Identification of a deletion variant in the gene encoding the human alpha(2A)-adrenergic receptor.

OBJECTIVE: The alpha(2)-adrenergic receptors are involved in the effects of catecholamines on energy metabolism. Of three known subtypes with differential expression, alpha(2A)-adrenergic receptors are also localized in adipose tissue where they counteract the lipolytic activity of beta-adrenergic receptors. This study was undertaken to assess whether variants in the alpha(2A)-adrenergic receptor gene are associated with body weight. DESIGN AND METHODS: Single strand conformation polymorphism (SSCP) screening and subsequent sequencing were applied to determine genetic variants in DNA samples from individuals with obesity, those of normal weight and those underweight. RESULTS: Analysis of the coding region resulted in the identification of an 18 bp deletion, with no other mutation found. Of 429 genotyped subjects, 7 carried the deletion, with no significant differences between lean and obese subjects. A previously identified polymorphism in the promoter of the alpha(2A)-adrenergic receptor gene also did not show an association with any of the tested body weight categories. CONCLUSION: Our data suggest that variants in the alpha(2A)-adrenergic receptor gene are unlikely to contribute to the predisposition for the lean or obese state.

Therapeutic dose of HIV-1 protease inhibitor saquinavir does not permanently influence early insulin signaling.

UNLABELLED: The introduction of HIV-1 protease inhibitor therapy has significantly improved the expectancy and quality of life for HIV-infected patients. Recent reports have highlighted the development of metabolic complications in patients taking protease inhibitors, including abnormalities in glucose metabolism such as impaired glucose tolerance and type 2 diabetes. The mechanisms by which protease inhibitors induce these metabolic syndromes are not well understood. The aim of this study was to determine whether treatment with the HIV-1 protease inhibitor, saquinavir, influences the early insulin signaling cascade in insulin-sensitive cell lines. METHODS: Insulin-stimulated phosphorylation of insulin receptor (IR-beta), insulin receptor substrates (IRS-1 and IRS-2), association of phosphatidylinositol 3-kinase (PI 3-kinase), Ser 473-phosphorylation of Akt and Thr202/Tyr204-phosphorylated p44/42 MAP kinase in 3T3L1 adipocytes and FAO hepatoma cells incubated with increasing concentrations of saquinavir for 24, 36 hours, 2, 3 and 6 days were measured. Results. Phosphorylation of IR-beta, IRS-1 and IRS-2 was not permanently affected by incubation with therapeutic doses (2.5 microM) of saquinavir for 36 hours. After 24 hours we observed an increase of IR-beta and IRS-1 phosphorylation. However, this initial stimulation of IR-beta and IRS-1 phosphorylation was not permanent and did not result in an increased PI 3-kinase association. Phosphorylation of IRS-2 and MAP kinase as well as glucose transport activity was not altered by therapeutic doses. Doses of 10, 25 and 50 microM of saquinavir altered the early insulin signaling events in a dose-dependent manner. However, this effect was primarily due to the cytotoxic effect of higher saquinavir doses. Glucose transport activity was not significantly reduced in 3T3L1 cells treated with 2.5 microM saquinavir in comparison to the control cells stimulated with insulin. CONCLUSION: Early insulin signaling cascade, essential for normal glucose metabolism, is not affected by therapeutic doses of saquinavir. The reduction of insulin-induced phosphorylation in higher concentrations is primarily related to cytotoxic effects. Other mechanisms than early insulin signaling must be primarily responsible for the metabolic alterations during saquinavir therapy.

Immune-mediated activation of the endocannabinoid system in visceral adipose tissue in obesity.

The aim of the study was to investigate if the endocannabinoid system (ECS) is activated in visceral adipose tissue and if adipose tissue inflammation affects the ECS activation state. Therefore, expression of fatty acid amide hydrolase (FAAH), cannabinoid receptor 1 (Cb1), adiponectin, and tumor necrosis factor (TNF)-alpha was compared in visceral adipose tissue from 10 normal-weight (BMI 24.4+/-1.1 kg/m2) and 11 obese subjects (BMI 37.6+/-13.6 kg/m2) using quantitative RT-PCR, and gene expression changes were analyzed after in vitro stimulation of visceral adipose tissue with TNF-alpha. The data demonstrate that the ECS is activated in obese visceral adipose tissue as shown by decreased FAAH, Cb1, and adiponectin expression. Obesity-related ECS activation is accompanied by elevated expression of the pro-inflammatory cytokine TNF-alpha, which in turn stimulates ECS activation in vitro. Our data show a strong association between adipose tissue inflammation and ECS activation in obesity, and indicate that a pro-inflammatory state may directly activate the ECS.

TNF-alpha alters visfatin and adiponectin levels in human fat.

