Lamar Galloway

Protein Kinase C-ζ as a Downstream Effector of Phosphatidylinositol 3-Kinase during Insulin Stimulation in Rat Adipocytes POTENTIAL ROLE IN GLUCOSE TRANSPORT

Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-ζ (PKC-ζ) in rat adipocytes. Concomitantly, insulin provoked increases in32P labeling of PKC-ζ both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-ζ; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-ζ pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-ζ was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-ζ enzyme activity. Also, PI-3,4-(PO4)2, PI-3,4,5-(PO4)3, and PI-4,5-(PO4)2 directly stimulated enzyme activity and autophosphoralytion in control PKC-ζ immunoprecipitates to levels observed in insulin-treated PKC-ζ immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-ζ enzyme activity in vitro by both the PKC-ζ pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigen-tagged GLUT4, co-transfection of wild-type or constitutive PKC-ζ stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-ζ partially inhibited it. Our findings suggest that insulin activates PKC-ζ through PI 3-kinase, and PKC-ζ may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.

Activation of Protein Kinase C (α, β, and ζ) by Insulin in 3T3/L1 Cells TRANSFECTION STUDIES SUGGEST A ROLE FOR PKC-η IN GLUCOSE TRANSPORT

We presently studied (a) insulin effects on protein kinase C (PKC) and (b) effects of transfection-induced, stable expression of PKC isoforms on glucose transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin provoked increases in membrane PKC enzyme activity and membrane levels of PKC-α and PKC-β. However, insulin-induced increases in PKC enzyme activity were apparent in both non-down-regulated adipocytes and adipocytes that were down-regulated by overnight treatment with 5 μM phorbol ester, which largely depletes PKC-α, PKC-β, and PKC-ε, but not PKC-η. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-η. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-α, PKC-β1, and PKC-β2 failed to influence basal or insulin-stimulated glucose transport (2-deoxyglucose uptake) in fibroblasts and adipocytes, despite inhibiting insulin effects on glycogen synthesis. In contrast, stable overexpression of wild-type PKC-η increased, and a dominant-negative mutant form of PKC-η decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-η, as well as PKC-α and β; and (b) PKC-η is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.

Insulin Activates Protein Kinases C-ζ and C-λ by an Autophosphorylation-dependent Mechanism and Stimulates Their Translocation to GLUT4 Vesicles and Other Membrane Fractions in Rat Adipocytes

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-ζ/λ activity in plasma membranes and microsomes and (b) immunoreactive PKC-ζ and PKC-λ in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-ζ and PKC-λ were also increased by insulinin situ and phosphatidylinositol 3,4,5-(PO4)3 (PIP3) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-ζ and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-ζ pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-ζ/λ but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-ζ. Also, insulinin situ and PIP3 in vitro activated and stimulated autophosphorylation of a PKC-ζ mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-ζ that lacks the regulatory (presumably PIP3-binding) domain; this may reflect PIP3effects on PDK-1 or transphosphorylation by endogenous full-length PKC-ζ. Our findings suggest that insulin activates both PKC-ζ and PKC-λ in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP3, PDK-1-dependent phosphorylation of activation loop sites in PKC-ζ and λ, and subsequent autophosphorylation and/or transphosphorylation.

Evidence for Involvement of Protein Kinase C (PKC)-ζ and Noninvolvement of Diacylglycerol-Sensitive PKCs in Insulin-Stimulated Glucose Transport in L6 Myotubes1

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.

Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/zucker rats : A mechanism for inhibiting glycogen synthesis

We examined the possibility that protein kinase C (PKC) is chronically activated and may contribute to impaired glycogen synthesis and insulin resistance in soleus muscles of hyperinsulinemic type II diabetic Goto-Kakizaki (GK) rats. Relative to nondiabetic controls, PKC enzyme activity and levels of immunoreactive PKC-α, β, є, and delta were increased in membrane fractions and decreased cytosolic fractions of GK soleus muscles. In addition, PKC-θ levels were decreased in both membrane and cytosol fractios, whereas PKC-ζ levels were not changed in either fraction in GK soleus muscles. These increases in membrane PKC (α, β, є, and δ) could not be accounted for by alterations in PKC mRNA or total PKC levels but were associated with increases in membrane diacylglycerol (DAG) and therefore appeared to reflect translocative activation of PKC. In evaluation of potential causes for persistent PKC activation, membrane PKC levels were decreased in soleus muscles of hyperglycemic streptozotocin (STZ)-induced diabetic rats; thus, a role for simple hyperglycemia as a cause of PKC activation in GK rats was not evident in the STZ model. In support of the possibility that hyperinsulinemia contributed to PKC activation in GK soleus muscles, we found that DAG levels were increased, and PKC was translocated, in soleus muscles of both (1) normoglycemic hyperinsulinemic obese/aged rats and (2) mildly hyperglycemic hyperinsulinemic obese/Zucker rats. In keeping with the possibility that PKC activation may contribute to impaired glycogen synthase activation in GK muscles, phorbol esters inhibited, and a PKC inhibitor, RO 31-8220, increased insulin effects on glycogen synthesis in soleus muscles incubated in vitro. Our findings suggested that: (1) hyperinsulinemia, as observed in type II diabetic GK rats and certain genetic and nongenetic forms of obesity in rats, is associated with persistent translocation and activation of PKC in soleus muscles, and (2) this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance.

