Biomedical subjects

M Fasshauer

Publications and source records attributed to M Fasshauer.

25 records · Page 2Linked to original sources

PubMed2001

Isoproterenol inhibits resistin gene expression through a G(S)-protein-coupled pathway in 3T3-L1 adipocytes.

Resistin was recently identified as a hormone secreted by adipocytes which leads to insulin resistance in vivo and in vitro and might therefore be an important link between obesity and diabetes. To clarify the regulation of resistin gene expression, 3T3-L1 adipocytes were treated with various agents known to modulate insulin sensitivity, and resistin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, isoproterenol treatment reduced the level of resistin mRNA to 20% of non-treated control cells. This effect was dose-dependent with significant inhibition occurring at concentrations as low as 10 nM isoproterenol. Moreover, pretreatment of adipocytes with the beta-adrenergic antagonist propranolol almost completely reversed the inhibitory effect of isoproterenol, whereas addition of the alpha-adrenergic antagonist phentolamine did not have any effect. Furthermore, the effect of isoproterenol could be mimicked by activation of G(S)-proteins and adenylyl cyclase. Thus, both cholera toxin and forskolin decreased resistin mRNA expression in a dose-dependent fashion by up to 90% of control levels. Taken together, these results suggest that resistin gene expression is regulated by a protein kinase A-dependent pathway in 3T3-L1 adipocytes.

3T3 Cells↗

PubMed2001

Essential role of insulin receptor substrate 1 in differentiation of brown adipocytes.

The most widely distributed members of the family of insulin receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of IRS-1 in adipocyte biology, we established brown adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals. Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and lipid accumulation (10 to 40%) compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO cells showed decreased expression of adipogenic marker proteins, such as peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha (C/EBPalpha), fatty acid synthase, uncoupling protein-1, and glucose transporter 4. The differentiation deficit in the KO cells could be reversed almost completely by retrovirus-mediated reexpression of IRS-1, PPARgamma, or C/EBPalpha but not the thiazolidinedione troglitazone. Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various stages of the differentiation process revealed a strong and transient activation in IRS-1, IRS-2, and phosphotyrosine-associated PI 3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed impaired phosphotyrosine-associated PI 3-kinase activation, all of which was associated with IRS-2. Akt phosphorylation was reduced in parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase with LY294002 blocked differentiation of wild-type cells. Thus, IRS-1 appears to be an important mediator of brown adipocyte maturation. Furthermore, this signaling molecule appears to exert its unique role in the differentiation process via activation of PI 3-kinase and its downstream target, Akt, and is upstream of the effects of PPARgamma and C/EBPalpha.

Adipocytes↗

PubMed2000

Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes.

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p < 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.

Adipocytes↗

PubMed2000

Insulin and the beta3-adrenoceptor differentially regulate uncoupling protein-1 expression.

Cross-talk between insulin and the adrenergic system is important in the regulation of energy homeostasis. In cultured, differentiated mouse brown adipocytes, beta3-adrenergic stimulation induced a 4.5-fold increase in uncoupling protein-1 (UCP-1) expression, which was diminished by 25% in the presence of insulin. Beta3-adrenergic stimulation also activated mitogen-activated protein (MAP) kinase by 3.5-fold and caused a decrease in basal phosphoinositide (PI) 3-kinase activity detected in p110gamma- and Gbeta-subunit-immunoprecipitates in a time-dependent manner, whereas insulin stimulated p110alpha- and phosphotyrosine-associated PI 3-kinase activity. Inhibition of MAP kinase or PI 3-kinase potentiated the beta3-adrenergic effect on UCP-1 expression, both alone and in the presence of insulin. Thus, insulin inhibits beta3-adrenergic stimulation of UCP-1, and both MAP kinase and PI 3-kinase are negative regulatory elements in the beta3-adrenergic control of UCP-1 expression. Cross-talk between the adrenergic and insulin signaling systems and impaired regulation of UCP-1 might contribute to the development of a reduced energy balance, resulting in obesity and insulin resistance.

Adipocytes↗

PubMed1999

beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes.

