RESUMO
The renin-angiotensin system is an important regulator of blood pressure, and blockade of this system improves blood pressure in obesity and type 2 diabetes. Recently, components of the system have been described in adipose tissue. However, to date no study has investigated the influence of varying insulin concentrations on angiotensinogen (AGT) protein expression in human subcutaneous abdominal fat. Isolated subcutaneous adipocytes were treated with insulin (1-1000 nm) for 48 h. As part of the studies, a novel AGT antibody was developed and validated by Western blotting and immunohistochemistry. Western blotting was performed on the protein extracted from the adipocytes treated with insulin to determine AGT expression. Increasing doses of insulin raised AGT protein expression in a dose-dependent manner (control 1.0 +/- 0.0 (mean +/- s.e.) - protein expression standardized relative to control; 1 nm insulin: 2.64 +/- 0.0.32 upward arrow ***; 100 nm insulin: 4.37 +/- 0.57 upward arrow ***; 1000 nm insulin: 6.50 +/- 0.97 upward arrow ***; ***p < 0.001, n = 3). In conclusion, increasing insulin doses stimulates AGT production. In this study, protein analysis suggests that hyperinsulinaemia may be an important factor in obesity-related hypertension.
Assuntos
Adipócitos/efeitos dos fármacos , Angiotensinogênio/metabolismo , Insulina/farmacologia , Abdome , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adulto , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Rim/metabolismo , Fígado/metabolismo , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: The aim of this study was to investigate the effect of insulin and an insulin-sensitizing agent, rosiglitazone (RSG), on the production of plasminogen-activator inhibitor-1 (PAI-1) in isolated subcutaneous abdominal adipocytes. Human tissue-type plasminogen activator (t-PA) was also measured to assess changes in overall thrombotic risk. METHODS: The mean depot-specific expression of PAI-1 and t-PA mRNA (n = 42) in subcutaneous abdominal (n = 21), omental (n = 10) and thigh (n = 11) adipose tissue depots was examined. Furthermore, subcutaneous adipocytes were treated with insulin, RSG and insulin in combination with RSG (10-8 m) for 48 h. Conditioned media were collected and enzyme-linked immunosorbent assays performed for PAI-1 and t-PA (n = 12) antigen. PAI-1 and t-PA mRNA levels were also assessed. RESULTS: PAI-1 mRNA levels were significantly higher in subcutaneous and omental abdominal tissue than in thigh fat (p = 0.037 and p = 0.014). No change in t-PA mRNA expression between the adipose tissue depots was observed. Insulin stimulated PAI-1 protein secretion in a concentration-dependent manner in adipocytes (control: 68.3 +/- 1.2 ng/ml (s.e.m.); 10 nm insulin: 73.7 +/- 3.8 ng/ml upward arrow; 100 nm insulin: 86.8 +/- 4.1 ng/ml upward arrow **; 1000 nm insulin: 102.0 +/- 4.8 ng/ml upward arrow ***; **p < 0.01, ***p < 0.001). In contrast, insulin + RSG (10-8 m) reduced PAI-1 production relative to insulin alone (***p < 0.001), whilst RSG alone reduced PAI-1 protein secretion in a concentration-dependent manner (RSG at 10-10 m: 50.4 +/- 2.87 ng/ml downward arrow ***; RSG at 10-5 m: 30.3 +/- 2.0 ng/ml downward arrow ***; p < 0.001). No difference was observed between control and treatments for t-PA secretion (range 7-11 ng/ml). CONCLUSIONS: Insulin stimulated PAI-1 secretion, whilst RSG reduced both PAI-1 secretion alone and in combination with insulin. These data suggest that adipose tissue may contribute significantly to the elevated circulating PAI-1 in obesity. Therefore, RSG's effects on PAI-1 production in adipose tissue may contribute to the fall in circulating PAI-1 levels observed in patients receiving RSG therapy.
