Stefan Welte

Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

ABSTRACT Caveolae and caveolin-containing detergent-insoluble glycolipid-enriched rafts (DIG) have been implicated to function as plasma membrane microcompartments or domains for the preassembly of signaling complexes, keeping them in the basal inactive state. So far, only limited in vivo evidence is available for the regulation of the interaction between caveolae-DIG and signaling components in response to extracellular stimuli. Here, we demonstrate that in isolated rat adipocytes, synthetic intracellular caveolin binding domain (CBD) peptide derived from caveolin-associated pp59Lyn (10 to 100 μM) or exogenous phosphoinositolglycan derived from glycosyl-phosphatidylinositol (GPI) membrane protein anchor (PIG; 1 to 10 μM) triggers the concentration-dependent release of caveolar components and the GPI-anchored protein Gce1, as well as the nonreceptor tyrosine kinases pp59Lyn and pp125Fak, from interaction with caveolin (up to 45 to 85%). This dissociation, which parallels redistribution of the components from DIG to non-DIG areas of the adipocyte plasma membrane (up to 30 to 75%), is accompanied by tyrosine phosphorylation and activation of pp59Lyn and pp125Fak (up to 8- and 11-fold) but not of the insulin receptor. This correlates well to increased tyrosine phosphorylation of caveolin and the insulin receptor substrate protein 1 (up to 6- and 15-fold), as well as elevated phosphatidylinositol-3′ kinase activity and glucose transport (to up to 7- and 13-fold). Insulin-mimetic signaling by both CBD peptide and PIG as well as redistribution induced by CBD peptide, but not by PIG, was blocked by synthetic intracellular caveolin scaffolding domain (CSD) peptide. These data suggest that in adipocytes a subset of signaling components is concentrated at caveolae-DIG via the interaction between their CBD and the CSD of caveolin. These inhibitory interactions are relieved by PIG. Thus, caveolae-DIG may operate as signalosomes for insulin-independent positive cross talk to metabolic insulin signaling downstream of the insulin receptor based on redistribution and accompanying activation of nonreceptor tyrosine kinases.

Identification of a novel AS160 splice variant that regulates GLUT4 translocation and glucose-uptake in rat muscle cells

AS160 (AKT substrate of 160 kDa) is an important mediator of GLUT4 (glucose transporter 4) translocation and glucose-uptake in adipocytes and muscle cells. In our study we have identified a novel splice variant of AS160 (variant 2 of AS160, AS160_v2) that lacks exon 11 and 12. The protein is phosphorylated in response to insulin via the PI3K/AKT pathway. Expression of this splice variant in human tissues from different donors was examined with quantitative RT-PCR. Our data reveal a tissue specific distribution pattern of both isoforms with highest overall expression of AS160_v2. To investigate the function of the novel splice variant we established the doxycycline-inducible expression of the protein in a rat myoblast cell line co-expressing GLUT4-myc. In contrast to data reported for the full-length AS160 protein, over expression and activation of transcript variant 2 in this cell line increased GLUT4 translocation and glucose-uptake rates in response to insulin and IGF-1 but not in response to AICAR or metformin. Immunofluorescence based studies indicated a direct association of AS160_v2 with GLUT4 under basal but not under insulin-stimulated conditions. Additionally, over expression of AS160_v2 slightly improved glucose-uptake rates in a model of insulin resistance but was not able to fully prevent induction of insulin resistance. This was accompanied with decreased phosphorylation of AS160_v2 and AKT. Taken together, our data suggest a tissue specific distribution of full-length AS160 and the novel AS160 splice variant (AS160_v2) indicating different functions. In contrast to full-length AS160, transcript variant 2 of AS160 seems to be a novel regulator of glucose transport that positively influences glucose-uptake rates.