RESUMO
BACKGROUND: Lipolysis regulates energy homeostasis through the hydrolysis of intracellular triglycerides and the release of fatty acids for use as energy substrates or lipid mediators in cellular processes. Genes encoding proteins that regulate energy homeostasis through lipolysis are thus likely to play an important role in determining susceptibility to metabolic disorders. METHODS: We sequenced 12 lipolytic-pathway genes in Old Order Amish participants whose fasting serum triglyceride levels were at the extremes of the distribution and identified a novel 19-bp frameshift deletion in exon 9 of LIPE, encoding hormone-sensitive lipase (HSL), a key enzyme for lipolysis. We genotyped the deletion in DNA from 2738 Amish participants and performed association analyses to determine the effects of the deletion on metabolic traits. We also obtained biopsy specimens of abdominal subcutaneous adipose tissue from 2 study participants who were homozygous for the deletion (DD genotype), 10 who were heterozygous (ID genotype), and 7 who were noncarriers (II genotype) for assessment of adipose histologic characteristics, lipolysis, enzyme activity, cytokine release, and messenger RNA (mRNA) and protein levels. RESULTS: Carriers of the mutation had dyslipidemia, hepatic steatosis, systemic insulin resistance, and diabetes. In adipose tissue from study participants with the DD genotype, the mutation resulted in the absence of HSL protein, small adipocytes, impaired lipolysis, insulin resistance, and inflammation. Transcription factors responsive to peroxisome-proliferator-activated receptor γ (PPAR-γ) and downstream target genes were down-regulated in adipose tissue from participants with the DD genotype, altering the regulation of pathways influencing adipogenesis, insulin sensitivity, and lipid metabolism. CONCLUSIONS: These findings indicate the physiological significance of HSL in adipocyte function and the regulation of systemic lipid and glucose homeostasis and underscore the severe metabolic consequences of impaired lipolysis. (Funded by the National Institutes of Health and others).
Assuntos
Diabetes Mellitus Tipo 2/genética , Mutação da Fase de Leitura , Predisposição Genética para Doença , Lipólise/genética , Esterol Esterase/genética , Adulto , Idoso , Amish/genética , Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/genética , Dislipidemias/metabolismo , Feminino , Heterozigoto , Humanos , Resistência à Insulina/genética , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , LinhagemRESUMO
Thromboxane A2, an arachidonic acid-derived eicosanoid generated by thromboxane synthase (TBXAS), plays critical roles in hemostasis and inflammation. However, the contribution of thromboxane A2 to obesity-linked metabolic dysfunction remains incompletely understood. Here, we used in vitro and mouse models to better define the role of TBXAS in metabolic homeostasis. We found that adipose expression of Tbxas and thromboxane A2 receptor (Tbxa2r) was significantly upregulated in genetic and dietary mouse models of obesity and diabetes. Expression of Tbxas and Tbxa2r was detected in adipose stromal cells, including macrophages. Furthermore, stimulation of macrophages with interferon-γ or resistin factors known to be upregulated in obesity induced Tbxas and Tbxa2r expression. Mice lacking Tbxas had similar weight gain, food intake, and energy expenditure. However, loss of Tbxas markedly enhanced insulin sensitivity in mice fed a low-fat diet. Improvement in glucose homeostasis was correlated with the upregulated expression of multiple secreted metabolic regulators (Ctrp3, Ctrp9, and Ctrp12) in the visceral fat depot. Following a challenge with a high-fat diet, Tbxas deficiency led to attenuated adipose tissue fibrosis and reduced circulating IL-6 levels without adipose tissue macrophages being affected; however, these changes were not sufficient to improve whole body insulin action. Together, our results highlight a novel, diet-dependent role for thromboxane A2 in modulating peripheral tissue insulin sensitivity and adipose tissue fibrosis.
