THIAZOLIDINEDIONES, AS ACTIVATORS of PPAR s,

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1 /02/$15.00/0 Endocrinology 143(5): Printed in U.S.A. Copyright 2002 by The Endocrine Society Cbl, IRS-1, and IRS-2 Mediate Effects of Rosiglitazone on PI3K, PKC-, and Glucose Transport in 3T3/L1 Adipocytes MARY L. STANDAERT, YOSHINORI KANOH, MINI P. SAJAN, GAUTAM BANDYOPADHYAY, AND ROBERT V. FARESE J. A. Haley Veterans Hospital Research Service and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida The thiazolidenedione, rosiglitazone, increases basal and/or insulin-stimulated glucose transport in various cell types by diverse but uncertain mechanisms that may involve insulin receptor substrate (IRS)-1-dependent PI3K. Presently, in 3T3/L1 adipocytes, rosiglitazone induced sizable increases in basal glucose transport that were: dependent on PI3K, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and PKC- ; accompanied by increases in tyrosine phosphorylation of Cbl and Cbl-dependent increases in PI3K and PKC- activity; but not accompanied by increases in IRS-1/2-dependent PI3K or protein kinase B Abbreviations: CAP, Cbl-associated protein; Cbl , forms of Cbl in which there are deletions of amino acids 437 to 647; Cbl , forms of Cbl in which there are deletions of amino acids 648 to the 906 end; Cbl-Y8F, Cbl in which eight C-terminal tyrosine residues have been mutated to phenylalanine; GLUT, glucose transporter; HA, hemagglutinin antigen; IRS, insulin receptor substrate; KI, kinase inactive; KI/AR, kinase inactive/activation resistant; KRP, Krebs Ringer phosphate medium; MOI, multiplicity of infection or viral particles per cell; PDK-1, protein kinase-1; PIP 3, PI-3,4,5-(PO 4 ) 3 ; PKB, protein kinase B. activity. Additionally, rosiglitazone increased IRS-1 and IRS-2 levels, thereby enhancing insulin effects on IRS-1- and IRS-2-dependent PI3K and downstream signaling factors PKC- and protein kinase B. Our findings suggest that Cbl participates in mediating effects of rosiglitazone on PI3K, PDK-1, and PKC- and the glucose transport system and that this Cbl-dependent pathway complements the IRS-1 and IRS-2 pathways for activating PI3K, PDK-1, and PKC- during combined actions of rosiglitazone and insulin in 3T3/L1 cells. (Endocrinology 143: , 2002) THIAZOLIDINEDIONES, AS ACTIVATORS of PPAR s, serve as important insulin-sensitizing agents for treating type 2 diabetes mellitus (1). The therapeutic efficacy of thiazolidinediones stems largely from their ability to stimulate glucose transport and subsequent storage of glucose in glycogen, particularly in response to insulin (2); however, the mechanism of action of thiazolidinediones remains uncertain. Although there are still large gaps in our understanding of how glucose transport is regulated by insulin, it is now generally accepted that PI3K is essential and is to a large extent activated through insulin receptor-mediated phosphorylation of specific tyrosine residues of insulin receptor substrates IRS-1 and IRS-2. It is further thought that downstream effectors of PI3K, protein kinase B (PKB) (3 7), and PKC- / (8 13) serve as major distal regulators of glucose transport during insulin action. Of further note, both PKB and PKC- / are activated at least partly through PI3Kdependent increases in PI-3,4,5-(PO 4 ) 3 (PIP 3 ) and subsequent action of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which phosphorylates critical threonine residues in the activation loops of PKB (3, 14) and PKC- / (15, 16). In addition, PIP 3 also activates PKC- / by increasing its autophosphorylation and allosterically negating pseudosubstrate-dependent autoinhibition (17, 18). Recently we reported that treatment of nondiabetic and type 2 diabetic rats in vivo for 7 14 d with the thiazolidinedione, rosiglitazone, led to increases in both basal and insulinstimulated phosphorylation and enzymatic activation of PKC- / in isolated adipocytes (19). Surprisingly, these rosiglitazone-induced increases in PKC- / phosphorylation/ activity in the rat adipocyte were not accompanied by significant increases in either insulin receptor substrate (IRS)- 1/2-dependent PI3K or PKB activity, and the mechanism used by rosiglitazone to increase basal and insulin-stimulated PKC- / activity was uncertain. It was also uncertain whether increases in PKC- / activity in fact contributed to observed increases in glucose transport in adipocytes of rosiglitazone-treated rats and, for that matter, whether increases in glucose transport and PKC- / activation in rat adipocytes were because of direct effects of rosiglitazone in these adipocytes. To gain insight into these questions, we presently examined the actions of rosiglitazone in 3T3/L1 adipocytes. Interestingly, we found that rosiglitazone activated a PI3K activity that was associated with Cbl, which, like IRS-1 and -2, via tyrosine phosphorylated residues, i.e. pyxxm, is known to activate p85 subunits of PI3K (20). Of further note, this Cbl-associated PI3K appeared to be required for rosiglitazone-induced activation of PKC- and glucose transport. In addition to increasing Cbl py content and Cbl-dependent PI3K activity, rosiglitazone increased the levels of IRS-1 and IRS-2 and enhanced insulin-induced increases in IRS-1- and -2-dependent PI3K activities. The activation of Cbl provided an explanation for the activation of PI3K, PKC-, and glucose transport during simple rosiglitazone treatment, and this Cbl 1705

