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Biomedical subjects

L Galloway

Publications and source records attributed to L Galloway.

16 recordsLinked to original sources
PubMed1999

Insulin activates protein kinases C-zeta and C-lambda 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)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta 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-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.

Adipocytes↗
PubMed1999

Effects of knockout of the protein kinase C beta gene on glucose transport and glucose homeostasis.

The beta-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 PKCbeta 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 PKCbeta gene. PKCbeta-/- knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCbeta+/+ wild-type offspring in both the fasting state and 30 min after i.p. 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 PKCbeta knockout mice in fasting and fed states and after i.p. glucose administration. In keeping with a tendency for slightly lower serum glucose and/or insulin levels in PKCbeta knockout mice, insulin-stimulated 2-deoxyglucose (2-DOG) uptake was enhanced by 50-100% in isolated adipocytes; basal and insulin-stimulated epitope-tagged GLUT4 translocations in adipocytes were increased by 41% and 27%, respectively; and basal 2-DOG uptake was mildly increased by 20-25% in soleus muscles incubated in vitro. The reason for increased 2-DOG uptake and/or GLUT4 translocation in these tissues was uncertain, as there were no significant alterations in phosphatidylinositol 3-kinase activity or activation or in levels of GLUT1 or GLUT4 glucose transporters or other PKC isoforms. On the other hand, increases in 2-DOG uptake may have been partly caused by the loss of PKCbeta1, rather than PKCbeta2, as transient expression of PKCbeta1 selectively inhibited insulin-stimulated translocation of epitope-tagged GLUT4 in adipocytes prepared from PKCbeta knockout mice. Our findings suggest that 1) PKCbeta is not required for insulin-stimulated glucose transport; 2) overall glucose homeostasis in vivo is mildly enhanced by knockout of the PKCbeta gene; 3) glucose transport is increased in some tissues in PKCbeta knockout mice; and 4) increased glucose transport may be partly due to loss of PKCbeta1, which negatively modulates insulin-stimulated GLUT4 translocation.

Adipocytes↗
PubMed1998

Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport.

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS 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 GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS 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 GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. 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 GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS 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-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.

ADP Ribose Transferases↗
PubMed1997

Protein kinase C-zeta 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-zeta (PKC-zeta) in rat adipocytes. Concomitantly, insulin provoked increases in 32P labeling of PKC-zeta both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-zeta; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-zeta pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-zeta 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-zeta 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-zeta immunoprecipitates to levels observed in insulin-treated PKC-zeta immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-zeta enzyme activity in vitro by both the PKC-zeta 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-zeta stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-zeta partially inhibited it. Our findings suggest that insulin activates PKC-zeta through PI 3-kinase, and PKC-zeta may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.

Adipocytes↗
PubMed1997

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.

ADP-Ribosylation Factors↗
PubMed1997

Activation of protein kinase C (alpha, beta, and zeta) by insulin in 3T3/L1 cells. Transfection studies suggest a role for PKC-zeta 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-alpha and PKC-beta. 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 microM phorbol ester, which largely depletes PKC-alpha, PKC-beta, and PKC-epsilon, but not PKC-zeta. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-zeta. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-alpha, PKC-beta1, and PKC-beta2 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-zeta increased, and a dominant-negative mutant form of PKC-zeta decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-zeta, as well as PKC-alpha and beta; and (b) PKC-zeta is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.

3T3 Cells↗
PubMed1997

Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes.

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.

Animals↗
PubMed1996

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.

Adipocytes↗
PubMed1996

Insulin translocates PKC-epsilon and phorbol esters induce and persistently translocate PKC-beta 2 in BC3H-1 myocytes.

