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Insulin acutely up-regulates p85␣ phosphatidylinositol 3-kinase (p85␣PI 3-K) mRNA levels in human skeletal muscle (Laville, M., Auboeuf, D., Khalfallah, Y., Vega, N., Riou, J. P., and Vidal, H. (1996) J. Clin. Invest. 98, 43-49). In the present work, we attempted to elucidate the mechanism of action of insulin in primary cultures of human muscle cells. Insulin (10 ؊7 M, 6 h of incubation) induced a 2-fold increase in p85␣PI 3-K mRNA abundances (118 ؎ 12 versus 233 ؎ 35 amol/g total RNA, n ‫؍‬ 5, p < 0.01) without changing the expression levels of insulin receptor, IRS-1, glycogen synthase, and Glut 4 mRNAs in differentiated myotubes from healthy subjects. The effect is most probably due to a transcriptional activation of the p85␣PI 3-K gene because the half-life of the mRNA was not affected by insulin treatment (4.0 ؎ 0.8 versus 3.1 ؎ 0.4 h). PD98059 (50 M) did not modify the insulin response but increased p85␣PI 3-K mRNA levels in the absence of insulin, suggesting that the mitogen-activated protein kinase pathway exerts a negative effect on p85␣PI 3-K mRNA expression in the absence of the hormone. On the other hand, the insulin effect was totally abolished by LY294002 (10 M) and rapamycin (50 nM). In addition, overexpression of a constitutively active protein kinase B increased p85␣PI 3-K mRNA levels. These results indicate that the phosphatidylinositol 3-kinase/PKB/p70S6 kinase pathway is required for the stimulation by insulin of p85␣PI 3-K gene expression in human muscle cells. Phosphatidylinositol (PI)1 3-kinase (EC 2.7.1.67) is one of the key components of insulin signaling (1, 2). This lipid kinase is activated when the SH2 domains of its p85 regulatory subunit bind to the insulin receptor substrates (IRS) on specific tyrosine-phosphorylated sites (1-3). PI 3-kinase phosphorylates the D-3 position of the inositol ring of phosphoinositides (1, 2), generating potential second messengers that participate in the activation of protein kinase B (PKB) and p70 S6 kinase (p70S6K) (4, 5). Mainly using inhibitors, like LY294002 and wortmannin (inhibitors of PI 3-kinase) and rapamycin (inhibitor of p70S6K) (6 -8), the PI 3-kinase pathway was demonstrated to participate in a variety of insulin effects. These include (but are not limited to) the regulation of glucose uptake, the activation of glycogen synthesis, the inhibition of lipolysis, the control of protein synthesis, the inhibition of apoptosis, and the insulin-induced membrane ruffling (1, 2).The PI 3-kinase involved in insulin actions belongs to the class 1a of heterodimeric p85/p110 PI 3-kinases (9). The p85 regulatory subunit is an adaptator protein that links the p110 catalytic subunit to upstream signaling molecules. Two different genes coding highly homologous p85 (␣ and ␤) have been identified in mammalians, but the p85␣ regulatory subunit (p85␣PI 3-K) has been studied in more detail (2, 9). In cultured cells, expression of mutants of p85␣PI 3-K dramatically altered the response to insulin, supporting a major role of this subunit in the transduction of ...

Section: Discussionsupporting

confidence: 61%

Section: Phosphatidylinositol (Pi)mentioning

confidence: 99%

A Phosphatidylinositol 3-Kinase/p70 Ribosomal S6 Protein Kinase Pathway Is Required for the Regulation by Insulin of the p85α Regulatory Subunit of Phosphatidylinositol 3-Kinase Gene Expression in Human Muscle Cells

Roques

Vidal

1999

“…Like in skeletal muscle (11,32) insulin usually causes a doubling in surface GLUT4myc in L6-GLUT4myc muscle cells (26), out of a compartment that is largely segregated away from constitutively recycling proteins (26,33). In the present study, we implement the use of membrane lawns to quantify surfaceassociated GLUT4myc.…”

Section: Resultsmentioning

confidence: 99%

Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

Ishiki

Randhawa

Poon

et al. 2005

Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) to intact cells significantly increased lawnassociated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P 3 causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.Insulin promotes the uptake of glucose into muscle and fat tissues through a rapid gain in surface-bound glucose transporters (1-3). The muscle-and fat-specific glucose transporter GLUT4 1 cycles continuously between the plasma membrane and intracellular stores, with the steady-state distribution largely favoring the latter. Insulin changes this steady-state resulting in a net gain in surface GLUT4 (4 -6) largely as a result of enhancing the exocytic rate of GLUT4 cycling (7,8). Of significance, insulin resistance and diabetes are accompanied by defective GLUT4 gain at the plasma membrane of muscle and fat cells (9 -11).It is well established that signaling from class IA phosphatidylinositol (PI) 3-kinase is required for the insulin-dependent net gain in surface GLUT4 (12-15), but the specific step(s) in GLUT4 cycling that are regulated are not elucidated. We have recently shown that class IA PI 3-kinase is required for the insulin-dependent acceleration of GLUT4 transit through the recycling endosome (16). A second input of class IA PI 3-kinase in muscle cells is the spatial-temporal actin remodeling and its possible contribution to segregating specific signaling molecules (17, 18). However, it is not known whether fusion of insulin-sensitive GLUT4 vesicles with the plasma membrane is a regulated step, nor w...

“…Although the levels of GLUT4 protein are unaffected in states of insulin resistance, there is a marked impairment in the ability of insulin to induce its translocation from intracellular storage sites to the cell surface membrane (48). At the cellular level, multiple studies have suggested that insulin resistance involves decreases in insulin receptor-tyrosine kinase activity, IRS tyrosine phosphorylation, and/or activation of PI 3-kinase activity (25)(26)(27)(28)49). Other defects reported in muscle from obese and type 2 diabetic muscle include decreases in insulin-stimulated PKC/ activities as well as reduction in AS160 phosphorylation (30 -32, 50, 51).…”

Section: Discussionmentioning

confidence: 99%

“…In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25,26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27)(28)(29)(30)(31)(32)(33).…”

mentioning

confidence: 99%

Insulin Resistance in Striated Muscle-specific Integrin Receptor β1-deficient Mice

Zong

Bastie

et al. 2009

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin ␤1 subunit in striated muscle results in a near complete loss of integrin ␤1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin ␤1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemic-hyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulinstimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR⅐Rictor⅐LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin ␤1 signaling events in mediating cross-talk to that of insulin action.