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Comparative Effects of GTPγS and Insulin on the Activation of Rho, Phosphatidylinositol 3-Kinase, and Protein Kinase N in Rat Adipocytes

RELATIONSHIP TO GLUCOSE TRANSPORT*
Open AccessPublished:March 27, 1998DOI:https://doi.org/10.1074/jbc.273.13.7470
      Electroporation of rat adipocytes with guanosine 5′-3-O-(thio)triphosphate (GTPγS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPγS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPγS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPγS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPγS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-ζ (PKC-ζ) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-ζ, GTPγS activated PKN but not PKC-ζ. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPγS on HA-GLUT4 translocation. Our findings suggest that (a) GTPγS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-ζ may contribute to increases in glucose transport during insulin, but not GTPγS, action.
      GTPγS,
      The abbreviations used are: GTPγS, guanosine 5′-3-O-(thio)triphosphate; PI, phosphatidylinositol; PKC, protein kinase C; KRP, Krebs Ringer phosphate; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; IRS, insulin receptor substrate; 2-DOG, 2-[3H]deoxyglucose.
      1The abbreviations used are: GTPγS, guanosine 5′-3-O-(thio)triphosphate; PI, phosphatidylinositol; PKC, protein kinase C; KRP, Krebs Ringer phosphate; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; IRS, insulin receptor substrate; 2-DOG, 2-[3H]deoxyglucose.
      like insulin, has been found to activate GLUT4 translocation and/or glucose transport in rat adipocytes (
      • Baldini G.
      • Hohman R.
      • Charron M.J.
      • Lodish H.F.
      ,
      • Izawa T.
      • Saitou S.
      • Mochizuki T.
      • Komabayashi T.
      ) and 3T3/L1 adipocytes (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ). The mechanisms whereby GTPγS and insulin activate GLUT4 translocation and glucose transport, however, are unclear. In 3T3/L1 cells, unlike insulin, GTPγS was not found to activate cytosolic phosphatidylinositol (PI) 3-kinase (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ), and this suggested that (a) PI 3-kinase was not essential for the activation of glucose transport and (b) GTPγS may operate through different or more distal processes. In keeping with the latter possibility, small G-proteins in the Rab group are present in GLUT4 vesicles, appear to translocate or mobilize in response to insulin stimulation (
      • Cormont M.
      • Tanti J.F.
      • Zahraoui A.
      • Van Obberghen E.
      • Tavitian A.
      • Le Marchand-Brustel Y.
      ), and could act as direct mediators for GTPγS stimulation of glucose transport; accordingly, GTPγS-stimulated glucose transport is only partly inhibited by the PI 3-kinase inhibitor, wortmannin, in 3T3/L1 adipocytes (
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ), and GTPγS therefore appears to act, at least partially, independently of PI 3-kinase in 3T3/L1 cells. On the other hand, we have recently found that the small G-protein, RhoA, is activated by insulin in rat adipocytes, and, based uponClostridium botulinum C3 transferase sensitivity, Rho appears to be required for insulin-stimulated glucose transport in these cells (
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ). Further, it seems clear that Rho is directly activated by GTPγS in rat adipocytes, since it was found that the addition of GTPγS to rat adipocyte homogenates stimulates the translocation of Rho to plasma membranes and Rho-dependent activation of phospholipase D (
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ). Of further note, it has been reported that GTP-Rho directly activates PI 3-kinase in some (
      • Zhang J.
      • King W.G.
      • Dillon S.
      • Hall A.
      • Feig L.
      • Rittenhouse S.E.
      ), but not all (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ), cell-free systems. Presently, we examined the possibility that GTPγS activates PI 3-kinase via Rho in rat adipocytes. We also examined the role of Rho, PI 3-kinase, and protein kinases that are known to be downstream of Rho and PI 3-kinase (e.g. PKN and protein kinase C-ζ (PKC-ζ)) in the activation of glucose transport during treatment of rat adipocytes with GTPγS or insulin. Our findings suggested that (a) both GTPγS and insulin, albeit by different mechanisms, activate Rho, PI 3-kinase, and PKN; and (b) each of these factors may be required for, and may contribute to, the activation of GLUT4 translocation and glucose transport in rat adipocytes.

