Biomedical subjects
Publications and source records attributed to Alan R Saltiel.
When chronically elevated, insulin increases hepatic lipogenesis and VLDL synthesis. However, the hormone reduces liver lipids when acutely elevated. Work in this issue of Cell Metabolism (Najjar at al., 2005) suggests a new mechanism for the inhibition of the rate-limiting enzyme in liver, fatty acid synthase.
3T3-L1 preadipocytes require cAMP for maximal differentiation. Microarray analysis reveals that the integrins alpha5 and alpha6 are coordinately regulated by cAMP. alpha5 expression is gradually diminished during adipogenesis, whereas alpha6 is increased. Overexpression of alpha5 in preadipocytes results in enhanced proliferation and attenuated differentiation. Conversely, alpha6 overexpression is without effect. The GTPase Rac is normally inhibited during differentiation. However, overexpression of integrin alpha5 increases Rac activity. Constitutively active but not dominant-negative Rac inhibits differentiation when overexpressed in preadipocytes, implying its role downstream of alpha5 integrin in maintaining preadipocytes in an undifferentiated state. Moreover, alpha6 integrin is critically involved in clustering growth-arrested preadipocytes on basement membrane Matrigel. Perturbation of such clustering enhances Rho activity and promotes growth-arrested preadipocytes to reenter the cell cycle. These findings demonstrate a role for integrin alpha6 in connecting morphogenesis with signaling processes leading to terminal differentiation.
BACKGROUND: Diabetes mellitus is an independent risk factor for early postoperative mortality and complications after coronary artery bypass grafting (CABG). We sought to compare the cardiac gene expression responses to cardiopulmonary bypass (CPB) and cardioplegic arrest (C) in patients with and without diabetes. METHODS AND RESULTS: Twenty atrial myocardium samples were harvested from 5 type II insulin-dependent diabetic and 5 matched nondiabetic patients undergoing CABG, before and after CPB/C. Oligonucleotide microarray analyses of 12625 genes were performed on the 10 sample pairs using matched pre-CPB tissues as controls. Array results were validated with Northern blotting and immunoblotting. Compared with pre-CPB/C, post-CPB/C myocardial tissues revealed 851 upregulated and 480 downregulated genes with a threshold P< or =0.025 (signal-to-noise ratio, 4.04) in the diabetic group, compared with 480 upregulated and 626 downregulated genes (signal-to-noise ratio, 3.04) in the nondiabetic group (P<0.001). There were 18 genes that were upregulated >4-fold in diabetic and nondiabetic patients (including inflammatory/transcription activators FOS, CYR 61, and IL-6, apoptotic gene NR4A1, stress gene DUSP1, and glucose-transporter gene SLC2A3). However, 28 genes showed such marked upregulation in the diabetic group exclusively (including inflammatory/transcription activators MYC, IL8, IL-1beta, growth factor vascular endothelial growth factor, amphiregulin, and glucose metabolism-involved gene insulin receptor substrate 1), and 27 genes in the nondiabetic group only, including glycogen-binding subunit PPP1R3C. CONCLUSIONS: Gene expression profile after CPB/C is quantitatively and qualitatively different in patients with diabetes. These results have important implications for the design of tailored myocardial protection and operative strategies for diabetic patients undergoing CPB/C.
Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCzeta/lambda) to plasma membrane lipid raft microdomains. This redistribution of PKCzeta/lambda was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCzeta/lambda to the plasma membrane through an indirect association with the Par6-Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCzeta. Although PI 3-kinase activation also resulted in PKCzeta/lambda phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3beta phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways. Together, these data demonstrate that PKCzeta/lambda can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.
Explore the source record for details and available documents.
Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation.
Insulin stimulates glucose uptake in fat and muscle cells via the translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the cell surface. The signaling pathways linking the insulin receptor to GLUT4 translocation in adipocytes involve activation of the Rho family GTPases TC10alpha and beta. We report here the identification of TCGAP, a potential effector for Rho family GTPases. TCGAP consists of N-terminal PX and SH3 domains, a central Rho GAP domain and multiple proline-rich regions in the C-terminus. TCGAP specifically interacts with cdc42 and TC10beta through its GAP domain. Although it has GAP activity in vitro, TCGAP is not active as a GAP in intact cells. TCGAP translocates to the plasma membrane in response to insulin in adipocytes. The N-terminal PX domain interacts specifically with phos phatidylinositol-(4,5)-bisphosphate. Overexpression of the full-length and C-terminal fragments of TCGAP inhibits insulin-stimulated glucose uptake and GLUT4 translocation. Thus, TCGAP may act as a downstream effector of TC10 in the regulation of insulin-stimulated glucose transport.