Activation and Translocation of Rho (and ADP Ribosylation Factor) by Insulin in Rat Adipocytes APPARENT INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE

Insulin reportedly (Standaert, M. L., Avignon, A., Yamada, K., Bandyopadhyay, G., and Farese, R. V. (1996) Biochem. J. 313, 1039-1046) activates phospholipase D (PLD)-dependent hydrolysis of phosphatidylcholine (PC) in plasma membranes of rat adipocytes by a mechanism that may involve wortmannin-sensitive phosphatidylinositol (PI) 3-kinase. Because Rho and ADP ribosylation factor (ARF) activate PC-PLD, we questioned whether these small G-proteins are regulated by insulin and PI 3-kinase. We found that insulin provoked a rapid translocation of both Rho and ARF to the plasma membrane and increased GTP loading of Rho. Wortmannin and LY294002 inhibited Rho translocation in intact adipocytes, and the polyphosphoinositide, PI 4,5-(PO4)2, stimulated Rho translocation in adipocyte homogenates. On the other hand, wortmannin did not block GTP loading of Rho. Guanosine 5′-3-O-(thio)triphosphate stimulated both Rho and ARF translocation and activated PC-PLD in homogenates. C3 transferase, which inhibits and depletes Rho, inhibited PC-PLD activation by insulin in intact adipocytes. C3 transferase also inhibited insulin stimulation of [3H]2-deoxyglucose uptake. Our findings suggest that: (a) insulin translocates Rho by a PI 3-kinase-dependent mechanism, but another factor is responsible for GTP loading of Rho; (b) both Rho and ARF may contribute to PC-PLD activation during insulin action; and (c) Rho may be required for insulin stimulation of glucose transport.

Effects of Knockout of the Protein Kinase C β Gene on Glucose Transport and Glucose Homeostasis1

The β-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resistance as well as for contributing to the pathogenesis of diabetic complications. Presently, we evaluated the effects of knockout of the PKCβ gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeostasis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCβ gene. PKCβ−/− knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCβ+/+ wild-type offspring in both the fasting state and 30 min after ip injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/6–129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6–129/SV hybrid ...

Effects of phorbol esters on insulin-induced activation of phosphatidylinositol 3-kinase, glucose transport, and glycogen synthase in rat adipocytes

In rat adipocytes, phorbol ester‐induced activation of PKC did not inhibit insulin signalling through IRS‐1‐dependent phosphatidylinositol (PI) 3‐kinase activation. Moreover, phorbol esters alone provoked an increase in membrane PI 3‐kinase activity. These findings may be relevant to the failure of phorbol esters to inhibit insulin effects on glucose transport and glycogen synthesis in rat adipocytes.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

Insulin translocates PKC-ϵ and phorbol esters induce and persistently translocate PKC-β2 in BC3H-1 myocytes

Abstract Initial studies suggested that insulin increases diacylglycerol and activates protein kinase C (PKC) in BC3H-1 myocytes. In these earlier studies, insulin was found to translocate PKC-β, but the presence of PKC-ϵ was not appreciated. More recently, the presence of PKC-ϵ was documented, but PKC-β was not detected, and it was questioned whether insulin activates PKC in BC3H-1 myocytes [Stumpo, D.J., Haupt, D.M. and Blackshear, P.J. (1994)J. Biol. Chem. 269:21184–21190]. We questioned whether insulin translocates PKC-ϵ in BC3H-1 myocytes, and re-evaluated the question of whether myocytes truly contain a PKC-β isoform whose existence can be verified by its response to phorbol ester treatment. We found that PKC-ϵ was acutely translocated by insulin and phorbol esters from the cytosol to the membrane fraction in BC3H-1 myocytes; in addition, PKC-ϵ, like PKC-α, was depleted by chronic phorbol ester treatment. We also found that BC3H-I myocytes containing a 76,000 Mr PKC-β isoform that is acutely translocated and subsequently depleted by phorbol esters. Moreover, chronic phorbol ester treatment induced an 84,000 Mr PKC-β2 isoform that appeared to be persistently translocated and activated, as suggested by studies of myristoylated arginine-rich C kinase substrate (MARCKS) phosphorylation. We conclude that: (1) insulin acutely translocates PKC-ϵ, as well as PKC-β, in BC3H-1 myocytes; and (2) PKC-β is not truly downregulated by phorbol esters in BC3H-1 myocytes.