2 1706 Endocrinology, May 2002, 143(5): Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC pathway complemented IRS-1- and -2-dependent pathways for activating PI3K and PKC- during combined actions of rosiglitazone and insulin. Materials and Methods Cell culture, incubations, and treatments 3T3/L1 adipocytes were cultured and differentiated as described previously (9). Insulin was withdrawn from fully differentiated adipocytes 48 h before experimentation. Where indicated, adipocytes were incubated in DMEM containing FBS (Sigma, St. Louis, MO) for 48 h with indicated concentrations of rosiglitazone (kindly supplied by Smith- Kline Beecham, Worthing, West Sussex, UK) and/or adenovirus alone or adenovirus encoding kinase-inactive (KI) PKC- (kindly supplied by Dr. Masato Kasuga, see Ref. 12) or other proteins (see below). The cells were then incubated in serum-free DMEM for 3 4 h and finally incubated for 30 min in glucose-free Krebs Ringer phosphate medium (KRP) containing 1% BSA before intense treatment with insulin and other agents for indicated times. PKC- activation PKC- activity was measured as described previously (9 11, 17 19). In brief, PKC- was immunoprecipitated from salt/detergent-treated cell lysates with a rabbit polyclonal antiserum (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) that recognizes the C termini of both PKC- and PKC- (3T3/L1 adipocytes contain only PKC- ), collected on Sepharose-AG beads (Santa Cruz Biotechnologies, Inc.), and incubated for 8 min at 30 C in 100 l buffer containing 50 mm Tris/HCl (ph 7.5), 100 m Na 3 VO 4, 100 m Na 4 P 2 O 7,1mm NaF, 100 m PMSF, 4 g phosphatidylserine (Sigma), 50 m [ - 32 P]ATP (NEN Life Science Products, Boston, MA), 5 mm MgCl 2 and, as substrate, 40 m serine analog of the PKC- pseudosubstrate (Biosource Technologies, Inc., Camarillo, CA). After incubation, 32 P-labeled substrate was trapped on P-81 filter paper and counted. PKC- activation was also assessed by immunoblotting for phosphorylation of threonine-411 in its activation loop as described (17 19). For this purpose, 1 mg lysate protein was subjected to immunoprecipitation with anti-pkc- / antiserum (Santa Cruz Biotechnologies, Inc.), resolution on SDS-PAGE, and blotting with specific anti-phospho-threonine-411 antibodies (see below). Note that immunoprecipitation was not altered by rosiglitazone or insulin treatments. PKB activation PKB enzyme activity was measured using a kit (Upstate Biotechnologies, Inc., Lake Placid, NY), as described previously (13, 17, 19). In brief, PKB or, in some cases, PKB (using larger amounts of lysates because of apparently lesser recovered enzyme activity of the isoform) was immunoprecipitated with sheep polyclonal antisera raised to PKB or PKB (both from Upstate Biotechnologies, Inc.), collected on Sepharose-AG beads, and assayed as per directions in the PKB assay kit. PKB (presumably plus ) activation was also assessed by immunoblotting for phosphorylation of threonine-308 and serine-473 as described (17, 19; also see below). PI3K activation Immunoprecipitable IRS-1-dependent (rabbit polyclonal antiserum kindly supplied by Dr. Alan Saltiel), IRS-2-dependent (rabbit polyclonal antiserum kindly supplied by Dr. Morris White), p85 subunit-dependent (rabbit polyclonal antiserum from Upstate Biotechnologies, Inc.), py-dependent (mouse monoclonal py-99 antibodies from Santa Cruz Biotechnologies, Inc.) and Cbl-dependent (rabbit polyclonal antiserum from Santa Cruz Biotechnologies, Inc.) PI3K activity was determined as described previously (19). 2-Deoxyglucose uptake and glucose transporter (GLUT) 4/GLUT 1 translocation Adipocytes were incubated in glucose-free KRP medium for 30 min with or without insulin, before measurement of plasma membrane and microsomal levels of immunoreactive GLUT4 and GLUT 1, or [ 3 H]2- deoxyglucose (50 m) over 5 min, as described previously (9). Immunoblotting Western analyses were conducted as described previously (8 11, 13, 17 19), using the following antibodies/antisera: (1) rabbit polyclonal anti-pkc- / C-terminal antisera (Santa Cruz Biotechnologies, Inc.); (2) sheep polyclonal anti-pkb antiserum (Upstate Biotechnologies, Inc.); (3) rabbit polyclonal anti-phospho-serine-473-pkb antiserum (New England Biolabs, Inc., Beverly, MA); (4) rabbit polyclonal anti-phosphothreonine-308-pkb (Biosource Technologies, Inc.); (5) rabbit polyclonal anti-glut 1 antiserum (kindly provided by Dr. Ian Simpson); (6) mouse monoclonal anti-glut 4 antibodies (Biogenesis, Bournemouth, UK); (7) rabbit polyclonal anti-p85/pi3k antiserum (Upstate Biotechnologies, Inc.); (8) rabbit polyclonal anti-pdk-1 antiserum (Upstate Biotechnologies, Inc.); (9) rabbit polyclonal anti-phospho-threonine-410-pkc- antiserum (the same PDK-1-dependent activation loop site in PKC- is threonine-411) (kindly supplied by Dr. Alex Toker, 17 19); and (10) rabbit polyclonal anti-irs-1 and anti-irs-2 antisera (kindly supplied by Dr. Morris White). Blots were quantified by measurement of extended chemiluminescence in a PhosphorImager/Chemiluminescence imaging system using a Molecular Analyst Program (Bio-Rad Laboratories, Inc., Richmond, CA). Adenoviral constructs Adenovirus encoding KI PKC- was provided by Dr. Masato Kasuga and used as described (12); expression of this protein inhibits the activation of PKC- but not PKB or PKB (12). Adenoviruses encoding KI PDK-1 and KI/activation-resistant (KI/AR) PKB, a triple A (K197A/ T308A/S473A) mutant, were constructed using plasmids encoding these mutants (10, 21) and an Adeno-X expression kit (CLONTECH Laboratories, Inc., Palo Alto, CA) (22). All final constructs were sequenced to ensure that the mutations were maintained through the preparative measures. Fully differentiated adipocytes were