulina , Macrófagos/metabolismo , Masculino , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/genética , Obesidade/metabolismo , Adulto JovemRESUMO
OBJECTIVE: Factors responsible for the variability in outcomes after lower extremity vein bypass grafting (LEVBG) are poorly understood. Recent evidence has suggested that a single nucleotide polymorphism (SNP) in the promoter region of the p27(Kip1) gene, a cell-cycle regulator, is associated with coronary in-stent restenosis. We hypothesized an association with vein graft patency. METHODS: This was a retrospective genetic association study nested within a prospective cohort of 204 patients from three referral centers undergoing LEVBG for claudication or critical ischemia. The main outcome measure was primary vein graft patency. RESULTS: All patients were followed up for a minimum of 1 year with duplex graft surveillance (median follow-up, 893 days; interquartile range, 539-1315). Genomic DNA was isolated and SNP analysis for the p27(Kip1)-838C>A variants was performed. Allele frequencies were correlated with graft outcome using survival analysis and Cox proportional hazards modeling. The p27(Kip1)-838C>A allele frequencies observed were CA, 53%; CC, 30%; and AA, 17%, satisfying Hardy-Weinberg equilibrium. Race (P = .025) and history of coronary artery disease (P = .027) were different across the genotypes; all other baseline variables were similar. Primary graft patency was greater among patients with the -838AA genotype (75% AA vs 55% CA/CC at 3 years; P = .029). In a Cox proportional hazards model including age, sex, race, diabetes, critical limb ischemia, redo (vs primary) bypass, vein type, and baseline C-reactive protein level, the p27(Kip1)-838AA genotype was significantly associated with higher graft patency (hazard ratio for failure, 0.4; 95% confidence interval, 0.17-0.93). Genotype was also associated with early (0-1 month) changes in graft lumen diameter by ultrasound imaging. CONCLUSIONS: These data suggest that the p27(Kip1)-838C>A SNP is associated with LEVBG patency and, together with previous reports, underscore a central role for p27(Kip1) in the generic response to vascular injury.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Oclusão de Enxerto Vascular/genética , Claudicação Intermitente/cirurgia , Isquemia/cirurgia , Extremidade Inferior/irrigação sanguínea , Doença Arterial Periférica/cirurgia , Polimorfismo de Nucleotídeo Único , Enxerto Vascular/efeitos adversos , Grau de Desobstrução Vascular/genética , Veias/transplante , Idoso , Estado Terminal , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Oclusão de Enxerto Vascular/diagnóstico por imagem , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Claudicação Intermitente/genética , Claudicação Intermitente/fisiopatologia , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Doença Arterial Periférica/genética , Doença Arterial Periférica/fisiopatologia , Fenótipo , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia Doppler Dupla , Estados Unidos , Veias/diagnóstico por imagem , Veias/fisiopatologiaRESUMO
BACKGROUND: Overfeeding amino acids (AAs) increases cellular exposure to advanced glycation end-products (AGEs), a mechanism for protein intake to worsen diabetic kidney disease (DKD). This study assessed receptor for AGE (RAGE)-mediated apoptosis and inflammation in glomerular cells exposed to metabolic stressors characteristic of high-protein diets and/or diabetes in vitro with proof-of-concept appraisal in vivo. METHODS: Mouse podocytes and mesangial cells were cultured under control and metabolic stressor conditions: (i) no addition; (ii) increased AAs (4-6-fold>control); (iii) high glucose (HG, 30.5 mM); (iv) AA/HG combination; (v) AGE-bovine serum albumin (AGE-BSA, 300 µg/mL); (vi) BSA (300 µg/mL). RAGE was inhibited by blocking antibody. Diabetic (streptozotocin) and nondiabetic mice (C57BL/6J) consumed diets with protein calories of 20 or 40% (high) for 20 weeks. People with DKD and controls provided 24-h urine samples. RESULTS: In podocytes and mesangial cells, apoptosis (caspase 3/7 activity and TUNEL) increased in all metabolic stressor conditions. Both inflammatory mediator expression (real-time reverse transcriptase-polymerase chain reaction: serum amyloid A, caspase-4, inducible nitric oxide synthase, and monocyte chemotactic protein-1) and RAGE (immunostaining) also increased. RAGE inhibition prevented apoptosis and inflammation in podocytes. Among mice fed high protein, podocyte number (WT-1 immunostaining) decreased in the diabetic group, and only these diabetic mice developed albuminuria. Protein intake (urea nitrogen) correlated with AGE excretion (carboxymethyllysine) in people with DKD and controls. CONCLUSIONS: High-protein diet and/or diabetes-like conditions increased glomerular cell death and inflammation, responses mediated by RAGEs in podocytes. The concept that high-protein diets exacerbate early indicators of DKD is supported by data from mice and people.