Assuntos
Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Insulina/metabolismo , Tromboxano-A Sintase/genética , Células 3T3-L1 , Animais , Células Cultivadas , Citocinas/farmacologia , Feminino , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Tromboxano-A Sintase/deficiênciaRESUMO
Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Neovascularização Patológica , Aldeído Redutase/metabolismo , Inibidores da Angiogênese/farmacologia , Antioxidantes/farmacologia , Sítios de Ligação , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Metionina Sulfóxido Redutases/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Fenótipo , Transdução de Sinais , Especificidade por Substrato , Fatores de TempoRESUMO
The thiazolidedione (TZD) class of drugs is clinically approved for the treatment of type 2 diabetes. The therapeutic actions of TZDs are mediated via activation of peroxisome proliferator-activated receptor γ (PPARγ). Despite their widespread use, concern exists regarding the safety of currently used TZDs. This has prompted the development of selective PPARγ modulators (SPPARMs), compounds that promote glucose homeostasis but with reduced side effects due to partial PPARγ agonism. However, this also results in partial agonism with respect to PPARγ target genes promoting glucose homeostasis. Using a gene expression-based screening approach we identified N-acetylfarnesylcysteine (AFC) as both a full and partial agonist depending on the PPARγ target gene (differential SPPARM). AFC activated PPARγ as effectively as rosiglitazone with regard to Adrp, Angptl4, and AdipoQ, but was a partial agonist of aP2, a PPARγ target gene associated with increased adiposity. Induction of adipogenesis by AFC was also attenuated compared with rosiglitazone. Reporter, ligand binding assays, and dynamic modeling demonstrate that AFC binds and activates PPARγ in a unique manner compared with other PPARγ ligands. Importantly, treatment of mice with AFC improved glucose tolerance similar to rosiglitazone, but AFC did not promote weight gain to the same extent. Finally, AFC had effects on adipose tissue remodeling similar to those of rosiglitazone and had enhanced antiinflammatory effects. In conclusion, we describe a new approach for the identification of differential SPPARMs and have identified AFC as a novel class of PPARγ ligand with both full and partial agonist activity in vitro and in vivo.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína/análogos & derivados , Cisteína/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Hipoglicemiantes/farmacologia , PPAR gama/agonistas , Células 3T3-L1 , Acetilcisteína/química , Acetilcisteína/farmacologia , Animais , Cisteína/química , Homeostase/efeitos dos fármacos , Hipoglicemiantes/química , Ligantes , Camundongos , Camundongos Knockout , PPAR gama/metabolismo , Ligação Proteica , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
µ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6-147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, ß-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that µ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of µ-crystallin are associated with a shift toward greater fat metabolism.
RESUMO
Postprandial triglyceridemia is an emerging risk factor for cardiovascular disease. However, most of the genes that influence postprandial triglyceridemia are not known. We evaluated whether a common nonsynonymous SNP rs1260326/P446L in the glucokinase regulatory protein (GCKR) gene influenced variation in the postprandial lipid response after a high-fat challenge in seven hundred and seventy participants in the Amish HAPI Heart Study who underwent an oral high-fat challenge and had blood samples taken in the fasting state and during the postprandial phase at 1, 2, 3, 4, and 6 h. We found that the minor T allele at rs1260326 was associated with significantly higher fasting TG levels after adjusting for age, sex, and family structure (P (a) = 0.06 for additive model, and P (r) = 0.0003 for recessive model). During the fat challenge, the T allele was associated with significantly higher maximum TG level (P (a) = 0.006), incremental maximum TG level (P (a) = 0.006), TG area under the curve (P (a) = 0.02) and incremental TG area under the curve (P (a) = 0.03). Our data indicate that the rs1260326 T allele of GCKR is associated with both higher fasting levels of TG as well as the postprandial TG response, which may result in higher atherogenic risk.