Assuntos
Adipócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Tiazolidinedionas/farmacologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adulto , Células Cultivadas , Técnicas de Cultura , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/farmacologia , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Resistin, an adipocyte-derived cytokine, causes insulin resistance and glucose intolerance in mice. We investigated whether resistin expression was higher in human abdominal adipose tissue than other adipose tissue depots. We extracted RNA from 32 adipose tissue samples (13 subcutaneous abdominal, seven omentum, six thigh, and six breast). Quantitative PCR was used to determine resistin mRNA expression. Resistin mRNA concentrations were similar in both the subcutaneous abdominal and omental depots. The abdominal depots showed a 418% increase in resistin mRNA expression compared with the thigh. Increased resistin expression in abdominal fat could explain the increased risk of type 2 diabetes associated with central obesity.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hormônios Ectópicos/genética , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intercelular , Obesidade/metabolismo , RNA Mensageiro/genética , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/isolamento & purificação , ResistinaRESUMO
Adiponectin is an adipocyte-derived hormone associated with insulin sensitivity and atherosclerotic risk. As central rather than gluteofemoral fat is known to increase the risk of type 2 diabetes and cardiovascular disease, we investigated the mRNA and protein expression of adiponectin in human adipose tissue depots. RNA was extracted from 46 human adipose tissue samples from non-diabetic subjects aged 44.33 +/- 12.4 with a BMI of 28.3 +/- 6.0 (mean +/- SD). The samples were as follows: 21 abdominal subcutaneous, 13 omentum, 6 thigh; samples were also taken from diabetic subjects aged 66.6 +/- 7.5 with BMI 28.9 +/- 3.17; samples were: 6 abdominal subcutaneous; 3 thigh. Quantitative PCR and Western analysis was used to determine adiponectin content. Protein content studies determined that when compared with non-diabetic abdominal subcutaneous adipose tissue (Abd Sc AT) (values expressed as percentage relative to Abd Sc AT -100 %). Adiponectin protein content was significantly lower in non-diabetic omental AT (25 +/- 1.6 %; p < 0.0001, n = 6) and in Abd Sc AT from diabetic subjects (36 +/- 1.5 %; p < 0.0001, n = 4). In contrast, gluteal fat maintained high adiponectin protein content from non-diabetic patients compared with diabetic patients. An increase in BMI was associated with lower adiponectin protein content in obese ND Abd Sc AT (25 +/- 0.4 %; p < 0.0001). These findings were in agreement with the mRNA expression data. In summary, this study indicates that adiponectin protein content in non-diabetic subjects remains high in abdominal subcutaneous fat, including gluteal fat, explaining the high serum adiponectin levels in these subjects. Omental fat, however, expresses little adiponectin. Furthermore, abdominal and gluteal subcutaneous fat appears to express significantly less adiponectin once diabetic status is reached. In conclusion, the adipose tissue depot-specific expression of adiponectin may influence the pattern of serum adiponectin concentrations and subsequent disease risk.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Obesidade , Proteínas/metabolismo , Adipócitos/metabolismo , Adiponectina , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Omento/citologia , Omento/metabolismo , RNA Mensageiro/análise , Tela Subcutânea/metabolismo , Coxa da Perna , Distribuição TecidualRESUMO
11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) inactivates cortisol to cortisone. In the placenta 11beta-HSD2 activity is thought to protect the fetus from the deleterious effects of maternal glucocorticoids. Patients with apparent mineralocorticoid excess owing to mutations in the 11beta-HSD2 gene invariably have reduced birth weight, and we have recently shown reduced placental 11beta-HSD2 activity in pregnancies complicated by intrauterine growth restriction. This is reflected in the literature by evidence of hypercortisolemia in the fetal circulation of small babies. In this study we have determined the levels of placental 11beta-HSD2 mRNA expression across normal gestation (n = 86 placentae) and in pregnancies complicated by intrauterine growth restriction (n = 19) and evaluated the underlying mechanism for any aberrant 11beta-HSD2 mRNA expression in intrauterine growth restriction. 11beta-HSD2 mRNA expression increased more than 50-fold across gestation, peaking at term. Placental 11beta-HSD2 mRNA levels were significantly decreased in intrauterine growth restriction pregnancies when compared with gestationally matched, appropriately grown placentae [e.g. at term DeltaCt (11beta-hydroxysteroid dehydrogenase type 2/18S) 12.8 +/- 0.8 (mean +/- SE) vs. 10.2 +/- 0.2, respectively, P < 0.001]. These differences were not attributable to changes in trophoblast mass in intrauterine growth restriction placentae, as assessed by parallel analyses of cytokeratin-8 mRNA expression. No mutations were found in the 11beta-HSD2 gene in the intrauterine growth restriction cohort, and imprinting analysis revealed that the 11beta-HSD2 gene was not imprinted. Although the underlying cause is unknown, 11beta-HSD2 gene expression is reduced in intrauterine growth restriction pregnancies. These data highlight the important role of 11beta-HSD2 in regulating fetal growth, a known factor in determining fetal morbidity but also the subsequent development of cardiovascular disease in adulthood.