Assuntos
Apoptose , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/etiologia , Dieta , Proteínas Alimentares/farmacologia , Inflamação/etiologia , Animais , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Produtos Finais de Glicação Avançada/genética , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Masculino , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/metabolismo , Podócitos/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.
Assuntos
Camundongos Endogâmicos/genética , Animais , Bases de Dados Genéticas , CamundongosRESUMO
Endothelial activation, which is characterized by upregulation of cellular adhesion molecules and pro-inflammatory chemokines and cytokines, and consequent monocyte recruitment to the arterial intima are etiologic factors in atherosclerosis. Redox-active transition metal ions, such as copper and iron, may play an important role in endothelial activation by stimulating redox-sensitive cell signaling pathways. We have shown previously that copper chelation by tetrathiomolybdate (TTM) inhibits LPS-induced acute inflammatory responses in vivo. Here, we investigated whether TTM can inhibit atherosclerotic lesion development in apolipoprotein E-deficient (apoE-/-) mice. We found that 10-week treatment of apoE-/- mice with TTM (33-66 ppm in the diet) reduced serum levels of the copper-containing protein, ceruloplasmin, by 47%, and serum iron by 26%. Tissue levels of "bioavailable" copper, assessed by the copper-to-molybdenum ratio, decreased by 80% in aorta and heart, whereas iron levels of these tissues were not affected by TTM treatment. Furthermore, TTM significantly attenuated atherosclerotic lesion development in whole aorta by 25% and descending aorta by 45% compared to non-TTM treated apoE-/- mice. This anti-atherogenic effect of TTM was accompanied by several anti-inflammatory effects, i.e., significantly decreased serum levels of soluble vascular cell and intercellular adhesion molecules (VCAM-1 and ICAM-1); reduced aortic gene expression of VCAM-1, ICAM-1, monocyte chemotactic protein-1, and pro-inflammatory cytokines; and significantly less aortic accumulation of M1 type macrophages. In contrast, serum levels of oxidized LDL were not reduced by TTM. These data indicate that TTM inhibits atherosclerosis in apoE-/- mice by reducing bioavailable copper and vascular inflammation, not by altering iron homeostasis or reducing oxidative stress.