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Gorduras na Dieta/administração & dosagem , Glucoquinase/genética , Lipídeos/análise , Polimorfismo de Nucleotídeo Único/genética , Período Pós-Prandial/genética , Adulto , Jejum , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores de Risco , Triglicerídeos/metabolismoRESUMO
The Randle cycle, which has been invoked to explain the reciprocal relationship between fatty acid oxidation and glucose oxidation, has long been implicated as a potential mechanism for hyperglycemia and type 2 diabetes mellitus (T2DM). Now genetic, functional genomic, and transgenic approaches have identified PPARgamma coactivators (PGC-1alpha and PGC-1beta) as key regulators of mitochondrial number and function. They regulate adaptive thermogenesis as well as glucose and fat oxidation in muscle and fat tissue, gluconeogenesis in liver, and even glucose-regulated insulin secretion in beta cells. PGC-1alpha and PGC-1beta mRNA levels and the mitochondrial genes they regulate are decreased in muscle of people with prediabetes and T2DM. A new report indicates that PGC-1alpha and PGC-1beta mRNA levels decrease with age in individuals with a genetic variant in PGC-1alpha, and these decreases correlate with alterations in whole-body glucose and fatty acid oxidation. These findings provide insights into how aging modifies genetic susceptibility to alterations in oxidative phosphorylation and T2DM.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Genes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores Etários , Glicemia/metabolismo , Dieta , Gorduras/metabolismo , Ácidos Graxos/metabolismo , Variação Genética , Gluconeogênese , Humanos , Hiperglicemia/etiologia , Hiperglicemia/fisiopatologia , Insulina/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Oxirredução , Fosforilação Oxidativa , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , TermogêneseRESUMO
We performed a genome-wide linkage scan of plasma adiponectin levels in 569 nondiabetic participants in the Amish Family Diabetes Study. The highest logarithm of odds (LOD) score (2.13; P = 0.0009) occurred on chromosome 3q27 between markers D3S1602 and D3S1580, which flank APM1/ACDC, the adiponectin gene. The APM1 +2019 A/- insertion/deletion polymorphism in the 3' untranslated region (single nucleotide polymorphism [SNP] +2019; deletion allele frequency 0.30 in Amish) showed strong association with adiponectin levels in a dosage-dependent manner in a direction consistent with that reported in previous studies, with deletion heterozygosity increasing adiponectin levels by 1.3 +/- 0.5 microg/ml and deletion homozygosity increasing levels by 3.0 +/- 0.8 microg/ml (P < 0.0001). Two other SNPs, rs2241766 and rs1501299, showed moderate association. In a subset of 523 subjects genotyped for both SNP +2019 and rs2241766, including the APM1 SNP +2019 genotype as a covariate reduced the linkage signal at 3q27 by 1.26 LOD units (from 2.22 to 0.96) and including both SNPs reduced the signal by 1.51 LOD units (to 0.71). These findings, combined with a two-point LOD score of 2.35 for SNP +2019, provide evidence that variation in APM1 is responsible for linkage of adiponectin levels to 3q27 in the Old Order Amish.
Assuntos
Cromossomos Humanos Par 3 , Variação Genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Adiponectina , Mapeamento Cromossômico , Marcadores Genéticos , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Polimorfismo de Nucleotídeo Único , Sequências Repetitivas de Ácido NucleicoRESUMO
Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.
Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Inibidores Enzimáticos/metabolismo , Genômica , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Diacilglicerol O-Aciltransferase/genética , Drosophila/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epistasia Genética , Ácidos Graxos/metabolismo , Feminino , Humanos , Peroxidação de Lipídeos , Masculino , Camundongos , Fenótipo , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , Análise de Sequência de RNA , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Obesity is a risk factor for breast cancer progression. Breast cancer patients who are overweight or obese or have excess abdominal fat have an increased risk of local or distant recurrence and cancer-related death. Hormone depletion therapies can also cause weight gain, exacerbating the risk for these patients. To understand the effect of obesity on hormone-dependent human breast cancer tumors, we fed ovariectomized athymic nude mice a diet containing 45% kcal fat and 17% kcal sucrose (high fat sucrose diet (HFSD)), 10% kcal fat (low fat diet (LFD)), or a standard chow diet (chow). The mice fed the HFSD developed metabolic abnormalities consistent with the development of obesity such as weight gain, high fasting blood glucose, and impaired glucose tolerance. These mice also developed hyperinsulinemia and insulin resistance. The obese mice also had a higher tumor growth rate compared to the lean mice. Furthermore, the obese mice showed a significantly reduced responsiveness to letrozole. To understand the role of obesity in this reduced responsiveness, we examined the effect of insulin on the growth of MCF-7Ca cells in response to estrogen or letrozole. The presence of insulin rendered MCF-7Ca cells less responsive to estrogen and letrozole. Exogenous insulin treatment of MCF-7Ca cells also resulted in increased p-Akt as well as ligand-independent phosphorylation of ERα. These findings suggest that diet-induced obesity may result in reduced responsiveness of tumors to letrozole due to the development of hyperinsulinemia. We conclude that obesity influences the response and resistance of breast cancer tumors to aromatase inhibitor treatment.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Nitrilas/uso terapêutico , Obesidade/metabolismo , Triazóis/uso terapêutico , Tecido Adiposo/metabolismo , Animais , Aromatase/genética , Aromatase/metabolismo , Glicemia/análise , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Dieta , Modelos Animais de Doenças , Estrogênios/farmacologia , Humanos , Insulina/sangue , Letrozol , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Obesidade/sangue , Ovariectomia , Carga TumoralRESUMO
OBJECTIVE: To determine whether super-activation of PPARγ can reprogram human myoblasts into brown-like adipocytes and to establish a new cell model for browning research. METHODS: To enhance the PPARγ signaling, M3, the transactivation domain of MyoD, was fused to PPARγ. PPARγ and M3-PPARγ-lentiviral vectors were used to convert human myoblasts into adipocytes. Brown adipocyte markers of the reprogrammed adipocytes were assessed by qPCR and protein analyses. White adipocytes differentiated from subcutaneous stromal vascular cells and perithyroid brown fat tissues were used as references. RESULTS: In transient transfections, M3-PPARγ had a stronger constitutive activity than PPARγ by reporter assay. Although the transduction of either PPARγ or M3-PPARγ induced adipogenesis in myoblasts, M3-PPARγ drastically induced the brown adipocyte markers of UCP1, CIDEA, and PRDM16 by 1,050, 2.4, and 5.0 fold, respectively and increased mitochondria contents by 4 fold, compared to PPARγ. CONCLUSIONS: Super-activation of PPARγ can effectively convert human myoblasts into brown-like adipocytes and a new approach to derive brown-like adipocytes.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , PPAR gama/metabolismo , Adipogenia/fisiologia , Diferenciação Celular/fisiologia , Humanos , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Células Estromais/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The purpose of this study was to determine the effects of 6-month aerobic exercise training + weight loss (AEX + WL) on basal and insulin activation of glycogen synthase, basal citrate synthase activity, and Akt and AS160 phosphorylation in older, overweight/obese insulin-resistant men (n = 14; 63 ± 2 years; body mass index, 32 ± kg/m(2)). Muscle samples of the vastus lateralis were collected before and during a 3-hour 80 mU/m(2)/min hyperinsulinemic-euglycemic clamp. AEX + WL increased VO2max by 11% (p < .05) and decreased body weight (-9%, p < .001). AEX + WL increased basal citrate synthase activity by 46% (p < .01) and insulin activation of independent (2.9-fold) and fractional (2.3-fold) activities (both p < .001) of glycogen synthase. AEX + WL had no effect on phosphorylation of Akt or AS160. Glucose utilization (M) improved 25% (p < .01), and the change tended to be related to the increase in insulin activation of glycogen synthase fractional activity (r = .50, p = .08) following AEX + WL. In summary, AEX + WL has a robust effect on insulin activation of skeletal muscle glycogen synthase activity that likely contributes to improved glucose utilization in older insulin-resistant men.