Adiponectin and visfatin are newly discovered adipokines that are strongly expressed in human visceral adipose tissue. To identify new regulatory mechanisms in fat, the effect of TNF-alpha (TNF) on adiponectin, on its two receptors, and on visfatin was investigated by incubating human visceral adipose tissue from patients without diabetes mellitus with TNF for 24, 48 and 72 hours. The mRNA expression of visfatin, adiponectin, and its two receptors, as well as the protein expression of adiponectin were determined. A decrease of adiponectin mRNA expression of 97% after incubation with TNF (5.75 nmol/l) for 24 hours, a decrease of 91% after 48 hours, and a decrease of 96% after 72 hours were measured. The reduction of protein expression was measured to be 42% after 24 hours, 28% after 48 hours, and 39% after 72 hours of incubation with TNF (5.75 nmol/l). The mRNA level of adiponectin receptor 1 (AdipoR1) was elevated about 72% after 48 hours of incubation and 67% after 72 hours of incubation, whereas the mRNA expression of adiponectin receptor 2 (AdipoR2) was not altered significantly. The visfatin mRNA level was found to be highly increased by 255% after 24 hours and 335% after 48 hours and 341% after 72 hours of incubation with TNF (5.75 nmol/l). Our results support the concept of visceral adipose tissue as an endocrine organ. We demonstrate that TNF has regulatory functions on adiponectin, AdipoR1 and on visfatin in human visceral adipose tissue. TNF levels are elevated in states of obesity and insulin resistance. Due to this fact TNF could be the reason that there is a decrease in the level of adiponectin, whereas there is an increase in the level of visfatin in states of obesity and insulin resistance.

Neural driven angiogenesis by overexpression of nerve growth factor.

Mechanisms regulating angiogenesis are crucial in adjusting tissue perfusion on metabolic demands. We demonstrate that overexpression of nerve growth factor (NGF) in brown adipose tissue (BAT) of NGF-transgenic mice elevates both mRNA and protein levels of vascular endothelial growth factor (VEGF) and VEGF-receptors. Increased vascular permeability, leukocyte-endothelial interactions (LEI), and tissue perfusion were measured using intravital microscopy. NGF-stimulation of adipocytes and endothelial cells elevates mRNA expression of VEGF and its receptors, an effect blocked by NGF neutralizing antibodies. These data suggest an activation of angiogenesis as a result of both: stimulation of adipozytes and direct mitogenic effects on endothelial cells. The increased nerve density associated with vessels strengthened our hypothesis that tissue perfusion is regulated by neural control of vessels and that the interaction between the NGF and VEGF systems is the critical driver for the activated angiogenic process. The interaction of VEGF- and NGF-systems gives new insights into neural control of organ vascularization and perfusion.

Met326Ile aminoacid polymorphism in the human p85 alpha gene has no major impact on early insulin signaling in type 2 diabetes.

Class I alpha phosphatidylinositol (PI) 3-kinase is an important enzyme in the early insulin signaling cascade, and plays a key role in insulin-mediated glucose transport. Despite extensive investigation, the genes responsible for the development of the common forms of type 2 diabetes remain unknown. This study was performed to identify variants in the coding region of p85 alpha, the regulatory subunit of PI 3-kinase. Fibroblasts from skin biopsies from type 2 diabetics and controls were established to address this issue. P85 alpha cDNA was sequenced, and a single point mutation at codon 326 was found. This mutation resulted in a homozygous missense amino acid change Met --> Ile in one subject with type 2 diabetes and heterozygous variant in two other diabetic patients and one with severe insulin resistance. Interestingly, those patients revealed an impaired insulin-mediated insulin receptor substrate (IRS)-1 binding to p85 alpha without any alteration in IRS-2/p85 alpha association. Furthermore, IRS-1, IRS-2, p85 alpha and MAPK protein contents were not significantly changed, and neither were MAPK or Akt phosphorylation. We conclude from our data that this variant may have only minor impact on signaling events; however, in combination with variants in other genes encoding signaling proteins, this may have a functional impact on early insulin signaling.

A new model of primary human adipocytes reveals reduced early insulin signalling in type 2 diabetes.

The aim of this study was to establish a diabetic model of primary human adipocytes for investigating potential defects in early insulin signalling. Specimens of human subcutaneous adipose tissue were obtained during orthopaedic surgical procedures. Preadipocytes were isolated and differentiated to adipocytes. Western blot analysis and immunoprecipitation were performed to determine protein content of IRS-1, IRS-2, p85, phosphorylation of IRS-1, IRS-2, Akt and MAPK as well as association between p85 and IRS-1/IRS-2. In addition to short-term insulin stimulation, the effect of hyperinsulinaemia was investigated by treating cells with insulin over a period of 36 hours. We found a significantly reduced basal expression of IRS-1 (54 +/- 15%) in adipocytes from type 2 diabetic subjects compared to controls with a further significant reduction in expression after long-term treatment (30 +/- 12%) compared to short-term treatment. IRS-2 expression also showed a significant reduction under hyperinsulinaemic conditions (20 +/- 2%) in diabetics vs. controls. Furthermore, long-term treatment with insulin in diabetic adipocytes led to a significant reduction in the phosphorylation of IRS-1(68 +/- 11%), IRS-2 (82 +/- 11%), Akt (42 +/- 2%), and MAPK (92 +/- 12%) and in the subsequent association between p85 to IRS-1 and IRS-2 (100 +/- 16% and 96 +/- 12%) in comparison to controls. Investigating glucose uptake diabetic adipocytes revealed a significant reduction of 90 +/- 2%. In this study, we were able to establish a new diabetic model of primary human adipocytes. A defect in early insulin signalling in type 2 diabetic patients under hyperinsulinaemic conditions was determined. These results might help to give further insights in early insulin action; additionally, this human model represents a useful target for the study of new therapeutic approaches.