routinely infected with 10 MOI (multiplicity of infection or viral particles per cell) KI PKC-,10 MOI KI PDK-1, or 150 MOI KI/AR PKB -AAA, which, after a 48-h incubation, increased levels of total cellular PKC-, PDK-1, or PKB by approximately 2- to 3-fold, and these increases resulted in nearly complete inhibition of the activation of total cellular (endogenous plus expressed) PKC-, PDK-1, and PKB /, respectively (see below and Refs. 12 and 22). Note that, as reported previously (10, 12, 22), the expression of KI atypical PKCs does not inhibit the activation of PKB, and, viceversa, we presently found (see below) that expression of KI/AR PKB - AAA did not inhibit the activation of PKC-, despite the fact that PDK-1 activates both atypical PKCs and PKB. Thus, it may be surmised that, in the present experimental conditions, PDK-1 availability did not become rate limiting for phosphorylation of activation loops of atypical PKCs and PKB during expression of the opposite KI PKB and PKC-, respectively. Cbl plasmids The pcmv2 plasmids encoding hemagglutinin antigen (HA)-tagged wild-type Cbl, HA-tagged tyrosine phosphorylation-defective Cbl in which eight C-terminal tyrosine residues (at positions 552, 674, 700, 731 [note that this 731 site initiates a YEAM motif that, when phosphorylated, can presumably activate SH2 domains of the p85 subunit of PI3K], 735, 774, 869, and 871; Cbl-Y8F) have been mutated to phenylalanine, and FLAG-tagged forms of Cbl, in which there are deletions of amino acids 437 to 647 (Cbl ) and 648 to the 906 end (Cbl ), were kindly provided by Dr. Tomoyuki Shishido (23, 24). Note that the Cbl-8F mutant diminished by approximately 50% a rosiglitazone-insensitive, i.e. apparently constitutive, interaction between Cbl and the p85 subunit of PI3K; this suggests the presence of a tyrosine-dependent interaction between proline-rich or other sequences in the C terminus of Cbl and SH3 or other domains of the p85 subunit of PI3K or Cblactivating kinases such as Abl (23, 24). Accordingly, the Cbl deleted form of Cbl would also lack these C-terminal interaction sites. Also note that the Cbl deleted form of Cbl is missing prolinerich sequences that may interact with SH3 domains of PI3K or other proteins, including Src nonreceptor tyrosine kinases (20, 23, 24).

3 Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC Endocrinology, May 2002, 143(5): Results Effects of rosiglitazone on glucose transport Treatment of 3T3/L1 adipocytes for 48 h with increasing concentrations of rosiglitazone resulted in progressive increases in both basal (i.e. noninsulin-stimulated) and insulinstimulated 2-deoxyglucose uptake (Fig. 1A). Rosiglitazoneinduced increases in basal glucose uptake were in most cases comparable in magnitude with those of insulin alone, but increases in glucose transport were greatest with combined rosiglitazone and insulin treatment (Figs. 1 and 2 and see Fig. 6). Dose-related effects of insulin on 2-deoxyglucose uptake in rosiglitazone-treated 3T3/L1 adipocytes are shown in Fig. 1E. Effects of rosiglitazone on PKC- activity Rosiglitazone-induced increases in 2-deoxyglucose uptake were accompanied by similar dose-dependent increases in both basal and insulin-stimulated PKC- activity (Figs. 1B and 2, A and B). Increases in PKC- activity could not be explained by changes in levels of immunoreactive PKC-, which did not change, or, if anything, diminished with rosiglitazone treatment (e.g. Fig. 1C). Effects of rosiglitazone on glucose transporters In addition to increasing basal 2-deoxyglucose uptake, rosiglitazone provoked significant increases in plasma membrane levels of both GLUT 4 (961% 174%, mean se, n 4; P 0.005, paired t test) and GLUT1 (478% 79%, mean se, n 4; P 0.01, paired t test) glucose transporters, whereas microsomal levels of GLUT 4 and GLUT 1 transporters diminished by 47% 7% and 51 8% (both n 4 and P 0.01, paired t tests) (blots shown in Fig. 1D). Rosiglitazone did not significantly alter cellular levels of immunoreactive GLUT 4 (e.g. see Fig. 1C), although, in a few instances, rosiglitazone appeared to increase GLUT 1 levels, in keeping with a report that the thiazolidinedione, troglitazone, induces increases of 1.5- to 2-fold in GLUT 1 levels (25). However, increases in GLUT 1 were not consistently observed, and, in most instances (six of nine comparisons), rosiglitazone did not increase GLUT 1 levels (e.g. see Fig. 1C). The reason for this variability was uncertain, and, although it is possible that increases in total cellular GLUT 1 levels may have contributed to increases in 2-deoxyglucose uptake, note that rosiglitazone-induced increases in both basal and insulin-stimulated 2-deoxyglucose uptake were completely de- FIG. 1. Effects of rosiglitazone on basal and insulin-stimulated [ 3 H]2-deoxyglucose uptake (A and E), PKC- activity (B), and levels of immunoreactive PKC-, GLUT 4 and GLUT 1 (C and D) in 3T3/L1 adipocytes. Cells were treated for 48 h with indicated concentrations of rosiglitazone (RSGZ) in all panels and then incubated in glucose-free KRP medium without (control) or with 100 nm insulin for 30 min in A or with 100 nm insulin for 10 min in B or with increasing concentrations of insulin for 30 min in E. Values are mean SE of (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between rosiglitazone-treated samples and corresponding rosiglitazone-untreated controls that were either insulin-treated or basal insulin-untreated samples. See text for results of multiple determinations of alterations in plasma membrane and microsomal levels of GLUT 4 and GLUT 1 glucose transporters.