      DISCUSSION

      Our findings suggested that GTPγS activates PI 3-kinase through a Rho-dependent mechanism in intact rat adipocytes. GTP-Rho has also been found to activate PI 3-kinase in platelet homogenates (
      • Zhang J.
      • King W.G.
      • Dillon S.
      • Hall A.
      • Feig L.
      • Rittenhouse S.E.
      ) but, for uncertain reasons, not in other cell-free systems (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Our findings with wortmannin and LY294002 also suggested that PI 3-kinase is required for the activation of glucose transport during GTPγS stimulation of rat adipocytes. Thus, GTPγS, as presently used, did not appear to activate glucose transport in rat adipocytes simply by activating small G-proteins such as Rab that are thought to function distal to PI 3-kinase in regulating Glut4 translocation (
      • Cormont M.
      • Tanti J.F.
      • Zahraoui A.
      • Van Obberghen E.
      • Tavitian A.
      • Le Marchand-Brustel Y.
      ).
      Whereas GTPγS appeared to activate PI 3-kinase through Rho, insulin effects on PI 3-kinase were largely independent of Rho. Thus, although insulin activates Rho (
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ), Rho was not a major contributor to insulin-stimulated PI 3-kinase activation, which probably occurs largely through tyrosine phosphorylation of IRS-1 and/or other proteins (
      • White M.F.
      • Kahn C.R.
      ). Along these lines, it is pertinent to note that PI 3-kinase is required for the translocation, but not GTP loading, of Rho during insulin action (
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ); thus, PI 3-kinase can operate upstream (e.g. during insulin action), as well as downstream (e.g. during GTPγS activation), of Rho. During insulin action, Rho may translocate to specific sites of PI 3-kinase-induced increases in polyphosphoinositides (accordingly, we have found that Rho avidly binds to artificial phosphatidylcholine vesicles containing 5% PI-3, 4-(PO4)2, PI-3,4,5-(PO4)3, or PI-4,5-(PO4)2),
      M. L. Standaert, G. Bandyopadhyay, L. Galloway, Y. Ono, and R. V. Farese, unpublished results.
      and this may coordinate certain actions of PI 3-kinase and Rho. During GTPγS action, GTPγS stimulates the translocation of Rho to plasma and microsomal membranes (
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ), and this may explain how membrane-activated PI 3-kinase is activated by GTP-Rho.
      In addition to requirements for Rho and PI 3-kinase, our findings with RO 31-8220 suggested a requirement for one or more protein kinases in the activation of glucose transport by GTPγS as well as by insulin. In the case of insulin, the required protein kinase(s) appears to operate distally to, or in parallel with, PI 3-kinase, since RO 31-8220 does not inhibit insulin-induced activation of either PI 3-kinase (
      • Standaert M.L.
      • Avignon A.
      • Arnold T.
      • Saba-Siddique S.
      • Cooper D.R.
      • Watson J.
      • Zhou X.
      • Galloway L.
      • Farese R.V.
      ) or PI 3-kinase-dependent PKB activation2; presumably, the same situation pertains during GTPγS action,i.e. the RO 31-8220-sensitive protein kinase(s) required for glucose transport is distal or parallel to PI 3-kinase. Although the identity of the protein kinase is uncertain, note that both PKC-ζ and PKN are activated by PI 3-kinase lipid products (i.e.polyphosphoinositides) (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Giff R.
      • Parker P.J.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ), and PKN is directly activated by GTP-Rho (
      • Watanabe G.
      • Saito Y.
      • Madaule P.
      • Ishizaki T.
      • Fujisawa K.
      • Morii N.
      • Mukai H.
      • Ono Y.
      • Kakisuka A.
      • Narumiya S.
      ,
      • Amano M.
      • Mukai H.
      • Ono Y.