Mesenchymal stem cells can differentiate into muscle or fat cells, depending on the exposure to growth factors. The Rho GTPase appears to play a crucial role in this decision of cellular fate, promoting myogenesis and inhibiting adipogenesis.
Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.
The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.
Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.
Explore the source record for details and available documents.
To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.
Protein targeting to glycogen (PTG) is a scaffolding protein that targets protein phosphatase 1alpha (PP1alpha) to glycogen, and links it to enzymes involved in glycogen synthesis and degradation. We generated mice that possess a heterozygous deletion of the PTG gene. These mice have reduced glycogen stores in adipose tissue, liver, heart, and skeletal muscle, corresponding with decreased glycogen synthase activity and glycogen synthesis rate. Although young PTG heterozygous mice initially demonstrate normal glucose tolerance, progressive glucose intolerance, hyperinsulinemia, and insulin resistance develop with aging. Insulin resistance in older PTG heterozygous mice correlates with a significant increase in muscle triglyceride content, with a corresponding attenuation of insulin receptor signaling. These data suggest that PTG plays a critical role in glycogen synthesis and is necessary to maintain the appropriate metabolic balance for the partitioning of fuel substrates between glycogen and lipid.
The GTPase TC10 plays a critical role in insulin-stimulated glucose transport. We report here the identification of the TC10-interacting protein CIP4/2 (Cdc42-interacting protein 4/2) as an effector in this pathway. CIP4/2 localizes to an intracellular compartment under basal conditions and translocates to the plasma membrane on insulin stimulation. Overexpression of constitutively active TC10 brings CIP4/2 to the plasma membrane, whereas overexpression of an inhibitory form of TC10 blocks the translocation of CIP4/2 produced by insulin. Overexpression of mutant forms of CIP4/2 containing an N-terminal deletion or with diminished TC10 binding inhibits insulin-stimulated Glut4 translocation. These data suggest that CIP4/2 may play an important role in insulin-stimulated glucose transport as a downstream effector of TC10.
Our previous studies revealed that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3L1 adipocytes. To explore the mechanisms involved in this event, we evaluated the association of the insulin receptor with caveolin. The receptor was detected in a Triton-insoluble low density fraction, co-sedimenting with caveolin and flotillin on sucrose density gradients. We also detected the receptor in caveolin-enriched rosette structures by immunohistochemical analysis of plasma membrane sheets from 3T3L1 adipocytes. Insulin stimulated the phosphorylation of caveolin-1 on Tyr(14). This effect of the hormone was not blocked by overexpression of mutant forms of the Cbl-associated protein that block the translocation of phospho-Cbl to the caveolin-enriched, lipid raft microdomains. Moreover, this phosphorylation event was also unaffected by inhibitors of the MAPK and phosphatidylinositol 3-kinase pathways. Although previous studies demonstrated that the Src family kinase Fyn was highly enriched in caveolae, an inhibitor of this kinase had no effect on insulin-stimulated caveolin phosphorylation. Interestingly, overexpression of a mutant form of caveolin that failed to interact with the insulin receptor did not undergo phosphorylation. Taken together, these data indicate that the insulin receptor directly catalyzes the tyrosine phosphorylation of caveolin.
Insulin stimulates glucose transport via phosphatidylinositol 3-kinase-dependent and -independent pathways. The phosphatidylinositol 3-kinase-independent pathway involves activation of the G protein TC10. A cDNA encoding the mouse homolog of TC10 was cloned, and its gene was mapped at the distal end of chromosome 17. Additionally, a second gene was discovered with approximately 70% sequence identity to TC10. We refer to this gene as TC10beta. Both isoforms of TC10 were activated by insulin upon transfection in 3T3L1 adipocytes. Cotransfection of cells with TC10alpha or beta plus a dominant negative form of the c-cbl-associated protein CAP prevented the activation by insulin, implicating the CAP/Cbl pathway. Interestingly, both forms of TC10 were also localized in lipid raft fractions in transfected adipocytes. However, although overexpression of TC10alpha completely blocked glucose transport, TC10beta only partially inhibited this process. Furthermore, TC10alpha overexpression disrupted adipocyte cortical actin, whereas TC10beta had little if any effect. Thus, there are two isoforms of this key signaling intermediate, both of which are activated by insulin, but they may play different roles in initiating downstream effectors that influence glucose transport.