Assuntos
Retardo do Crescimento Fetal/etiologia , Hidroxiesteroide Desidrogenases/genética , Placenta/enzimologia , RNA Mensageiro/análise , 11-beta-Hidroxiesteroide Desidrogenases , Desenvolvimento Embrionário e Fetal , Feminino , Retardo do Crescimento Fetal/enzimologia , Humanos , Hidroxiesteroide Desidrogenases/fisiologia , GravidezRESUMO
AIMS: Whilst HLA-DRB1 and HLA-DQ alleles contribute to IDDM1, the major determinant of genetic susceptibility to Type 1 diabetes mellitus, other major histocompatibility complex (MHC)-encoded genes may also be involved. The LMP7 (large multifunctional proteasome 7) gene is a potential candidate. The aim of this study was to assess whether LMP7 confers susceptibility to Type 1 diabetes independently of linkage disequilibrium with HLA-DRB1 and HLA-DQ. METHODS: The diallelic LMP7 polymorphism (LMP7*A or *B) was determined in 142 multiplex families from the British Diabetic Association Warren Repository. At least one parent was heterozygous for LMP7 in 112 families and these were informative for calculation of the statistic Tsp. This gives a valid chi2 test of the null hypothesis of no association or no linkage. RESULTS: An excess of transmissions of LMP7*A was observed from parents to affected offspring and the Tsp statistic was significant for association in the presence of linkage. LMP7*A was in positive, and LMP7*B in negative, linkage disequilibrium with the HLA-DRB1*03-DQ2, DRB1*04-DQ8 (group of all DRB1*04 subtypes), DRB1*0401-DQ8 and DRB1*0404-DQ8 haplotypes, although the linkage disequilibrium coefficient (delta) value was not statistically significant for DRB1*0404-DQ8. Analysis of HLA-DR-DQ-LMP7 haplotypes and Tsp analysis of HLA-matched-homozygous parents showed no association between LMP7 alleles and Type I diabetes independent of linkage disequilibrium with HLA-DR-DQ haplotypes associated with increased risk of disease. A contribution of LMP7 alleles to susceptibility to Type 1 diabetes in subjects with low-risk HLA-DR-DQ haplotypes could not be excluded. CONCLUSIONS: LMP7 alleles do not contribute to genetic susceptibility to Type 1 diabetes in subjects with high-risk-associated HLA-DR-DQ haplotypes.
Assuntos
Cisteína Endopeptidases , Diabetes Mellitus Tipo 1/genética , Complexos Multienzimáticos , Polimorfismo Genético , Proteínas/genética , Ligação Genética , Predisposição Genética para Doença , Genótipo , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Haplótipos , Heterozigoto , Desequilíbrio de Ligação , Complexo de Endopeptidases do Proteassoma , Fatores de Risco , Reino UnidoRESUMO
The HLA-DP genes (HLA-DPA1 and -DPB1) are encoded by the major histocompatibility complex (MHC) on human chromosome 6. They are involved in the presentation of antigen to CD4+ T cells as part of the class II antigen-presentation pathway. During a small study of Oriental subjects (11 Chinese and 26 Japanese subjects), one Chinese subject was identified as having numerous heterozygous sites within the second exon of both DPA1 and DPB1. These were further analysed using novel codon-specific primers. Sequencing analysis using these primers determined the subject to have DPA1*0103/*02015 and DPB1*0501/*8401; these new alleles have been submitted to GenBank and assigned the accession numbers AF098794 and AF077015, respectively.
Assuntos
Alelos , Antígenos HLA-DP/genética , Sequência de Bases , China , Cadeias alfa de HLA-DP , Cadeias beta de HLA-DP , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Alinhamento de SequênciaRESUMO
The HLA-DM genes (DMA and DMB) are encoded in the major histocompatibility complex (MHC) on human chromosome 6. They are involved in the loading of antigenic peptide onto class II molecules as part of the class II antigen-processing and -presentation pathway. During a study of Oriental subjects (11 Chinese and 26 Japanese subjects), one Chinese subject was identified as having a new DMB allele using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This was confirmed with sequencing. The new allele was found to have Glu (GAG) and Thr (ACT) at positions 144 and 179 respectively. This was a previously unpublished combination of polymorphic sites, and was submitted to GenBank and assigned the accession number AF072680, and named HLA-DMB*0106.