      • Chihara K.
      • Matsui T.
      • Hamajima Y.
      • Okawa K.
      • Iwamatsu A.
      • Kaibuchi K.
      ). Also, as reported for other bisindolemaleimides (see Refs.
      • Wilkinson S.E.
      • Parker P.J.
      • Nixon J.S.
      and
      • Martiny-Baron G.
      • Kazanietz M.G.
      • Mischak H.
      • Blumberg P.M.
      • Kochs G.
      • Hug H.
      • Marme D.
      • Schachtele C.
      ), we have found (
      • Standaert M.L.
      • Galloway L.
      • Karnam P.
      • Bandyopadhyay G.
      • Moscat J.
      • Farese R.V.
      ) that RO 31-8220 inhibits recombinant conventional (α, β, and γ) and novel (δ, ε, and η) PKCs at relatively low concentrations (IC50 values of 15–100 nm) and the atypical PKC, PKC-ζ, at relatively high concentrations (IC50 of 1–4 μm). Presently, we found that RO 31-8220, at relatively low concentrations (IC50 = 30 nm), inhibited immunoprecipitated PKN. Presumably, inhibitory effects of RO 31-8220 on PKC and PKN reflect homology in the catalytic domains of PKN and most PKCs (
      • Palmer R.H.
      • Ridden J.
      • Parker P.J.
      ).
      With respect to PKC-ζ and PKN as RO 31-8220-inhibitable protein kinases that may be required for glucose transport during the actions of GTPγS and insulin, the following are germane. First, PKC-ζ was activated by insulin, but not by GTPγS; thus, PKC-ζ seems unlikely to be involved in the action of GTPγS but may play a role during insulin action. Second, as in other systems in which GTP-Rho directly activates PKN (
      • Watanabe G.
      • Saito Y.
      • Madaule P.
      • Ishizaki T.
      • Fujisawa K.
      • Morii N.
      • Mukai H.
      • Ono Y.
      • Kakisuka A.
      • Narumiya S.
      ,
      • Amano M.
      • Mukai H.
      • Ono Y.
      • Chihara K.
      • Matsui T.
      • Hamajima Y.
      • Okawa K.
      • Iwamatsu A.
      • Kaibuchi K.
      ), we found that GTPγS activated both Rho and PKN, and insulin activated PKN by a Rho-dependent mechanism; accordingly, PKN is probably activated via Rho during the actions of both GTPγS and insulin in rat adipocytes. On the other hand, our studies suggested that different RO 31-8220-sensitive protein kinases were required for glucose transport effects of GTPγS and insulin in rat adipocytes; thus, insulin required a protein kinase sensitive to higher (IC50 of 4–5 μm) concentrations of RO 31-8220, e.g. PKC-ζ, whereas GTPγS required a protein kinase sensitive to lower (IC50 < 1 μm) concentrations of RO 31-8220, e.g. PKN. Although these findings with RO 31-8220 might suggest that PKN is required for glucose transport effects of GTPγS, but not insulin, note that expression of dominant-negative PKN partially inhibited (a) the effects of insulin as well as GTPγS on the translocation of HA-GLUT4 to the plasma membrane in rat adipocytes and (b) insulin effects on glucose transport in 3T3/L1 fibroblasts. It is presently uncertain if these seemingly divergent findings reflect shortcomings in our experimental approaches (e.g. effective local concentrations of inhibitors such as RO 31-8220 at specific enzyme sites in situ are uncertain, and transfections of dominant-negative proteins may cause untoward effects).
      As discussed, our findings suggested that Rho and PKN contributed to the activation of glucose transport during GTPγS action. However, activating effects of GTP-Rho on PKN would not explain the sensitivity of GTPγS-stimulated 2-DOG uptake to wortmannin and LY294002, unless PI 3-kinase, as well as Rho, needed to be co-activated, perhaps to further activate or correctly localize PKN. Accordingly, (a) PI-4,5-(PO4)2 and PI-3,4,5-(PO4)3 directly activate PKN (also known as PKC-related kinase-1 or 2 (PRK-1 or 2)) (
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Giff R.