Assuntos
Anti-Inflamatórios/farmacologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Quelantes/farmacologia , Cobre/metabolismo , Inflamação/prevenção & controle , Molibdênio/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Biomarcadores/sangue , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Ceruloplasmina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Ferro/sangue , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismoRESUMO
Laminin-ß2 (LAMB2) is a critical component of the glomerular basement membrane as content of LAMB2 in part determines glomerular barrier permeability. Previously, we reported that high concentrations of glucose reduce expression of this laminin subunit at the translational level. The present studies were undertaken to further define systems that control Lamb2 translation and the effect of high glucose on those systems. Complementary studies were performed using in vitro differentiation of cultured podocytes and mesangial cells exposed to normal and elevated concentrations of glucose, and tissues from control and diabetic rats. Together, these studies provide evidence for regulation of Lamb2 translation by IMP2, an RNA binding protein that targets Lamb2 mRNA to the actin cytoskeleton. Expression of Imp2 itself is regulated by the transcription factor HMGA2, which in turn is regulated by the microRNA let-7b. Elevated concentrations of glucose increase let-7b, which reduces HMGA2 expression, in turn reducing IMP2 and LAMB2. Correlative changes in kidney tissues from control and streptozotocin-induced diabetic rats suggest these control mechanisms are operative in vivo and may contribute to proteinuria in diabetic nephropathy. To our knowledge, this is the first time that translation of Lamb2 mRNA has been linked to the actin cytoskeleton, as well as to specific RNA-binding proteins. These translational control points may provide new targets for therapy in proteinuric disorders such as diabetic nephropathy where LAMB2 levels are reduced.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Glomérulos Renais/metabolismo , Laminina/genética , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Diabetes Mellitus Experimental/genética , Humanos , Glomérulos Renais/citologia , Laminina/metabolismo , Masculino , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-DawleyRESUMO
Lymphotoxin-α (LTα) is secreted by lymphocytes and acts through tumor necrosis factor-α receptors and the LTß receptor. Our goals were to determine whether LT has a role in obesity and investigate whether LT contributes to the link between obesity and adipose tissue lymphocyte accumulation. LT deficient (LT(-/-)) and wild-type (WT) mice were fed standard pelleted rodent chow or a high-fat/high-sucrose diet (HFHS) for 13 wk. Body weight, body composition, and food intake were measured. Glucose tolerance was assessed. Systemic and adipose tissue inflammatory statuses were evaluated by quantifying plasma adipokine levels and tissue macrophage and T cell-specific gene expression in abdominal fat. LT(-/-) mice were smaller (20%) and leaner (25%) than WT controls after 13 wk of HFHS diet feeding. LT(-/-) mice showed improved glucose tolerance, suggesting that, in WT mice, LT may impair glucose metabolism. Surprisingly, adipose tissue from rodent chow- and HFHS-fed LT(-/-) mice exhibited increased T lymphocyte and macrophage infiltration compared with WT mice. Despite the fact that LT(-/-) mice exhibited an enhanced inflammatory status at the systemic and tissue level even when fed rodent chow, they were protected from enhanced diet-induced obesity and insulin resistance. Thus, LT contributes to body weight and adiposity and is required to modulate the accumulation of immune cells in adipose tissue.
Assuntos
Tecido Adiposo Branco/imunologia , Linfotoxina-alfa/metabolismo , Macrófagos/imunologia , Obesidade/imunologia , Linfócitos T/imunologia , Adipocinas/sangue , Adiposidade , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Expressão Gênica , Regulação da Expressão Gênica , Intolerância à Glucose/sangue , Intolerância à Glucose/etiologia , Intolerância à Glucose/imunologia , Intolerância à Glucose/metabolismo , Resistência à Insulina , Linfotoxina-alfa/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/etiologia , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Linfócitos T/metabolismo , Aumento de PesoRESUMO