Assuntos
Exercício Físico/fisiologia , Glicogênio Sintase/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Redução de Peso/fisiologia , Idoso , Envelhecimento/fisiologia , Composição Corporal , Dieta Redutora , Proteínas Ativadoras de GTPase/metabolismo , Glucose/metabolismo , Técnica Clamp de Glucose , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The purpose of this study is to compare central obesity, insulin sensitivity, and cardiovascular disease risk factors between premenopausal and postmenopausal women with a history of gestational diabetes mellitus (GDM), controls, and women with type 2 diabetes (T2DM). Subjects were 73 overweight/obese and sedentary women who had a history of GDM (n=31) and were either premenopausal (n=11, 44±1 years, X±s.e.m.), postmenopausal (n=20, 58±1 years), or without a history of GDM as healthy postmenopausal controls (n=27, 57±1 years) or postmenopausal with T2DM (n=16, 59±1 years). The premenopausal GDM women had higher maximal oxygen uptake and lower visceral fat than the other three groups (P<0.05). BMI, %body fat, subcutaneous abdominal fat, and intramuscular fat did not differ significantly among the four groups. Glucose utilization (M, 3âh 40âmU/m(2) per min hyperinsulinemic-euglycemic clamps) was 27% higher (P=0.05) in pre- than postmenopausal GDM and was not different between premenopausal GDM and postmenopausal controls. M was 28% lower (P=0.06) in postmenopausal GDM than controls and was not significantly different between postmenopausal GDM and T2DM groups. Thus, despite being younger and more physically fit, premenopausal women with prior GDM display similar central obesity, glucose, and metabolic profiles as postmenopausal controls. Postmenopausal women with prior GDM are more insulin resistant than controls of similar age, adiposity, and fitness levels and display comparable glucose utilization rates as similar as women with T2DM suggesting that a prior history of GDM may be an early manifestation of increased risk of later T2DM.
RESUMO
EIF2AK3 is a type I transmembrane protein that functions as an endoplasmic reticulum (ER) stress sensor to regulate global protein synthesis. Rare mutations in EIF2AK3 cause Wolcott-Rallison syndrome (OMIM 226980), an autosomal recessive disorder characterized by diabetes, epiphyseal dysplasia, osteoporosis, and growth retardation. To investigate the role of common genetic variation in EIF2AK3 as a determinant of bone mineral density (BMD) and osteoporosis, we sequenced all exons and flanking regions, then genotyped six potentially functional single nucleotide polymorphisms (SNPs) in this gene in 997 Amish subjects for association analysis, and attempted replication in 887 Mexican Americans. We found that the minor allele of a nonsynonymous SNP rs13045 had borderline associations with decreased forearm BMD in both discovery and replication cohorts (unadjusted p = 0.036 and ß = -0.007 for the Amish; unadjusted p = 0.031 and ß = -0.008 for Mexican Americans). A meta-analysis indicated this association achieved statistical significance in the combined sample (unadjusted p = 0.003; Bonferroni corrected p = 0.009). Rs13045 and three other potentially functional SNPs, a promoter SNP (rs6547787) and two nonsynonymous SNPs (rs867529 and rs1805165), formed two haplotypes: a low-BMD associated haplotype, denoted haplotype B [minor allele frequency (MAF) = 0.311] and a common haplotype A (MAF = 0.676). There were no differences in mRNA expression in lymphoblastoid cell lines between the two haplotypes. However, after treating lymphoblastoid cell lines with thapsigargin to induce ER stress, cell lines with haplotype B showed increased sensitivity to ER stress (p = 0.014) compared with cell lines with haplotype A. Taken together, our results suggest that common nonsynonymous sequence variants in EIF2AK3 have a modest effect on ER stress response and may contribute to the risk for low BMD through this mechanism.