      • Parker P.J.
      ); and (b) Rho is translocated by PI-4,5-(PO4)2 (Ref.
      • Karnam P.
      • Standaert M.L.
      • Galloway L.
      • Farese R.V.
      ) and PI-3,4-(PO4)2 and PI-3,4,5-(PO4)3 (see above), and PI 3-kinase lipid products may correctly localize Rho and, therefore, PKN to specific membrane compartments during the actions of both GTPγS and insulin.
      Our observation of activation of membrane-associated PI 3-kinase by GTPγS in rat adipocytes appears to differ from that of a previous report in which GTPγS failed to activate cytosolic PI 3-kinase in 3T3/L1 adipocytes (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ); this may reflect differences in cell types or the fact that we measured membrane, rather than cytosolic, PI 3-kinase activity. Along these lines, note that (a) we found that insulin and GTPγS activated membrane, but not cytosolic, PI 3-kinase in rat adipocytes; and (b) our failure to observe increases in cytosolic PI 3-kinase may reflect the large pool of insulin-independent PI 3-kinase that is activated indiscriminately during our assays of crude rat adipocyte cytosol. Although we did not examine the effects of GTPγS on membrane PI 3-kinase activity in 3T3/L1 adipocytes, we did find that overexpression of Rho activated membrane PI 3-kinase in these cells. Accordingly, it may be surmised that, as in rat adipocytes, GTPγS, by activating Rho, may activate PI 3-kinase in 3T3/L1 adipocytes; this could explain why GTPγS, at least partly (approximately 50%, as per wortmannin studies in Ref.
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ) requires PI 3-kinase for the activation of glucose transport in 3T3/L1 adipocytes; on the other hand, glucose transport effects of GTPγS that are independent of PI 3-kinase (also approximately 50%; see Ref.
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ) may be explained by direct activating effects of GTPγS on Rab (
      • Cormont M.
      • Tanti J.F.
      • Zahraoui A.
      • Van Obberghen E.
      • Tavitian A.
      • Le Marchand-Brustel Y.
      ) or other G-proteins that act distally to PI 3-kinase.
      Finally, it was of interest to find that, in addition to inhibitory effects of C3 transferase and dominant-negative forms of Rho and PKN on GTPγS- and insulin-stimulated glucose transport and/or GLUT4 translocation, transfected Rho (particularly if constitutively activated) and its downstream kinase, PKN, provoked increases in GLUT4 translocation and/or glucose transport in rat adipocytes and 3T3/L1 cells. It therefore may be conjectured that Rho is not only required for, but may actively participate in, the activation of GLUT4 translocation and glucose transport in the actions of insulin, GTPγS, and other agonists.
      In summary, like insulin, GTPγS provoked increases in 2-DOG uptake and HA-GLUT4 translocation in rat adipocytes. Also, like insulin, (a) GTPγS provoked increases in membrane-associated PI 3-kinase, and PI 3-kinase appeared to be required for GTPγS-induced activation of glucose transport; and (b) both Rho and an RO 31-8220-sensitive protein kinase appeared to be required for GTPγS-induced activation of glucose transport. In studies of RO 31-8220-sensitive protein kinases, both GTPγS and insulin activated PKN, and PKN appeared to be required for activation of GLUT4 translocation by GTPγS and insulin. Unlike insulin, however, GTPγS appeared to activate PI 3-kinase primarily through Rho, rather than through IRS-1; PKC-ζ was activated by insulin but not by GTPγS; and effects of GTPγS on glucose transport were inhibited by lower concentrations of RO 31-8220 than were effects of insulin. It may therefore be surmised that, although there are similarities in the signaling factors (i.e. Rho, PKN, and PI 3-kinase) that are used by GTPγS and insulin to activate glucose transport, these agents activate Rho and PI 3-kinase by different mechanisms and appear to use different distal protein kinases to activate glucose transport.

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