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-beta, but the presence of PKC-epsilon was not appreciated. More recently, the presence of PKC-epsilon was documented, but PKC-beta 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-epsilon in BC3H-1 myocytes, and re-evaluated the question of whether myocytes truly contain a PKC-beta isoform whose existence can be verified by its response to phorbol ester treatment. We found that PKC-epsilon was acutely translocated by insulin and phorbol esters from the cytosol to the membrane fraction in BC3H-1 myocytes; in addition, PKC-epsilon, like PKC-alpha, was depleted by chronic phorbol ester treatment. We also found that BC3H-1 myocytes containing a 76,000 Mr PKC-beta isoform that is acutely translocated and subsequently depleted by phorbol esters. Moreover, chronic phorbol ester treatment induced an 84,000 Mr PKC-beta 2 isoform that appeared to be persistently translocated and activated, as suggested by studies of myristoylated arginic-rich C kinase substrate (MARCKS) phosphorylation. We conclude that: (1) insulin acutely translocates PKC-epsilon, as well as PKC-beta, in BC3H-1 myocytes; and (2) PKC-beta is not truly downregulated by phorbol esters in BC3H-1 myocytes.

Amino Acid Sequence↗
PubMed1996

Insulin stimulates phospholipase D-dependent phosphatidylcholine hydrolysis, Rho translocation, de novo phospholipid synthesis, and diacylglycerol/protein kinase C signaling in L6 myotubes.

Previous studies have provided conflicting findings on whether insulin activates certain, potentially important, phospholipid signaling systems in skeletal muscle preparations. In particular, insulin effects on the hydrolysis of phosphatidylcholine (PC) and subsequent activation of protein kinase C (PKC) have not been apparent in some studies. Presently, we examined insulin effects on phospholipid signaling systems, diacylglycerol (DAG) production, and PKC translocation/activation in L6 myotubes. We found that insulin provoked rapid increases in phospholipase D (PLD)-dependent hydrolysis of PC, as evidenced by increases in choline release and phosphatidylethanol production in cells incubated in the presence of ethanol. In association with PC-PLD activation, Rho, a small G protein that is known to activate PC-PLD activation, translocated from the cytosol to the membrane fraction in response to insulin treatment. PC-PLD activation was also accompanied by increases in total DAG production and increases in the translocation of both PKC enzyme activity and DAG-sensitive PKC-alpha, -beta, -delta, and -epsilon from the cytosol to the membrane fraction. A potential role for PKC or a related protein kinase in insulin action was suggested by the finding that RO 31-8220 inhibited both PKC enzyme activity and insulin-stimulated [3H]2-deoxyglucose uptake. Our findings provide the first evidence that insulin stimulates Rho translocation and activates PC-PLD in L6 skeletal muscle cells. Moreover, this signaling system appears to lead to increases in DAG/PKC signaling, which, along with other related signaling factors, may regulate certain metabolic processes, such as glucose transport, in these cells.

Animals↗
PubMed1996

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-alpha, beta, epsilon, and delta were increased in membrane fractions and decreased cytosolic fractions of GK soleus muscles. In addition, PKC-theta levels were decreased in both membrane and cytosol fractios, whereas PKC-zeta levels were not changed in either fraction in GK soleus muscles. These increases in membrane PKC (alpha, beta, epsilon, and delta) 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.

Adipocytes↗
PubMed1994

Knowing the form.

This study set out to discover if midwives need to complete the antenatal record form (ANR) in its entirety, or if women could complete part of the form themselves. The study involved 33 pregnant women and eight midwives. Questionnaires were used for both the women and the midwives. In addition, a structured interview was employed for the participating women. These methods gave both qualitative and quantitative data. Thirty women (94%) completed the selected parts of the ANR form, either mostly or in full. Twenty-four women (67%) stated that they had completed this form by themselves. Midwives completed a questionnaire for each woman they booked. For 21 cases (62%) midwives found it useful when the woman completed part of the ANR form. Offering women the opportunity to complete part of the ANR form was considered to be advantageous by 19 (57%) mothers participating in the study. The conclusion is that midwives do not always need to complete the antenatal record form in its entirety.

Female↗
PubMed1985

Bupropion in the long-term treatment of cyclic mood disorders: mood stabilizing effects.

The effects of bupropion HCl in treating 11 patients with bipolar or schizoaffective disorder were examined in an open trial. Most patients had been intolerant of or showed minimal to moderate improvement on lithium, neuroleptics, antidepressants, or a combination of these drugs. All patients were maintained on bupropion alone or bupropion in combination with low-dose neuroleptics or anxiolytics for 1 year or more, with little or no relapse and few side effects.

Adult↗