4 1708 Endocrinology, May 2002, 143(5): Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC FIG. 2. Effects of rosiglitazone on basal and insulin-stimulated PKC- activity (A and B) and PKB activity (C and D) in 3T3/L1 adipocytes. Cells were treated with indicated concentrations of rosiglitazone for 48 h and then incubated in glucose-free KRP medium for 10 min in A and C or for indicated times in B and D, without (control or zero times) or with 100 nm insulin as indicated. The same cell lysates were analyzed for PKC- activity in A and B and for PKB activity in C and D. Values are mean SE of (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between rosiglitazone-treated samples and corresponding rosiglitazone-untreated controls that were either insulin-treated or basal insulin-untreated samples. pendent on activities of PI3K, PDK-1, and PKC- (see below). Accordingly, it seemed clear that that there was a strict requirement for active signaling through these factors for rosiglitazone-induced increases in glucose transport, and simple increases in GLUT 1 transporter levels in the absence of concomitant increases in signaling seemed unlikely to have altered glucose transport. Effects of rosiglitazone on PKB activity In contrast to increases in basal PKC- activity (Fig. 2, A and B), rosiglitazone did not provoke increases in basal PKB activity (Fig. 2, C and D) or alter contents of PKB or PKB (not shown). On the other hand, rosiglitazone treatment enhanced the effects of insulin on both PKC- (Fig. 2, A and B) and PKB activity (Fig. 2, C and D). As seen in Fig. 2, B and D, effects of rosiglitazone and insulin on activities of PKC- and PKB remained apparent throughout the course of a 20-min incubation period. Here again, note that, throughout this time course, rosiglitazone provoked increases in both basal and insulin-stimulated PKC- activity and insulinstimulated, but not basal, PKB activity. Effects of rosiglitazone on IRS-1 and IRS-2 and dependent PI3K activities Rosiglitazone alone had no significant effect on basal IRS-1- or IRS-2-dependent PI3K activity. On the other hand, rosiglitazone, at least at higher concentrations of 1 10 m, significantly enhanced insulin-dependent increases in IRS- 2-dependent, but not IRS-1-dependent, PI3K activity (Fig. 3). These increases in insulin-stimulated IRS-2-dependent PI3K activities appeared to reflect rosiglitazone-induced increases ( 150%) in levels of immunoreactive IRS-2 (Fig. 4). Rosiglitazone also induced increases ( 80%) levels of immunoreactive IRS-1 (Fig. 4). In contrast to increases in IRS-1 and -2 levels, we did not detect changes in levels of the p85 subunit of PI3K following rosiglitazone treatment (data not shown). Effects of rosiglitazone on phosphorylation of PKC- As seen in Fig. 5, A and B, rosiglitazone alone, at concentrations of nm, provoked relatively large increases in basal phosphorylation of threonine-411 in the activation loop of PKC-. Insulin alone also acutely increased threonine- 411 activation loop phosphorylation in PKC-, and insulin ef-

5 Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC Endocrinology, May 2002, 143(5): FIG. 3. Effects of rosiglitazone on basal and insulin-stimulated IRS-1-dependent (A and B) and IRS-2-dependent (C and D) PI3K activity in 3T3/L1 adipocytes. Cells were treated for 48 h with indicated concentrations of rosiglitazone (RSGZ) and then incubated in glucose-free KRP medium for 10 min without (control or minus signs) or with 100 nm insulin (insulin or plus signs) as indicated. Values in A and C are mean SE of (n) determinations. B and D show representative autoradiograms of 32 P-labeled PI-3-PO 4. Asterisks indicate significant increases, i.e. P 0.01, t test comparison, of insulin-stimulated values in the presence and absence 1 or 10 M rosiglitazone vs. insulin-stimulated values in the absence of rosiglitazone treatment. fects were further augmented by rosiglitazone treatment, however, to a level not greater than that of rosiglitazone alone. Effects of rosiglitazone on phosphorylation of PKB In contrast to PKC- phosphorylation, simple rosiglitazone treatment did not alter basal phosphorylation of serine- 473 or threonine-308 in PKB (Fig. 5C). On the other hand, rosiglitazone enhanced insulin-stimulated increases in phosphorylation of serine-473 and threonine-308 in PKB (Fig. 5C), in keeping with increases in insulin-stimulated increases in PKB enzyme activity. Requirements for signaling factors (PI3K, PDK-1, PKC-, and PKB) during rosiglitazone- and insulin-stimulated glucose transport The above-described findings suggested that alterations in PKC- activation could be important for alterations in both basal and insulin-stimulated 2-deoxyglucose uptake. To test this possibility, we used adenoviruses to express KI forms of PKC- and its upstream activator, PDK-1, and, for comparison, the KI/AR form of PKB -AAA. As reported previously (12, 22), the expression of KI PKC- by the presently used adenovirus effectively inhibits PKC-, but not PKB, activation by insulin. Similarly, we presently found that adenoviral-mediated expression of KI/AR-PKB -AAA inhibited insulin-stimulated activation of both PKB and PKB (Fig. 6, C and D) but not PKC- (Fig. 6A) and that adenoviralmediated expression of KI PDK-1 effectively inhibited insulin-stimulated activation of PKC- (Fig. 6A) and PKB (Fig. 6C). Note that, whereas the inhibitory effects of KI/AR PKB -AAA on total immunoprecipitatable PKB activity would reflect activity of combined endogenous wild-type active PKB and exogenous inactive KI/AR PKB -AAA, the precipitates of