The prevalence of obesity has reached epidemic proportions and is associated with several co-morbid conditions including diabetes, dyslipidemia, cancer, atherosclerosis and gallstones. Obesity is associated with low systemic inflammation and an accumulation of adipose tissue macrophages (ATMs) that are thought to modulate insulin resistance. ATMs may also modulate adipocyte metabolism and take up lipids released during adipocyte lipolysis and cell death. We suggest that high levels of free cholesterol residing in adipocytes are released during these processes and contribute to ATM activation and accumulation during obesity and caloric restriction. Db/db mice were studied for extent of adipose tissue inflammation under feeding conditions of ad libitum (AL) and caloric restriction (CR). The major finding was a marked elevation in epididymal adipose ABCG1 mRNA levels with obesity and CR (6-fold and 16-fold, respectively) over that seen for lean wild-type mice. ABCG1 protein was also elevated for CR as compared to AL adipose tissue. ABCG1 is likely produced by cholesterol loaded ATMs since this gene is not highly expressed in adipocytes and ABCG1 expression is sterol mediated. Our data supports the concept that metabolic changes in adipocytes due to demand lipolysis and cell death lead to cholesterol loading of ATMs. Based on finding cholesterol-loaded peritoneal leukocytes with elevated levels of ABCG1 in CR as compared to AL mice, we suggest that pathways for cholesterol trafficking out of adipose tissue involve ATM egress as well as ABCG1 mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Gordura Abdominal/metabolismo , Expressão Gênica , Lipoproteínas/metabolismo , Obesidade/metabolismo , Redução de Peso , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Gordura Abdominal/patologia , Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Restrição Calórica , Movimento Celular , Colesterol/metabolismo , Feminino , Lipólise , Lipoproteínas/genética , Macrófagos/enzimologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/dietoterapia , Obesidade/fisiopatologia , Triglicerídeos/metabolismoRESUMO
BACKGROUND: S100A9 is constitutively expressed in neutrophils, dendritic cells, and monocytes; is associated with acute and chronic inflammatory conditions; and is implicated in obesity and cardiovascular disease in humans. Most of the constitutively secreted S100A9 is derived from myeloid cells. A recent report demonstrated that mice deficient in S100A9 exhibit reduced atherosclerosis compared with controls and suggested that this effect was due in large part to loss of S100A9 in bone marrow-derived cells. METHODS AND RESULTS: To directly investigate the role of bone marrow-derived S100A9 in atherosclerosis and insulin resistance in mice, low-density lipoprotein receptor-deficient, S100A9-deficient bone marrow chimeras were generated. Neither atherosclerosis nor insulin resistance was reduced in S100A9-deficient chimeras fed a diet rich in fat and carbohydrates. To investigate the reason for this lack of effect, myeloid cells were isolated from the peritoneal cavity or bone marrow. S100A9-deficient neutrophils exhibited a reduced secretion of cytokines in response to toll-like receptor-4 stimulation. In striking contrast, S100A9-deficient dendritic cells showed an exacerbated release of cytokines after toll-like receptor stimulation. Macrophages rapidly lost S100A9 expression during maturation; hence, S100A9 deficiency did not affect the inflammatory status of macrophages. CONCLUSIONS: S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells. The effect of S100A9 deficiency on atherosclerosis and other inflammatory diseases is therefore predicted to depend on the relative contribution of these cell types at different stages of disease progression. Furthermore, S100A9 expression in nonmyeloid cells is likely to contribute to atherosclerosis.
Assuntos
Tecido Adiposo/patologia , Aterosclerose/etiologia , Calgranulina B/fisiologia , Células Dendríticas/fisiologia , Inflamação/etiologia , Macrófagos/fisiologia , Neutrófilos/fisiologia , Animais , Calgranulina A/fisiologia , Resistência à Insulina , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de LDL/fisiologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologiaRESUMO
OBJECTIVE: Nicotinic acetylcholine receptor α1 (nAChRα1) was recently identified as a functional cell receptor for urokinase, a potent atherogenic molecule. Here, we test the hypothesis that nAChRα1 plays a role in the pathogenesis of atherosclerosis. METHODS: Apolipoprotein E-deficient mice were initially fed a Western diet for 8 wks. Plasmid DNA encoding scramble RNA (pscr) or siRNA (psir2) for nAChRα1 was injected into the mice (n=16) using an aortic hydrodynamic gene transfer protocol. Four mice from each group were sacrificed 7 days after the DNA injection to confirm the nAChRα1 gene silencing. The remaining mice continued on a Western diet for an additional 16 wks. RESULTS: The nAChRα1 was up-regulated in aortic atherosclerotic lesions. A 78% knockdown of the nAChRα1 gene resulted in remarkably less severe aortic plaque growth and neovascularization at 16 wks (both P<0.05). In addition, significantly fewer macrophages (60% less) and myofibroblasts (80% less) presented in the atherosclerotic lesion of the psir2-treated mice. The protective mechanisms of the nAChRα1 knockdown may involve up-regulating interferon-γ/Y box protein-1 activity and down-regulating transforming growth factor-ß expression. CONCLUSIONS: The nAChRα1 gene plays a significant role at the artery wall, and reducing its expression decreases aortic plaque development.
Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Receptores Nicotínicos/genética , Actinas/biossíntese , Animais , Aorta/metabolismo , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Feminino , Inativação Gênica , Interferon gama/biossíntese , Macrófagos/metabolismo , Camundongos , Miofibroblastos/metabolismo , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Regulação para CimaRESUMO
OBJECTIVE: Insulin receptor (IR) translocates to the nucleus, but its recruitment to gene loci has not been demonstrated. Here, we tested the hypothesis that IR and its downstream mitogenic transducers are corecruited to two prototypic insulin-inducible genes: early growth response 1 (egr-1), involved in mitogenic response, and glucokinase (Gck), encoding a key metabolic enzyme. RESEARCH DESIGN AND METHODS: We used RNA and chromatin from insulin-treated rat hepatic tumor cell line expressing human insulin receptor (HTC-IR) and livers from lean and insulin-resistant ob/ob glucose-fed mice in quantitative RT-PCR and chromatin immunoprecipitation studies to determine gene expression levels and associated recruitment of RNA polymerase II (Pol II), insulin receptor, and cognate signaling proteins to gene loci, respectively. RESULTS: Insulin-induced egr-1 mRNA in HTC-IR cells was associated with corecruitment of IR signaling cascade (IR, SOS, Grb2, B-Raf, MEK, and ERK) to this gene. Recruitment profiles of phosphorylated IR, B-Raf, MEK, and Erk along egr-1 transcribed region were similar to those of elongating Pol II. Glucose-feeding increased Gck mRNA expression in livers of lean but not ob/ob mice. In lean mice, there was glucose feeding-induced recruitment of IR and its transducers to Gck gene synchronized with elongating Pol II. In sharp contrast, in glucose-fed ob/ob mice, the Gck recruitment patterns of active MEK/Erk, IR, and Pol II were asynchronous. CONCLUSIONS: IR and its signal transducers recruited to genes coupled to elongating Pol II may play a role in maintaining productive mRNA synthesis of target genes. These studies suggest a possibility that impaired Pol II processivity along genes bearing aberrant levels of IR/signal transducers is a previously unrecognized facet of insulin resistance.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glucoquinase/genética , Insulina/farmacologia , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologia , Ração Animal , Animais , DNA Complementar/genética , Quinases do Centro Germinativo , Glucose/farmacologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/genética , Proteínas Serina-Treonina Quinases/genética , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Magreza/genética , Transcrição GênicaRESUMO
Accumulation of cholesterol in arterial macrophages may contribute to diabetes-accelerated atherosclerotic cardiovascular disease. The ATP-binding cassette transporter ABCA1 is a cardioprotective membrane protein that mediates cholesterol export from macrophages. Factors elevated in diabetes, such as reactive carbonyls and free fatty acids, destabilize ABCA1 protein in cultured macrophages, raising the possibility that impaired ABCA1 plays an atherogenic role in diabetes. We therefore examined the modulation of ABCA1 in two mouse models of diabetes. We isolated peritoneal macrophages, livers, kidneys, and brains from type 1 non-obese diabetic (NOD) mice and mice made diabetic by viral-induced autoimmune destruction of pancreatic beta-cells, and we measured ABCA1 protein and mRNA levels and cholesterol contents. ABCA1 protein levels and cholesterol export activity were reduced by 40-44% (P<0.01) in peritoneal macrophages and protein levels by 48% (P<0.001) in kidneys in diabetic NOD mice compared with nondiabetic animals, even though ABCA1 mRNA levels were not significantly different. A similar selective reduction in ABCA1 protein was found in peritoneal macrophages (33%, P<0.05) and kidneys (35%, P<0.05) from the viral-induced diabetic mice. In liver and brain, however, diabetes had no effect or slightly increased ABCA1 protein and mRNA levels. The reduced ABCA1 in macrophages and kidneys was associated with increased cholesterol content. Impaired ABCA1-mediated cholesterol export could therefore contribute to the increased atherosclerosis and nephropathy associated with diabetes.