Protein Kinase C (PKC)- Activation Inhibits PKC- and Mediates the Action of PED/PEA-15 on Glucose Transport in the L6 Skeletal Muscle Cells Gerolama Condorelli, Giovanni Vigliotta, Alessandra Trencia, Maria Alessandra Maitan, Matilde Caruso, Claudia Miele, Francesco Oriente, Stefania Santopietro, Pietro Formisano, and Francesco Beguinot Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and im- pairs further insulin induction. Like glucose transport, protein kinase C (PKC)- and - are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6 PED ). PKC- features no basal change but completely loses insulin sensitivity in L6 PED . In these cells, blockage of PKC- and - additively returns 2-deoxy-D-glucose (2- DG) uptake to the levels of cells expressing only endog- enous PED (L6 WT ). Blockage of PKC- and - also restores insulin activation of PKC- in L6 PED cells, with that of PKC- sixfold more effective than PKC-. Simi- lar effects on 2-DG uptake and PKC- were also achieved by 50-fold overexpression of PKC- in L6 PED . In L6 WT , fivefold overexpression of PKC- or - increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor sub- strate phosphorylation. In these cells, overexpression of PKC- blocks insulin induction of PKC- activity. PKC- is 10-fold less effective than PKC- in inhibiting PKC- stimulation. Expression of the dominant-nega- tive K 281 3 W PKC- mutant simultaneously inhibits in- sulin activation of PKC- and 2-DG uptake in the L6 WT cells. We conclude that activation of classic PKCs, mainly PKC-, inhibits PKC- and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells. Diabetes 50:1244 –1252, 2001 P ED/PEA-15 is a 15-kDa cytosolic protein whose gene has been shown to be overexpressed in insulin-resistant type 2 diabetic subjects (1). PED protein has also been found to be overexpressed in skeletal muscle, adipose tissue, and fibroblasts from type 2 diabetic individuals (1). Muscle and adipose cells overexpressing PED to levels comparable to those occur- ring in type 2 diabetes feature slightly increased basal glucose uptake with a lack of further insulin-dependent uptake (1). The changes in glucose uptake in muscle and adipose cells overexpressing PED are accompanied by inhibition of insulin-dependent translocation of GLUT4, the major insulin-responsive glucose transporter (1). PED overexpression also impairs GLUT1 translocation in re- sponse to insulin, but increases GLUT1 content in the plasma membranes of basal cells (1). Antisense inhibition of endogenous PED expression in muscle cells and adipo- cytes significantly expands insulin sensitivity of glucose uptake (2). Thus, PED may represent a physiological regulator of glucose transporter trafficking and glucose transport in the major insulin-responsive skeletal muscle and adipose tissues. Furthermore, defective expression of the PED gene may contribute to insulin resistance in glucose transport in type 2 diabetes. Protein kinase C (PKC) comprises a multigene family that encodes at least 12 distinct isoforms differing in catalytic and regulatory properties (3– 6). These PKC (1) isoforms can be divided into the following three subgroups based on cofactor requirements: conventional PKCs (, , and ), which are dependent on Ca 2+ and diacylglycerol (DAG) for activity; novel PKCs (, , , and ), which are not dependent on Ca 2+ but are activated by DAG; and atypical PKCs (, , and ), which are not dependent on Ca 2+ and are not stimulated by DAG (7). PKC plays a pivotal role in controlling numerous cellular functions, including glucose transport (8). Indeed, current evidence indicates that DAG-dependent PKCs may control insulin- independent glucose transport in several different cell types (9,10). More recent work also indicates that different PKC isoforms have an important role in signaling insu- lin action on glucose transport (9 –13). However, at the molecular level, the role of specific PKC isoforms in controlling glucose transport as well as their regulatory mechanisms have been only partially elucidated. PED is a substrate of PKC (1,14). In previous work, we found that pharmacological inhibition of PKC reduces the high basal glucose uptake of muscle and adipose cells overexpressing PED and rescues most of the insulin effect in glucose uptake (1). These findings implied an important role of the PKC system in PED function, but whether PED is controlled by PKC phosphorylation or whether PKC is From the Dipartimento di Biologia e Patologia Cellulare e Molecolare and the Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Federico II University of Naples, Naples, Italy. Address correspondence and reprint requests to Francesco Beguinot, MD, PhD, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita ` di Napoli Federico II, Via S. Pansini 5, 80131 Naples, Italy. E-mail: beguino@unina.it. Received for publication 15 August 2000 and accepted in revised form 22 February 2001. G.C. and G.V. contributed equally to this article. 2-DG, 2-deoxy-D-glucose; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemiluminescence; IRS, insulin receptor substrate; PI, phosphatidylinositol; PKB, protein kinase B; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; TPA, 12-O-tetradecanoylphorbol-13- acetate. 1244 DIABETES, VOL. 50, JUNE 2001 RETRACTED