PKB would reflect only the activity of endogenous PKB, which is postulated to be the isoform required for insulin-stimulated glucose transport in 3T3/L1 adipocytes (4). Most importantly, using these adenoviruses, we found that expression of KI PKC- markedly inhibited rosiglitazone-stimulated increases in basal 2-deoxyglucose uptake (Fig. 7C) and PKC- activity (Fig. 6B), as well as insulinstimulated 2-deoxyglucose uptake (Fig. 7C) and PKC- activity (12, 22). Similarly, the PI3K inhibitor, wortmannin, like KI PKC-, markedly inhibited rosiglitazone-stimulated basal and insulin-stimulated 2-deoxyglucose uptake (Fig. 7D) and PKC- activation (Fig. 7B). Further, adenoviral-mediated expression of KI PDK-1 inhibited both rosiglitazone-stimulated and insulin-stimulated PKC- activation (Fig. 7A) and 2- deoxyglucose uptake (Fig. 7E). In contrast, adenoviralmediated expression of KI/AR PKB -AAA had only a slight inhibitory effect on rosiglitazone-stimulated and insulinstimulated 2-deoxyglucose uptake (Fig. 7E). Note that the inhibitory effects of KI PKC- on rosiglitazone-stimulated and insulin-stimulated glucose transport could not be ex-

6 1710 Endocrinology, May 2002, 143(5): Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC FIG. 4. Effects of rosiglitazone on levels of immunoreactive IRS-1 and IRS-2 in 3T3/L1 adipocytes. Cells were treated for 48 h with indicated concentrations of rosiglitazone (RSGZ), and cell lysates were subjected to SDS-PAGE and blotted for IRS-1 and IRS-2. Blot immunoreactivity was quantitated by measurement of chemiluminescence in a Molecular Analyst Chemiluminescence/PhosphorImager System (Bio-Rad Laboratories, Inc.). Values in A and B are mean SE of three determinations in which chemiluminescence values were compared with the rosiglitazone-untreated control values. Representative autoradiograms are shown in C. Asterisks indicate significant increases, i.e. P 0.05, paired t test comparison, of peak values at 1 or 10 M rosiglitazone vs. no rosiglitazone. plained by decreases in levels of glucose transporters because levels of GLUT1 (e.g. see Fig. 1C) and GLUT4 (data not shown) did not change in cells expressing KI-PKC-. Role of Cbl on rosiglitazone-induced activation of PI3K In view of the requirement for PI3K during rosiglitazoneinduced increases in basal PKC- activity and glucose transport, we evaluated the effects of rosiglitazone on PI3K activation in greater detail. As stated above, we did not see an effect of rosiglitazone alone on basal IRS-1-dependent or IRS-2-dependent PI3K activity. On the other hand, rosiglitazone alone provoked consistent increases in py-dependent PI3K activity (Fig. 8) that, like increases in phosphorylation of threonine-411 in the activation loop of PKC-, were readily apparent at low concentrations of rosiglitazone. Similarly, FIG. 5. Effects of rosiglitazone on basal and insulin-stimulated phosphorylation of threonine-411 in the activation loop of PKC- (A and B) and phosphorylation of the threonine-308 activation loop site and serine-473 in PKB in 3T3/L1 adipocytes. Cells were treated for 48 h with indicated concentrations of rosiglitazone (RSGZ) and then incubated for 10 min in glucose-free KRP medium without (control or minus signs) or with 100 nm insulin (insulin or plus signs), following which phospho-threonine-411 in PKC-, or phosphor-threonine-308 or phospho-serine-473 in PKB (presumably reflecting both and isoforms), was determined with phospho-specific antibodies as described in Materials and Methods. Representative immunoblots are shown in A and C. Values in B are mean SE of (n) determinations of relative chemiluminescence as described in Fig. 4. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between cells treated with rosiglitazone and/or insulin vs. untreated controls. rosiglitazone provoked increases in the activity of p85 subunit-associated PI3K activity (Fig. 8). Thiazolidinediones, troglitazone and rosiglitazone, are known to increase the expression of the Cbl-associated protein (CAP) in 3T3/L1 adipocytes, apparently by activating PPAR nuclear transcription factors (26). CAP in turn couples Cbl to the insulin receptor and other tyrosine kinases, e.g. src kinase fyn, that, via SH3 domains, binds to proline-rich regions and enhances the phosphorylation of tyrosine residues of Cbl (26 28), which, in turn, via pyxxm motifs at Y-371 and/or Y-731, may bind to and activate SH2 domains of the p85 subunit of PI3K (20). It was therefore of interest to find that rosiglitazone provoked increases in both the py content of Cbl (Fig. 9) and Cbl-dependent PI3K activity (Figs. 9 and 10). In this regard, note that rosiglitazone did not alter

7 Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC Endocrinology, May 2002, 143(5): FIG. 6. Effects of adenoviral-mediated expression of KI PDK-1 and KI/AR PKB triple alanine (AAA) mutant on insulin-induced activation of PKC- (A), PKB (B), and PKB (C) in 3T3/L1 adipocytes. Cells were incubated with indicated MOI (see vertical numbers in or near bars) ofadenovirus encoding the indicated mutant PDK-1 or PKB. After 48 h to allow time for expression (see text), cells were washed and incubated in glucosefree KRP medium for 10 min with or without 100 nm insulin, after which lysates were analyzed for immunoprecipitatable enzyme activities. Note that it was necessary to use four times as much cell lysate to observe a level of PKB activity that was comparable to that of PKB activity. Values are mean SE on (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between cells treated with rosiglitazone and/or insulin