Activity of the sympathetic nervous system is an important factor involved in the pathogenesis of insulin resistance and associated metabolic and vascular abnormalities. In this study, we investigate the molecular basis of cross-talk between beta(3)-adrenergic and insulin signaling systems in mouse brown adipocytes immortalized by SV40 T infection. Insulin-induced tyrosine phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and IRS-2 was reduced by prestimulation of beta(3)-adrenergic receptors (CL316243). Similarly, insulin-induced IRS-1-associated and phosphotyrosine-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity, but not IRS-2-associated PI 3-kinase activity, was reduced by beta(3)-adrenergic prestimulation. Furthermore, insulin-stimulated activation of Akt, but not mitogen-activated protein kinase, was diminished. Insulin-induced glucose uptake was completely inhibited by beta(3)-adrenergic prestimulation. These effects appear to be protein kinase A-dependent. Furthermore inhibition of protein kinase C restored the beta(3)-receptor-mediated reductions in insulin-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Together, these findings indicate cross-talk between adrenergic and insulin signaling pathways. This interaction is protein kinase A-dependent and, at least in part, protein kinase C-dependent, and could play an important role in the pathogenesis of insulin resistance associated with sympathetic overactivity and regulation of brown fat metabolism.

Adipocytes↗

PubMed1998

Synthesis of proto-oncogene proteins and cyclins depends on intact microfilaments.

It is well established that microfilament disintegration by cytochalasin D (CD) as well as latrunculin (LAT)-A and LAT-B causes an inhibition of S phase entry of various nontransformed cell lines. Our experiments extended these observations to human embryonal diploid fibroblasts (Wi-38). To investigate the question whether this stop of DNA synthesis is due to a decline of the synthesis of proteins that are necessary for G1 progression and S phase entry, we examined the expression of two proto-oncogenes (c-fos, c-jun) and three cyclins (D1, E, A) after altering the microfilament system. Disintegration of microfilaments by CD, LAT-A, or LAT-B of asynchronously growing fibroblasts caused a strong dose-dependent and time-dependent inhibition of total protein synthesis. Expression of c-jun, cyclins D1, E, and A decreased by about the same percentage as total protein synthesis. The strong induction of total protein synthesis after reactivating serum-starved fibroblasts by adding fetal calf serum was suppressed, when CD or LAT-A were added to the culture medium during this reactivation process. While expression of cyclin E as well as cyclin A decreased by about the same percentage as total protein synthesis, cyclin D1 was more suppressed after microfilament disintegration. After reactivating growth-arrested Wi-38 fibroblasts, cultured in suspension for 12 h, by transferring them to a rigid substratum they could adhere to, total protein synthesis was strongly induced. Again alteration of microfilaments by CD suppressed that increase. The expression of cyclin D1 was slightly more suppressed than total protein synthesis after addition of CD during that reactivation process. Our results suggest that alteration of microfilaments causes a strong decline of total protein synthesis accompanied by a decrease of the expression of proteins that are required for G1 progression and S phase entry. The diminished presence of proteins that are important for cell cycle progression could explain the inhibition of DNA synthesis after microfilament disintegration by various drugs.

Actin Cytoskeleton↗

PubMedDate not supplied

Direct peripheral effects of ghrelin include suppression of adiponectin expression.

The stomach-derived peptide, ghrelin, has recently been discovered as an important regulator of energy homeostasis. Central nervous system pathways involving stimulation of hypothalamic neuropeptides play a prominent role in mediating ghrelin's orexigenic effects. However, potential direct peripheral effects remain poorly understood. Using a brown adipocyte model, we tested ghrelin-mediated influences on adipose tissue. Chronic ghrelin stimulation of differentiating adipocytes did not affect the pattern or extent of fat accumulation. Furthermore, insulin-induced glucose uptake as a hallmark of adipocyte function was not altered by ghrelin pre-treatment. However, acute ghrelin treatment resulted in a significant time-dependent increase in p44/42 mitogen-activated protein kinase phosphorylation. There was no stimulation of phosphatidylinositol 3-kinase, JAK/STAT, or stress kinase signaling pathways. Furthermore, ghrelin did not significantly alter gene expression of the thermogenic uncoupling protein-1. By contrast, expression of the novel adipokine adiponectin, which has been implicated in the pathogenesis of insulin resistance and obesity, was strongly impaired. This inhibition occurred acutely, and was sustained for several hours. In summary, our data provide evidence for selective effects of ghrelin on adipocyte signaling and function and thus propose a role for adipose tissue as a novel mediator of ghrelin's effects on energy balance and glucose homeostasis.

Adipocytes↗