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.

Vascular alterations are the most common causes of morbidity and mortality in diabetic patients. Despite the impact of endothelial dysfunction on microcirculatory properties, little is known about the endothelial cell alteration during the development of diabetes and its correlation to the metabolic situation. For that reason we continuously monitored in vivo functional and morphological alterations of the microvasculature in hyperglycemic and hyperinsulinemic transgenic UCP1/DTA mice with brown fat deficiency, using a dorsal skin-fold chamber preparation and fluorescence microscopy. UCP1/DTA mice showed a dramatic decrease in vascular density due to a remarkable reduction of small vessels. Vascular permeability and leukocyte endothelial interactions (LEIs) significantly increased. The extent of vascular alteration correlated with the extent of metabolic dysfunction. Decreased tissue perfusion observed in UCP1/DTA mice might play a role in impaired wound healing observed in diabetes. The increased permeability in subcutaneous tissue may serve as predictor of vascular changes in early stages of diabetes. The increased LEI and serum tumor necrosis factor-␣ levels, which mirror the inflammatory process, support the growing evidence of the inflammatory component of diabetic disease. The results suggest that anti-inflammatory strategies might be able to prevent vascular deterioration in early stages of diabetes. Further investigations are required to evaluate the benefit of such therapeutic strategies. Diabetes 52:542-549, 2003 V ascular alterations are the most common causes of morbidity and mortality in diabetic patients. The microcirculation not only governs the efficacy of substrate delivery but also mediates adaptations to changing local requirements and metabolic conditions. Functional alterations of the microcirculation precede morphological changes and determine the resultant vascular morphology (1). Microvascular disease has been shown to have a high prevalence in diabetes (2,3). Several studies described endothelial dysfunction and functional alterations in the microcirculation of diabetic patients. Animal models of diabetes show increased vascular permeability (4), alterations in erythrocyte velocity (5), sequestration of leukocytes in the microcirculation (5-7), and morphological alterations such as altered vascular density (5). These alterations are mainly described as the result of hyperglycemia and advanced glycation end products (8,9) and develop sequentially. Functional alterations, such as increased microvascular permeability and increased entrapment of leukocytes, have been described as an early event in diabetes and in animal models and could be partially observed after only a few hours of hyperglycemia (4,7). Morphological alterations, such as altered microvascular density and diameter, appear later (5,10). However, mechanisms that lead to microangiopathies in diabetic patients remain only partially understood.Monitoring of microcirculatory alterations in patients is limited by the invasive chara...

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.

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.

Objective: The a 2 -adrenergic receptors are involved in the effects of catecholamines on energy metabolism. Of three known subtypes with differential expression, a 2A -adrenergic receptors are also localized in adipose tissue where they counteract the lipolytic activity of b-adrenergic receptors. This study was undertaken to assess whether variants in the a 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 a 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 a 2A -adrenergic receptor gene are unlikely to contribute to the predisposition for the lean or obese state.

We investigated whether the angiogenic profile, which is based on the local expression and systemic levels of angiogenic growth factors (VEGF, Ang-1, Ang-2, and the corresponding receptors), differs between rheumatoid arthritis (RA) and osteoarthritis (OA) patients. We determined the expression of VEGF, Ang-1, and Ang-2 together with its receptors (VEGFR-1/-2 and Tie2) in synovium tissue (ST) and muscular tissue (MT) from patients with RA and OA using quantitative PCR. Tissue samples were obtained from 15 RA and 19 OA patients during total knee arthroplasty. Control MT samples (n = 10) were obtained during spinal surgery. Results are correlated to VEGF and angiopoietin serum levels via ELISA measurements. The VEGF expressions in ST and serum levels were significantly higher in RA patients than in OA patients (P < 0.05). Furthermore, the VEGFR-1 and VEGFR-2 expression in ST from RA patients were significantly higher than in OA patients (P < 0.001 and P < 0.05). The relative concentration of angiopoietins (Ang-1/Ang-2 ratio) was significantly increased in RA (P < 0.01). Serum levels for Ang-2 showed no significant differences. Statistical analysis showed a significant higher level of Tie2 in RA patients (P < 0.001). Analysis of local levels of VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, and Tie2 in the muscular tissue showed no significant difference between RA and OA patients. These results underline the importance of pro-angiogenic growth factor levels for RA corroborating the assumption that VEGF and angiopoietins play an important role in the pathogenesis of RA.

Angiogenesis is essential for wound healing and proliferative processes such as bone formation and repair. Since increased expression of the vascular endothelial growth factor (VEGF) stimulates bone formation, it can be hypothesized that surgical procedures leading to a systemic increase of VEGF for instance during wound healing, influence enchondral ossification processes and might be responsible for observed growth phenomena during callus distraction. To study the mechanisms of angiogenesis in soft tissue during unilateral callus distraction, lengthening of the right tibia was performed in 12 beagles. After osteotomy, application of a ring fixator and after five latency days, distraction was started for 25 days. A control group of four additional beagles underwent no surgical procedure. Subsequent to the distraction period (Group A), muscle samples from six beagles were taken from the distracted side (ds) and the contralateral non-distracted side (n-ds), six beagles underwent an additional consolidation period of 25 days (Group B). Samples were analyzed for VEGF, VEGFR-1 and VEGFR-2 mRNA expression using real-time PCR and protein expression using Western Blot analysis. Muscles from both extremities showed significantly increased expression of VEGF and its cognate receptors VEGFR-112. Expression decreased significantly after the consolidation period, whereby the level at the nondistracted side decreased more than the level at the distracted side. Interestingly VEGF and VEGFR-I levels at the non-distracted side were significantly higher than at the distracted side. In contrast VEGFR-2, the receptor that mediates endothelial cell proliferation, showed higher levels at the distracted than at the non-distracted side. These findings indicate that callus distraction results not only in locally increased expression of VEGF and its receptors, but leads also to increased VEGF and VEGFR-112 levels at distant sides and might therefore be responsible for the observed growth phenomena during callus distraction.

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.