vs. adjacent untreated controls. Insets depict increases (i.e. relative to the basal levels) in levels of immunoreactive PDK-1, PKC-, and PKB seen after transfection with indicated MOI of KI PDK-1, KI PKC, and KI/AR PKB (i.e. the AAA mutant). Note that adenovirus alone (i.e. vector) was without effect, regardless of MOI concentrations ranging from10 to 150. Note that some cells in panel B were treated for 48 h with 1 M rosiglitazone. either the level of immunoreactive Cbl in 3T3/L1 adipocytes, as found in either total cell lysates (not shown) or immunoprecipitates (Fig. 9) or the apparently constitutive binding of the p85 subunit of PI3K to immunoprecipitated Cbl (not shown). In addition to chronic rosiglitazone treatment, insulin, which is known to rapidly increase tyrosine phosphorylation of Cbl (27, 28), provoked rapid increases in Cbl-associated PI3K activity (Fig. 10). These increases in insulin-stimulated Cbl-dependent PI3K, however, were relatively small, i.e. being only approximately 10 15% of insulin-stimulated increases in IRS-1-dependent PI3K activity (comparative data not shown). Effects of Cbl expression on rosiglitazone-induced activation of PKC- To evaluate the importance of Cbl in mediating effects of rosiglitazone on PKC-, we used plasmids to express wildtype Cbl and mutated and deleted forms of Cbl, viz., Cbl- Y8F, Cbl , and Cbl , that are known to interfere with binding Cbl-activating tyrosine kinases, including Src and Abl, and other proteins (20, 23, 24) and possibly inhibit activation of the SH2 domains of the p85 subunit of PI3K. Note that we initially documented that, as with endogenous Cbl, rosiglitazone increased PI3K activity in immunoprecipitates of HA-tagged wild-type Cbl, but not HA-tagged Cbl-Y8F, which, we found, bound p85 poorly and contained little or no PI3K activity (data not shown). As seen in Fig. 11, expression of each mutant or deleted form of Cbl inhibited rosiglitazone-induced, but not insulin-induced, increases in activity of epitope-tagged PKC-. In contrast, expression of wild-type Cbl, had no effect, or, if anything, increased rosiglitazone-induced activation of epitope-tagged PKC- (note that PKC- and PKC- function interchangeably; 11). Thus, these mutant and deleted forms of Cbl served effectively as dominant negatives for rosiglitazone-induced activation of PKC-. Discussion It was surprising to find that treatment of 3T3/L1 adipocytes with rosiglitazone induced sizable increases in basal, i.e. noninsulin-stimulated, PKC- activity. These rosiglitazone-induced increases in basal PKC- activity appeared to be largely responsible for the increases in basal 2-deoxyglucose uptake and plasma membrane levels of GLUT4 and GLUT 1 glucose transporters following simple rosiglitazone treatment. In support of this conclusion, expression of KI PKC- as well as inhibitors of upstream activators of PKC-,

8 1712 Endocrinology, May 2002, 143(5): Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC FIG. 7. Effects of KI PDK-1 (A and E), KI PKC- (C), wortmannin (Wort) (B and D), and KI/AR PKB on basal, insulin-stimulated, and rosiglitazone-stimulated [ 3 H]2-deoxyglucose uptake (C, D, and E) and PKC- activation (A and B) in 3T3/L1 adipocytes. Cells were treated for 48 h with or without 1 M rosiglitazone (RSGZ) (all panels) and 10 MOI adenovirus alone or adenovirus encoding KI PDK-1 (A), KI PKC- (C and E), or 150 MOI adenovirus alone or adenovirus encoding KI AA-PKB (E). Cells were then washed and equilibrated in glucose-free KRP medium and, in B and D, incubated for 15 min without or without 100 nm wortmannin, and then, in C, D, and E, incubated for 30 min with or without 100 nm insulin, following which uptake of [ 3 H]2-deoxyglucose over 5 min was measured and, in A and B, incubated for 10 min with or without 100 nm insulin, following which PKC- activity was measured. Values are mean SE of (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between cells treated with rosiglitazone and/or insulin vs. adjacent untreated controls. viz., expressed KI PDK-1 and the PI3K inhibitor, wortmannin, blocked rosiglitazone-induced increases in basal 2- deoxyglucose uptake, indicating that PKC-, PDK-1, and PI3K were needed to actively signal to factors that control glucose transport in the basal noninsulin-stimulated state. In contrast to PKC-, there were no significant increases in basal PKB phosphorylation or activation following simple rosiglitazone treatment. It therefore seemed unlikely that alterations in PKB contributed to the observed increases in basal 2-deoxyglucose uptake following rosiglitazone treatment. This was further confirmed by the failure of the KI and KI/AR PKB -AAA mutant to significantly diminish rosiglitazone-induced increases in basal glucose transport. It was, of course, surprising to find that basal PKB activity was not increased by simple rosiglitazone treatment, in view of the dependence of rosiglitazone-induced increases in PKC- activation on PI3K and PDK-1 and, as discussed further below, in view of increases in Cbl-dependent PI3K activity following simple rosiglitazone treatment. This dichotomy suggested that there may be cellular pools of PI3K and associated PDK-1 that selectively activate atypical PKCs or, as a perhaps less likely alternative, there may be phosphatases that selectively inactivate PKB during rosiglitazone treatment. With respect to the first alternative, such putative pools of PI3K/PDK-1 that may selectively activate atypical PKCs during simple rosiglitazone treatment are obviously

