Defective Insulin-Induced GLUT4 Translocation in Skeletal Muscle of High Fat–Fed Rats Is Associated With Alterations in Both Akt/Protein Kinase B and Atypical Protein Kinase C (/) Activities Fre ´de ´ ric Tremblay, 1,2 Charles Lavigne, 2,3 He ´le ` ne Jacques, 2,3 and Andre ´ Marette 1,2 The cellular mechanism by which high-fat feeding in- duces skeletal muscle insulin resistance was investi- gated in the present study. Insulin-stimulated glucose transport was impaired (40 – 60%) in muscles of high fat–fed rats. Muscle GLUT4 expression was significantly lower in these animals (40%, P < 0.05) but only in type IIa– enriched muscle. Insulin stimulated the trans- location of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abro- gated in the muscle of insulin-stimulated high fat–fed rats. High-fat feeding markedly decreased insulin recep- tor substrate (IRS)-1–associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS pro- teins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (40%, P < 0.01) in insulin-stimulated muscle of high fat–fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was ele- vated in muscle of high fat–fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chow- fed rats, the hormone failed to further increase the kinase activity in high fat–fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat–fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities. Diabetes 50:1901–1910, 2001 I nsulin resistance represents a major pathogenic impairment in the development of type 2 diabetes (1,2). In humans and rodents, skeletal muscle is the primary site of insulin-mediated glucose disposal (2). Insulin increases glucose uptake in muscle by eliciting GLUT4 translocation from an intracellular storage site to both the plasma membrane and the transverse (T)-tubules through a complex signaling cascade (3–5). Impaired GLUT4 translocation has been shown to be linked to reduced glucose utilization in muscle of insulin-resistant and type 2 diabetic subjects (6 – 8). However, the precise mechanism underlying the reduced stimulatory effect of insulin on glucose transport is still unclear. Both receptor and pos- treceptor defects have been observed in various models of insulin resistance (9). Insulin stimulates GLUT4 translocation by binding its receptor -subunits, leading to autophosphorylation of the transmembrane -subunits and intrinsic activation of re- ceptor tyrosine kinase activity. In skeletal muscle, the activated insulin receptor (IR) increases the tyrosine phos- phorylation of IR substrate (IRS)-1 and IRS-2, leading to activation of phosphatidylinositol (PI) 3-kinase (4). It is believed that PI 3-kinase activation by insulin is essential for the stimulation of GLUT4 translocation. Indeed, a large number of studies have shown that inhibitors of PI 3-ki- nase (wortmannin and LY294002) or overexpression of a mutated p85 adapter subunit lacking the ability to bind the p110 catalytic subunit fully inhibit insulin-mediated GLUT4 translocation and glucose transport in adipocytes and skeletal muscle cells (rev. in [10]). Downstream effector(s) of PI 3-kinase involved in the regulation of glucose transport have yet to be clearly identified. Candidate molecules of interest include the serine/threonine kinase Akt (also termed protein kinase B [PKB] or related to A and C [RAC] protein kinase) and atypical protein kinase C (aPKC) (/). Both aPKC and Akt lie in the PI 3-kinase/3-phosphoinositide-dependent kinase (PDK)-1 signaling pathway, giving rise to phosphorylation on Thr410 and Thr308, respectively (11,12). Full activation of Akt further requires phosphorylation on Ser473 by the putative PDK-2 (13), whereas aPKC activity appeared to be dependent on autophosphorylation by an as yet unknown mechanism (14). Evidence for the implication of Akt and aPKC in the insulin-dependent regulation of glucose trans- From the 1 Department of Physiology and 2 Lipid Research Unit, Laval Univer- sity Hospital Research Center; and the 3 Department of Food Science and Nutrition, Human Nutrition Research Group, Laval University, Ste-Foy, Que ´- bec, Canada. Address correspondence and reprint requests to Andre ´ Marette, Depart- ment of Physiology and Lipid Research Unit, Laval University Hospital Research Center, 2705, Laurier Blvd., Ste-Foy, Que ´ bec, Canada, G1V 4G2. Email: andre.marette@crchul.ulaval.ca. Received for publication 30 June 2000 and accepted in revised form 7 May 2001. aPKC, atypical protein kinase C; DTT, dithiothreitol; 2-[ 3 H]DG, 2-deoxy- D-[ 3 H]glucose; IR, insulin receptor; IRS, insulin receptor substrate; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; PDK, 3-phospho- inositide-dependent kinase; PI, phosphatidylinositol; PKB, protein kinase B; PKC, protein kinase C; PVDF, polyvinylidene difluoride; RAC, related to A and C; RDU, relative densitometric units; T, transverse; TNF-, tumor necrosis factor-. DIABETES, VOL. 50, AUGUST 2001 1901