Assuntos
Densidade Óssea/genética , Estresse do Retículo Endoplasmático/genética , Estudos de Associação Genética , Haplótipos/genética , eIF-2 Quinase/genética , Adulto , Alelos , Sequência de Aminoácidos , Amish/genética , Linhagem Celular , Feminino , Heterozigoto , Humanos , Desequilíbrio de Ligação/genética , Masculino , Americanos Mexicanos/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Transdução de Sinais/genética , eIF-2 Quinase/químicaRESUMO
We previously detected strong evidence for linkage of forearm bone mineral density (BMD) to chromosome 4p (lod=4.3) in a set of 29 large Mexican American families. Fibroblast growth factor binding protein 1 (FGFBP1) is a strong candidate gene for bone homeostasis in this region. We sequenced the coding region of FGFBP1 in a subset of our Mexican American study population and performed association studies with BMD on SNPs genotyped in the entire cohort. We then attempted to replicate these findings in an independent study cohort and performed in vitro functional studies on replicated, potentially functional polymorphisms using a luciferase reporter construct to evaluate influence on gene expression. Several SNPs spanning the gene, all in one large block of linkage disequilibrium, were significantly associated with BMD at various skeletal sites (n=872, p=0.001-0.04). The associations were then replicated in an independent population of European ancestry (n=972; p=0.02-0.04). Sex-stratified association analyses in both study populations suggest this association is much stronger in men. Subsequent luciferase reporter gene assays revealed marked differences in FGFBP1 expression among the three common haplotypes. Further experiments revealed that a promoter polymorphism, rs12503796, results in decreased expression of FGFBP1 and inhibits upregulation of the gene by testosterone in vitro. Collectively, these findings suggest that sequence variation in FGFBP1 may contribute to variation in BMD, possibly influencing osteoporosis risk.
Assuntos
Densidade Óssea/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica , Polimorfismo de Nucleotídeo Único/genética , Adulto , Sítios de Ligação , Feminino , Haplótipos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Desequilíbrio de Ligação/genética , Masculino , Americanos Mexicanos/genética , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , População Branca/genéticaRESUMO
Hemiparetic stroke leads to major skeletal muscle abnormalities, as illustrated by paretic leg atrophy, weakness, and spasticity. Furthermore, the hemiparetic limb muscle shifts to a fast-twitch muscle fiber phenotype with anaerobic metabolism. This study investigated whether skeletal muscle genes were altered in chronic hemiparetic stroke. The nonparetic leg muscle served as an internal control. We used Affymetrix microarray analysis to survey gene expression differences between paretic and nonparetic vastus lateralis muscle punch biopsies from 10 subjects with chronic hemiparetic stroke. Stroke latency was greater than 6 months. We found that 116 genes were significantly altered between the paretic and non paretic vastus lateralis muscles. These gene differences were consistent with reported differences after stroke in areas such as injury and inflammation markers, the myosin heavy chain profile, and high prevalence of impaired glucose tolerance and type 2 diabetes. Furthermore, while many other families of genes were altered, the gene families with the most genes altered included inflammation, cell cycle regulation, signal transduction, metabolism, and muscle contractile protein genes. This study is an early step toward identification of specific gene regulatory pathways that might lead to these differences, propagate disability, and increase vascular disease risk.
Assuntos
Extremidade Inferior/fisiopatologia , Músculo Esquelético/metabolismo , Paresia/genética , Acidente Vascular Cerebral/genética , Idoso , Idoso de 80 Anos ou mais , Ciclo Celular , Doença Crônica , Feminino , Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Contração Muscular/genética , Músculo Esquelético/fisiopatologia , Paresia/metabolismo , Paresia/fisiopatologia , Paresia/reabilitação , RNA/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Reabilitação do Acidente Vascular Cerebral , Transcrição GênicaRESUMO
OBJECTIVE: Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation-related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance. RESEARCH DESIGN AND METHODS: We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of diet-induced obese (DIO) mice were also measured. RESULTS: Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by approximately 2- to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P < 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency. CONCLUSIONS: These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes.
