Ceramide- and Oxidant-Induced Insulin Resistance Involve Loss of Insulin-Dependent Rac-Activation and Actin Remodeling in Muscle Cells

Abstract

In muscle cells, insulin elicits recruitment of the glucose transporter GLUT4 to the plasma membrane. This process engages sequential signaling from insulin receptor substrate (IRS)-1 to phosphatidylinositol (PI) 3-kinase and the serine/threonine kinase Akt. GLUT4 translocation also requires an Akt-independent but PI 3-kinase-and Racdependent remodeling of filamentous actin. Although IRS-1 phosphorylation is often reduced in insulin-resistant states in vivo, several conditions eliciting insulin resistance in cell culture spare this early step. Here, we show that insulin-dependent Rac activation and its consequent actin remodeling were abolished upon exposure of L6 myotubes beginning at doses of C2-ceramide or oxidant-producing glucose oxidase as low as 12.5 mol/l and 12.5 mU/ml, respectively. At 25 mol/l and 25 mU/ml, glucose oxidase and C2-ceramide markedly reduced GLUT4 translocation and glucose uptake and lowered Akt phosphorylation on Ser473 and Thr308, yet they affected neither IRS-1 tyrosine phosphorylation nor its association with p85 and PI 3-kinase activity. Small interfering RNA-dependent Rac1 knockdown prevented actin remodeling and GLUT4 translocation but spared Akt phosphorylation, suggesting that Rac and actin remodeling do not contribute to overall Akt activation. We propose that ceramide and oxidative stress can each affect two independent arms of insulin signaling to GLUT4 at distinct steps, Rac-GTP loading and Akt phosphorylation. Diabetes 56:394 -403, 2007 I nsulin promotes dietary glucose disposal into skeletal muscle through recruitment of GLUT4-containing vesicles to the cell surface. Signaling to GLUT4 requires tyrosine phosphorylation of the insulin receptor substrate (IRS)-1 isoform, which recruits and activates phosphatidylinositol (PI) 3-kinase (1). The latter triggers activation of several serine/threonine kinases, notably Akt, which, through its substrate AS160, regulates GLUT4 vesicle mobilization to and/or fusion with the plasma membrane (2).Along with proper signaling, GLUT4 translocation and stimulation of glucose uptake require dynamic changes in the actin cytoskeleton. Insulin induces actin filament remodeling in mature skeletal muscle (3) and muscle cells in culture (4) that manifest as mesh-like structures beneath the plasma membrane. Actin filament-disrupting drugs (e.g., cytochalasin D and latrunculin B) and actin-stabilizing drugs (e.g., jasplakinolide) inhibit GLUT4 translocation and its consequent glucose uptake in muscle (4,5) and adipose (6,7) cells, as do toxins that inhibit Rho family GTPases that control actin dynamics (8). Interestingly, under these conditions, IRS-1 phosphorylation and PI 3-kinase activity remain unaffected (9 -11). In muscle cells, actin remodeling was also prevented by wortmannin or expression of a dominant-negative mutant of the p85 subunit of class I PI 3-kinase (5), but not by a dominantnegative Akt mutant (12). Hence, insulin signaling bifurcates downstream of PI 3-kinase, one arm leading to actin remodeling and another to Akt activation, bo...

From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy

“…Similar results were obtained in adipose tissue and 3T3-L1 adipocytes [164,165]. Additional evidences revealed that, in L6 myotubes, oxidative stress prevents insulin-induced actin reorganization [166]. Consequently, the molecular traffic required for insulin action could be regulated in a redox-dependent manner.…”

Inhibition or Ablation of p21-activated Kinase (PAK1) Disrupts Glucose Homeostatic Mechanisms in Vivo

“…More recent data extend this by showing PAK1 to be downstream of Rac1, and a Rac1 knock-out mouse exhibits defective insulin stimulated GLUT4 translocation (22). Because there is no evidence for the participation of Cdc42 in insulin action in skeletal muscle, it is very likely that Rac1 is the Rho family GTPase that signals to PAK1 in this tissue (20,22). Insulin was recently shown to reduce the amount of phosphocofilin in L6 rat skeletal myoblasts (19).…”

Positive and Negative Regulation of Insulin Signaling by Reactive Oxygen and Nitrogen Species

“…In those, when assessed in total cell lysates (i.e., when subcellular compartments are disregarded; these will be discussed in sect. IVC), intact IR and IRS proteins tyrosine phosphorylation were observed, as well as an intact insulin-stimulated interaction between IRS proteins and PI 3-kinase (211,357,429). In fact, these steps following insulin stimulation were even reported to be exaggerated in response to preexposure to oxidants, possibly reflecting the oxidant-induced inactivation of cellular phosphatases, particularly PTP1B (see sect.…”