Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85␣ regulatory subunit yields three splicing variants, p85␣, AS53/p55␣, and p50␣. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110␣ catalytic subunit. PI 3-kinase activity associated with p50␣ was greater than that associated with p85␣ or AS53. Increasing the level of p85␣ or AS53, but not p50␣, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85␣ mutant lacking the p110-binding site (⌬p85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70 S6K ), was decreased in cells expressing p85␣ or AS53 but not in cells expressing p50␣. Similar inhibition of PI 3-kinase, Akt, and p70 S6K was observed, even when p110␣ was coexpressed with p85␣ or AS53. Expression of p110␣ alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110␣ was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.Upon stimulation, the activated insulin receptor tyrosine kinase phosphorylates several intracellular substrates, leading to stimulation of a wide variety of metabolic and mitogenic actions (20,37). This occurs via interaction between the phosphorylated insulin receptor substrate (IRS) proteins and a number of Src homology 2 (SH2) domain-containing proteins including Grb2, SHP2, and the class Ia phosphoinositide (PI) 3-kinase (37). A great deal of evidence has shown that PI 3-kinase plays a pivotal role in carbohydrate, lipid, and protein metabolism regulated by insulin (34). The mechanisms by which PI 3-kinase-dependent signaling mediates these metabolic effects are unclear, since these biological endpoints are quite specific for insulin, but ...

We have identified two novel alternatively spliced forms of the p85␣ regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus-produced human insulin receptor substrate 1. One form is identical to p85␣ throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85␣ on the 5 side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of ϳ53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85␣, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a ϳ7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85␣ itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85␣ regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.Insulin binding of the insulin receptor leads to the tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and Shc, which can then interact with and modulate the activity of a number of proteins via Src homology 2 (SH2) domains, including phosphatidylinositol (PI) 3-kinase, the protein tyrosine phosphatase SHPTP-2, and Grb2, which, acting through Sos can stimulate Ras GTPase activity (reviewed in references 6, 36, and 37). Recent studies have indicated an important role for PI 3-kinase, especially in insulin stimulation of glucose transport (18,20,22,28,39), p70 S6 kinase (20, 28), glycogen synthesis (27), and antilipolysis (22). PI 3-kinase is a heterodimer composed of an 85-kDa regulatory subunit (p85␣) and a 110-kDa catalytic subunit (p110␣). Isoforms of p85␣ and p110␣ have been identified and termed p85␤ (23) and p110␤. In addition, a G-protein-activated form of PI 3-kinase, termed p110␥, which is not dependent on the 85-kDa regulatory s...

PISCHON, TOBIAS, CHRISTOPH M. BAMBERGER, JÜ RGEN KRATZSCH, BIRGIT-CHRISTIANE ZYRIAX, PETRA ALGENSTAEDT, HEINER BOEING, AND EBERHARD WINDLER. Association of plasma resistin levels with coronary heart disease in women. Obes Res. 2005;13:1764 -1771. Objective: To examine the association between plasma resistin levels and the presence of coronary heart disease (CHD) in women. Research Methods and Procedures: Plasma resistin levels were measured in a case-control study including 185 women with angiographically confirmed CHD and 227 population-based female controls from the Coronary Risk Factors for Atherosclerosis in Women (CORA) study. Results: After adjustment for age, smoking, family history of myocardial infarction, retirement, education, physical activity, menopausal status, hormone replacement use, BMI, hypertension, diabetes, and dyslipidemia, the odds ratio for CHD for women in the highest compared with lowest quintile of plasma resistin levels was 3.19 (95% confidence interval, 1.44 to 7.10; p log trend, 0.001). After additional adjustment for plasma C-reactive protein levels, this association was substantially attenuated and no longer significant (odds ratio, 1.80; 95% confidence interval, 0.69 ti 4.69; p trend ϭ 0.23). Discussion: These results suggest that plasma resistin levels are significantly associated with the presence of CHD in women; however, this association can largely be explained by concomitant inflammatory processes. Further studies are needed to determine the causal role of resistin in the development of CHD in humans.

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...

3.

Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the EXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca 2؉ -ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca 2؉ -ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2-to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca 2؉ -ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.Insulin binding to the insulin receptor leads to the tyrosine phosphorylation of several insulin receptor substrates (IRS), 1 including IRS-1, IRS-2, IRS-3, Gab1, and Shc (1-3). These insulin receptor substrates then bind to a number of proteins in the cell via interaction of tyrosine phosphorylation motifs and specific domains of the target proteins termed SH2 (src homology 2) domains (4 -6). For IRS-1 and IRS-2, the SH2 proteins include the lipid-modifying enzyme phosphatidylinositol (PI) 3-kinase, the cytoplasmic tyrosine kinase Fyn, the protein tyrosine phosphatase SHP2, and the adaptor molecules Nck and Grb2 (7-12). Exactly how IRS binding to SH2 domain proteins results in signal transduction is still not understood, but for PI 3-kinase and SHP2, binding of phosphorylated IRS-1 results in stimulation of enzymatic activity (13,14). Grb2, on the other ...

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.