9 Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC Endocrinology, May 2002, 143(5): FIG. 8. Effects of rosiglitazone (RSGZ) on py-dependent and p85/pi3k subunit-dependent PI3K activity in 3T3/L1 adipocytes. Cells were treated with indicated concentrations of rosiglitazone for 48 h and then incubated in glucose-free KRP medium for 10 min with or without 100 nm insulin, as indicated. After incubation, lysates were examined for immunoprecipitatable (IP) py-dependent and p85-dependent PI3K activity. Representative autoradiograms are shown at left. Bargrams portray mean SE of (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between cells treated with rosiglitazone and/or insulin vs. untreated controls. different from the insulin-sensitive IRS-1- and IRS-2-dependent pools of PI3K/PDK-1, which apparently simultaneously activate both atypical PKCs and PKB. It may be noted that rosiglitazone-induced increases in basal noninsulin-stimulated PKC- activation loop phosphorylation did not correlate directly with rosiglitazoneinduced increases in either basal PKC- enzyme activity or basal glucose transport. On the other hand, there was reasonably good correlation between increases in PKC- enzyme activity and glucose transport observed after treatment with increasing concentrations of rosiglitazone. The lack of a strict correlation between basal activation loop phosphorylation and actual enzyme activity may reflect the fact that PKC- activity is regulated by at least three interrelated but clearly separable factors, including: (1) PDK-1-dependent/ PIP 3 -dependent activation loop phosphorylation; (2) PIP 3 - dependent autophosphorylation; and (3) phosphorylationindependent, PIP 3 -dependent conformational release from pseudosubstrate-dependent autoinhibition (17, 18). In this context, it is possible that although activation loop phosphorylation is required for activation, this phosphorylation may serve more as a trigger for activation but may not be as important as other factors (i.e. autophosphorylation and PIP 3 -dependent molecular unfolding) for actually increasing PKC- enzyme activity. As discussed above, it seemed clear that rosiglitazoneinduced increases in basal PKC- activity and glucose transport were dependent on both PI3K and PDK-1. It was therefore interesting to find that, despite increases in the levels of IRS-1 and IRS-2 proteins, activities of IRS-1-dependent and IRS-2-dependent PI3K were not increased by simple rosiglitazone treatment. On the other hand, rosiglitazone-induced increases in basal PI3K activity were dependent on a pydependent factor and the p85 subunit of PI3K. Our findings, moreover, suggested that Cbl served in this capacity because rosiglitazone increased both the tyrosine phosphorylation of Cbl and Cbl-dependent PI3K activity, and, most importantly, effects of rosiglitazone on activation of epitope-tagged atypical PKC were inhibited by expression of mutant and deleted forms of Cbl that might be expected to interfere with its binding to SH3 and SH2 domains of the p85 subunit of PI3K or Cbl-activating tyrosine kinases. In view of the above considerations, because rosiglitazone increases the expression of CAP in 3T3/L1 adipocytes (26), it seems plausible to suggest that rosiglitazone, via CAP, enhances the coupling of Cbl to a noninsulin tyrosine kinase, e.g. Src family members, e.g. Yes, or Abl (20, 23, 24), which in turn phosphorylate Cbl on tyrosine residues to generate one or both pyxxm motifs at Y-371 and Y-731, that, as reported (20), interact with and activate SH2 domains of the

10 1714 Endocrinology, May 2002, 143(5): Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC FIG. 9. Effects of rosiglitazone (RSGZ) on Cbl-dependent PI3K activity (A), and py content of immunoprecipitatable Cbl in 3T3/L1 adipocytes (B). Cells were treated for 48 h with indicated concentrations of rosiglitazone, following which they were incubated in glucosefree KRP medium for 30 min. After incubation, Cbl was immunoprecipitated (IP) from cell lysates and precipitates were analyzed for associated PI3K activity and contents of total Cbl (middle panel) and py in Cbl. Note that Cbl content in cell lysates and immunoprecipitates was not altered appreciably by rosiglitazone. NI, Nonimmune serum used for immunoprecipitation. Results are representative of three experiments. p85 subunit of PI3K, which, we presume via its SH3 or other domain, is constitutively bound to proline-rich or other sequences in Cbl. Suffice it to say, further work is needed to more specifically determine the exact amino acids needed for binding between proline-rich and pyxxm-containing sequences of Cbl and SH3 and SH2 domains of the p85 subunit of PI3K. Further work is also needed to identify the tyrosine kinase responsible for mediating CAP-dependent phosphorylation of Cbl in response to rosiglitazone treatment. As noted above, the acute effects of insulin on Cbl-dependent PI3K activity were relatively small in comparison with effects on IRS-1-dependent PI3K. Moreover, effects of insulin on epitope-tagged PKC- were not inhibited to any significant extent by expression of mutated or deleted forms of Cbl that effectively served as dominant negatives for rosiglitazone-induced increases in epitope-tagged PKC- activity. Accordingly, it appears that effects of insulin on Cbl-dependent PI3K are either redundant with those of IRS-1- and -2-dependent PI3K or functionally unimportant. However, with respect to the latter possibility, it should be noted that we were presently unable to use plasmid expression methods to test for effects of Cbl mutations and deletions on the glucose transport system (for this purpose, we are currently developing appropriate adenoviruses that encode mutated forms of Cbl). It should also be noted that rosiglitazoneinduced increases in Cbl-dependent PI3K were relatively small and comparable in magnitude with those of insulin FIG. 10. Effects of rosiglitazone (RSGZ) and insulin (INS), alone or in combination, on Cbl-dependent PI3K activity in 3T3/L1 adipocytes. Cells were incubated for 48 h with or without 1 M rosiglitazone and then washed and incubated in glucose-free KRP medium for 1 or 2 min with or without 100 nm insulin as indicated. After incubation, Cbl immunoprecipitates (IP) were prepared and analyzed for PI3K activity. Upper panel shows a representative autoradiogram. Lower panel shows mean SE of three determinations. ***, P (standard unpaired t test) for comparisons between cells treated with rosiglitazone and/or insulin vs. untreated controls. but, nevertheless, were associated with increases in PKC- activity and glucose transport that were sizable and, in fact, comparable with those of simple insulin treatment. Obviously, further work is needed to evaluate the importance of Cbl-dependent PI3K in insulin action. The finding that Cbl-dependent PI3K was most strongly activated by combined treatment with both rosiglitazone and insulin may be important for explaining at least certain insulin-sensitizing effects of rosiglitazone that are not dependent on IRS-1 and -2. Accordingly, the present findings may be relevant to the previously reported findings indicating that rosiglitazone can enhance the effects of insulin on the activation of atypical PKCs in adipocytes (19) and skeletal muscles (29) of nonobese type 2 diabetic Goto Kakizaki rats, without alteration in IRS-1 or IRS-2 levels, and without apparent activation of IRS-1 or -2-dependent PI3K or PKB. In this regard, we have, in fact, seen increases in Cbl-dependent

11 Standaert et al. Thiazolidinedione Signaling via Cbl, IRS, PI3K, and PKC Endocrinology, May 2002, 143(5): FIG. 11. Effects of expression of wild-type (WT) Cbl and dominant-negative (DN) forms of Cbl on activation of HA- and FLAG-tagged forms of PKC- in 3T3/L1 adipocytes. Cells were cotransfected in the presence of Lipofectamine with plasmids encoding HA-PKC- or FLAG-PKC- and indicated forms of Cbl or empty vector (VEC), incubated for 48 h with or without 1 M rosiglitazone (RSGZ), and finally washed and incubated for 15 min in glucose-free KRP medium before harvesting, preparation of cell lysates, immunoprecipitation of PKC- with anti-ha antibodies or anti-flag antiserum, and assay for PKC- activity. Values are mean SE of (n) determinations. *, **, and ***, P 0.05, 0.01, and 0.001, respectively (standard unpaired t test) for comparisons between samples treated with rosiglitazone or insulin and corresponding adjacent untreated controls. PI3K activity in these GK rat tissues following rosiglitazone and/or insulin treatment (our unpublished data). As may be surmised, further studies are needed to see whether alterations in Cbl-dependent PI3K and atypical PKCs presently observed in rosiglitazone-treated 3T3/L1 cells are also seen following rosiglitazone treatment of other cell types. With respect to this question, similarly, if not identical with what was presently observed in 3T3/L1 adipocytes, we have found in cultured human adipocytes (which, like mouse 3T3/L1 adipocytes, are derived by differentiation of preadipocytes) that rosiglitazone enhanced insulin-stimulated, but not basal, IRS-1-dependent PI3K activity and provoked increases in both basal and insulinstimulated Cbl-dependent PI3K activity, atypical PKC activity, and glucose transport. However, our findings in cultured L6 myotubes were partly different from our findings in 3T3/L1 adipocytes in that rosiglitazone provoked increases in both basal and insulin-stimulated activities of IRS-1-dependent PI3K, PKB, and atypical PKCs in L6 cells, and these alterations led to increases in PI3K/PKC- -dependent basal glucose transport but, surprisingly, for uncertain reasons did not enhance insulin-stimulated glucose transport. Similarly, Yonemitsu et al. (30) have observed increases in basal GLUT 4 translocation in L6 myotubes following troglitazone treatment in L6 myotubes, but, different from our findings, these investigators did not observe increases in PKB activity and noted only partial dependence of increases in basal GLUT 4 translocation on PI3K. It is presently unclear whether these differences in L6 myotubes reflect clonal variations or different effects of specific thiazolidinediones. To summarize, rosiglitazone induced increases in PKC- activity in 3T3/L1 adipocytes by several mechanisms, viz., increases in basal Cbl-dependent PI3K and increases in IRS-1 and IRS-2 levels and insulin-stimulated activation of IRS-1- and IRS-2-dependent PI3K. Moreover, these increases in basal Cbl-dependent PI3K and PKC- activities appeared to be required for simple rosiglitazone-induced increases in basal glucose transport, whereas increases in IRS-1- and -2-dependent, as well as Cbl-dependent, PI3K and PKC- activities appeared to be required for increases in insulinstimulated glucose transport following rosiglitazone treatment. Further studies are needed to identify the tyrosine kinase that is responsible for rosiglitazone-induced Cbl phosphorylation and further elucidate the mechanisms that Cbl uses to activate PI3K, PDK-1, and PKC-. Acknowledgments Received October 15, Accepted January 31, Address all correspondence and requests for reprints to: Robert V. 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