Assuntos
Proteínas de Transporte/fisiologia , Hepatócitos/metabolismo , Proteínas de Membrana/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cromatografia em Camada Fina , Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Hepatócitos/citologia , Immunoblotting , Imuno-Histoquímica , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipólise/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Perilipina-2 , Perilipina-3 , RNA Interferente Pequeno/genéticaRESUMO
Apolipoprotein C-III (apoC-III) inhibits triglyceride hydrolysis and has been implicated in coronary artery disease. Through a genome-wide association study, we have found that about 5% of the Lancaster Amish are heterozygous carriers of a null mutation (R19X) in the gene encoding apoC-III (APOC3) and, as a result, express half the amount of apoC-III present in noncarriers. Mutation carriers compared with noncarriers had lower fasting and postprandial serum triglycerides, higher levels of HDL-cholesterol and lower levels of LDL-cholesterol. Subclinical atherosclerosis, as measured by coronary artery calcification, was less common in carriers than noncarriers, which suggests that lifelong deficiency of apoC-III has a cardioprotective effect.
Assuntos
Apolipoproteína C-III/genética , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Doença da Artéria Coronariana/prevenção & controle , Lipídeos/sangue , Mutação , Triglicerídeos/sangue , Adulto , Apolipoproteína C-III/sangue , Colesterol/sangue , Cristianismo , Doença da Artéria Coronariana/genética , Gorduras na Dieta/administração & dosagem , Jejum , Feminino , Estudo de Associação Genômica Ampla , Haplótipos , Heterozigoto , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Linhagem , Pennsylvania , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Central obesity and the accumulation of visceral fat are risk factors for the development of type 2 diabetes and cardiovascular disease. Omentin is a protein expressed and secreted from visceral but not subcutaneous adipose tissue that increases insulin sensitivity in human adipocytes. To determine the impact of obesity-dependent insulin resistance on the regulation of two omentin isoforms, gene expression and plasma levels were measured in lean, overweight, and obese subjects. Omentin 1 was shown to be the major circulating isoform in human plasma. Lean subjects had significantly higher plasma omentin 1 levels than obese and overweight subjects. In addition, higher plasma omentin 1 levels were detected in women compared with men. Plasma omentin 1 levels were inversely correlated with BMI, waist circumference, leptin levels, and insulin resistance as measured by homeostasis model assessment and positively correlated with adiponectin and HDL levels. Both omentin 1 and omentin 2 gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. In summary, decreased omentin levels are associated with increasing obesity and insulin resistance. Therefore, omentin levels may be predictive of the metabolic consequences or co-morbidities associated with obesity.
Assuntos
Citocinas/sangue , Citocinas/genética , Regulação da Expressão Gênica , Lectinas/sangue , Lectinas/genética , Obesidade/sangue , Obesidade/genética , Adiponectina/sangue , Tecido Adiposo/metabolismo , Adulto , Citocinas/isolamento & purificação , Feminino , Proteínas Ligadas por GPI , Humanos , Lectinas/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than is peripheral [subcutaneous (sc)] obesity, but the underlying mechanism for this pathophysiological difference is largely unknown. To understand the molecular basis of this difference, we sequenced 10,437 expressed sequence tags (ESTs) from a human omental fat cDNA library and discovered a novel visceral fat depot-specific secretory protein, which we have named omentin. Omentin ESTs were more abundant than many known adipose genes, such as perilipin, adiponectin, and leptin in the cDNA library. Protein sequence analysis indicated that omentin mRNA encodes a peptide of 313 amino acids, containing a secretory signal sequence and a fibrinogen-related domain. Northern analysis demonstrated that omentin mRNA was predominantly expressed in visceral adipose tissue and was barely detectable in sc fat depots in humans and rhesus monkeys. Quantative real-time PCR showed that omentin mRNA was expressed in stromal vascular cells, but not fat cells, isolated from omental adipose tissue, with >150-fold less in sc cell fractions. Accordingly, omentin protein was secreted into the culture medium of omental, but not sc, fat explants. Omentin was detectable in human serum by Western blot analysis. Addition of recombinant omentin in vitro did not affect basal but enhanced insulin-stimulated glucose uptake in both sc (47%, n = 9, P = 0.003) and omental (approximately 30%, n = 3, P < 0.05) human adipocytes. Omentin increased Akt phosphorylation in the absence and presence of insulin. In conclusion, omentin is a new adipokine that is expressed in omental adipose tissue in humans and may regulate insulin action.