Yoshinori Kanoh

Activation of the ERK Pathway and Atypical Protein Kinase C Isoforms in Exercise- and Aminoimidazole-4-carboxamide- 1-β-d-riboside (AICAR)-stimulated Glucose Transport

Exercise increases glucose transport in muscle by activating 5′-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-ζ pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-ζ. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.

Glucose activates protein kinase C-zeta /lambda through proline-rich tyrosine kinase-2, extracellular signal-regulated kinase, and phospholipase D: a novel mechanism for activating glucose transporter translocation.

Insulin controls glucose uptake by translocating GLUT4 and other glucose transporters to the plasma membrane in muscle and adipose tissues by a mechanism that appears to require protein kinase C (PKC)-ζ/λ operating downstream of phosphatidylinositol 3-kinase. In diabetes mellitus, insulin-stimulated glucose uptake is diminished, but with hyperglycemia, uptake is maintained but by uncertain mechanisms. Presently, we found that glucose acutely activated PKC-ζ/λ in rat adipocytes and rat skeletal muscle preparations by a mechanism that was independent of phosphatidylinositol 3-kinase but, interestingly, dependent on the apparently sequential activation of the dantrolene-sensitive, nonreceptor proline-rich tyrosine kinase-2; components of the extracellular signal-regulated kinase (ERK) pathway, including, GRB2, SOS, RAS, RAF, MEK1 and ERK1/2; and, most interestingly, phospholipase D, thus yielding increases in phosphatidic acid, a known activator of PKC-ζ/λ. This activation of PKC-ζ/λ, moreover, appeared to be required for glucose-induced increases in GLUT4 translocation and glucose transport in adipocytes and muscle cells. Our findings suggest the operation of a novel pathway for activating PKC-ζ/λ and glucose transport.

Glucose Activates Mitogen-activated Protein Kinase (Extracellular Signal-regulated Kinase) through Proline-rich Tyrosine Kinase-2 and the Glut1 Glucose Transporter

Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463–468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469–492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463–468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK.

Rosiglitazone, Insulin Treatment, and Fasting Correct Defective Activation of Protein Kinase C-ζ/λ by Insulin in Vastus Lateralis Muscles and Adipocytes of Diabetic Rats1

Atypical protein kinases C (PKCs), zeta and lambda, and protein kinase B (PKB) are thought to function downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and regulate glucose transport during insulin action in skeletal muscle and adipocytes. Insulin-stimulated glucose transport is defective in type II diabetes mellitus, and this defect is ameliorated by thiazolidinediones and lowering of blood glucose by chronic insulin therapy or short-term fasting. Presently, we evaluated the effects of these insulin-sensitizing modalities on the activation of insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase, PKC-zeta/lambda, and PKB in vastus lateralis skeletal muscles and adipocytes of nondiabetic and Goto-Kakizaki (GK) diabetic rats. Insulin provoked rapid increases in the activity of PI 3-kinase, PKC-zeta/lambda, and PKB in muscles and adipocytes of nondiabetic rats, but increases in IRS-1-dependent PI 3-kinase and PKC-zeta/lambda, but not PKB, activity were substantially diminished in GK muscles and adipocytes. Rosiglitazone treatment for 10-14 days, 10-day insulin treatment, and 60-h fasting reversed defects in PKC-zeta/lambda activation in GK muscles and adipocytes and increased glucose transport in GK adipocytes, without necessarily increasing IRS-1-dependent PI 3-kinase or PKB activation. Our findings suggest that insulin-sensitizing modalities, viz. thiazolidinediones, chronic insulin treatment, and short-term fasting, similarly improve defects in insulin-stimulated glucose transport at least partly by correcting defects in insulin-induced activation of PKC-zeta/lambda.

DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase

We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 μM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-α, -β, and -γ. DHEA provoked the translocation of PKC-β and -ζ from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12- O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

Cbl, IRS-1, and IRS-2 Mediate Effects of Rosiglitazone on PI3K, PKC-λ, and Glucose Transport in 3T3/L1 Adipocytes

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-λ; accompanied by increases in tyrosine phosphorylation of Cbl and Cbl-dependent increases in PI3K and PKC-λ activity; but not accompanied by increases in IRS-1/2-dependent PI3K or 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-λ and protein kinase B. Our findings suggest that Cbl participates in mediating effects of rosiglitazone on PI3K, PDK-1, and PKC-λ and the glucose transport system and that this Cbl-dependent pathway complements the IRS-1 and I...

Glucocorticoid-induced insulin resistance associates with activation of protein kinase C isoforms

We studied glucocorticoid-induced insulin resistance and possible role of protein kinase C (PKC). Pretreatment with dexamethasone, prednisolone and corticosterone for 60 min decreased insulin-induced [3H] 2-deoxyglucose (DOG) uptake in isolated rat adipocytes. Preincubation with Go6976, LY379196 or myristoylated PKC pseudosubstrate, conventional PKC inhibitor, but not cycloheximide or RU38486, recovered dexamethasone-induced insulin resistance. Dexamethasone activated immunoprecipitates with anti-PKC alpha, beta, and zeta antibodies. PKC zeta activity in adipocytes increased to 163%, and 264% from basal level (100%) with dexamethasone and insulin treatment, respectively. Dexamethasone provoked redistribution of both PKC beta and zeta from the cytosol to the membrane. These results indicate that dexamethasone activates both conventional and atypical PKC. However, conventional PKC is more important in glucocorticoid-induced insulin resistance.

Effect of tumor necrosis factor-α on insulin signal transduction in rat adipocytes: relation to PKCβ and ζ translocation

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.

Protein Kinase C (PKC) β Modulates Serine Phosphorylation of Insulin Receptor Substrate‐1 (IRS‐1)—Effect of Overexpression of PKCβ on Insulin Signal Transduction

In vitro phosphorylation of 180‐kDa protein, obtained by immunoprecipitation of adipocyte homogenate with anti‐IRS‐1 antibody was increased with the addition of conventional PKC in the presence of Ca2 +, phosphatidylserine (PS) and diolein (DL). Human purified IRS‐1 was phosphorylated by purified conventional PKC (cPKC) in the presence of Ca2 +/PS/DL. These results suggest that PKC may have a role in the serine phosphorylation of IRS‐1. In order to clarify the inhibitory effect of cPKC on glucose transport mechanism, we examined the overexpression of PKCβ in cultured adipocytes. Overexpression of PKCβ in adipocytes markedly induced mobility shift and serine phosphorylation of IRS‐1, whereas overexpression of dominant negative PKCβ (DNPKCβ) blocked this mobility shift and serine phosphorylation of IRS‐1. Insulin (10 nM) increased [3H]2‐deoxyglucose (2‐DOG) uptake to 200% from basal level (100%) in cultured adipocytes transfected with a vector alone. Overexpression of PKCβ in adipocytes decreased insulin‐induced 2‐DOG uptake to 110%, whereas overexpression of DNPKCβ increased it to 230%. These results suggest that PKCβ negatively regulates glucose uptake via serine phosphorylation of IRS‐1 in rat adipocytes.

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.