PLCγ Participates in Insulin Stimulation of Glucose Uptake through Activation of PKCζ in Brown Adipocytes

Abstract Background Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3β), on the direct insulin stimulation of glucose oxidation. Methods We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3β is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3β between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA. Results Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin’s direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3β phosphorylation does not influence insulin-stimulated glucose oxidation. Conclusion We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

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Insulin resistance of glucose metabolism in isolated brown adipocytes of lactating rats. Evidence for a post-receptor defect in insulin action

Biochemical Journal ◽

10.1042/bj2650511 ◽

1990 ◽

Vol 265 (2) ◽

pp. 511-517 ◽

Cited By ~ 6

Author(s):

A F Burnol ◽

S Ebner ◽

J Kandé ◽

J Girard

Keyword(s):

Insulin Resistance ◽

Glucose Metabolism ◽

Tyrosine Kinase ◽

Pyruvate Dehydrogenase ◽

Kinase Activity ◽

Insulin Stimulation ◽

Tyrosine Kinase Activity ◽

Brown Adipocytes ◽

Insulin Receptor Tyrosine Kinase ◽

Stimulation Of

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.

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Diet-induced muscle insulin resistance in rats is ameliorated by acute dietary lipid withdrawal or a single bout of exercise: parallel relationship between insulin stimulation of glucose uptake and suppression of long-chain fatty acyl-CoA

Diabetes ◽

10.2337/diabetes.46.12.2022 ◽

1997 ◽

Vol 46 (12) ◽

pp. 2022-2028 ◽

Cited By ~ 24

Author(s):

N. D. Oakes ◽

K. S. Bell ◽

S. M. Furler ◽

S. Camilleri ◽

A. K. Saha ◽

...

Keyword(s):

Insulin Resistance ◽

Glucose Uptake ◽

Fatty Acyl ◽

Dietary Lipid ◽

Insulin Stimulation ◽

Muscle Insulin Resistance ◽

Single Bout ◽

Long Chain ◽

Stimulation Of

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Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.6971 ◽

1998 ◽

Vol 18 (12) ◽

pp. 6971-6982 ◽

Cited By ~ 269

Author(s):

Ko Kotani ◽

Wataru Ogawa ◽

Michihiro Matsumoto ◽

Tadahiro Kitamura ◽

Hiroshi Sakaue ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transporter ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Pleckstrin Homology Domain ◽

Dependent Manner ◽

Insulin Stimulation ◽

Negative Effects ◽

Stimulation Of

ABSTRACT Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ∼50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

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Resistance to Insulin Suppression of Plasma Free Fatty Acid Concentrations and Insulin Stimulation of Glucose Uptake in Noninsulin-Dependent Diabetes Mellitus*

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem-64-1-17 ◽

1987 ◽

Vol 64 (1) ◽

pp. 17-21 ◽

Cited By ~ 118

Author(s):

Y.-D. I. CHEN ◽

A. GOLAY ◽

A. L. M. SWISLOCKI ◽

G. M. REAVEN

Keyword(s):

Diabetes Mellitus ◽

Fatty Acid ◽

Free Fatty Acid ◽

Glucose Uptake ◽

Plasma Free Fatty Acid ◽

Dependent Diabetes Mellitus ◽

Insulin Stimulation ◽

Resistance To Insulin ◽

Stimulation Of

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Insulin-stimulated glucose transport in muscle. Evidence for a protein-kinase-C-dependent component which is unaltered in insulin-resistant mice

Biochemical Journal ◽

10.1042/bj2580141 ◽

1989 ◽

Vol 258 (1) ◽

pp. 141-146 ◽

Cited By ~ 34

Author(s):

J F Tanti ◽

N Rochet ◽

T Grémeaux ◽

E Van Obberghen ◽

Y Le Marchand-Brustel

Keyword(s):

Protein Kinase C ◽

Glucose Uptake ◽

Glucose Transport ◽

Obese Mice ◽

Insulin Stimulation ◽

Insulin Resistant ◽

Kinase C ◽

Dependent Component ◽

Pkc Activation ◽

Stimulation Of

The aim of our work was to investigate a possible role of protein kinase C (PKC) in insulin-stimulated glucose uptake in mouse skeletal muscle, and to search for a defect in PKC activation in insulin resistance found in obesity. In isolated soleus muscle of lean mice, insulin (100 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microM) acutely stimulated glucose uptake 3- and 2-fold respectively. The effects of insulin and TPA were not additive. When PKC activity was down-regulated by long-term (24 h) TPA pretreatment, before measurement of glucose transport, the TPA effect was abolished, but in addition insulin-stimulated glucose transport returned to basal values. Furthermore, polymyxin B, which inhibits PKC in muscle extracts, prevented insulin-stimulated glucose uptake in muscle. In muscle of obese insulin-resistant mice, glucose uptake evoked by insulin was decreased, whereas the TPA effect, expressed as a fold increase, was unaltered. Thus both agents stimulated glucose transport to the same extent. Furthermore, no difference was observed when PKC activation by TPA was measured in muscle from lean and obese mice. These results suggest that: (1) PKC is involved in the insulin effect on glucose transport in muscle; (2) PKC activation explains only part of the insulin stimulation of glucose transport; (3) the defect in insulin response in obese mice does not appear to be due to an alteration in the PKC-dependent component of glucose transport. We propose that insulin stimulation of glucose uptake occurs by a sequential two-step mechanism, with first translocation of transporters to the plasma membrane, which is PKC dependent, and second, activation of the glucose transporters. In obesity only the activation step was decreased, whereas the translocation step was unaltered.

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Insulin stimulation of iodothyronine 5′-deiodinase in rat brown adipocytes

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(87)90632-2 ◽

1987 ◽

Vol 143 (1) ◽

pp. 81-86 ◽

Cited By ~ 21

Author(s):

Ira Mills ◽

Renee M. Barge ◽

J.Enrique Silva ◽

P.Reed Larsen

Keyword(s):

Insulin Stimulation ◽

Brown Adipocytes ◽

Stimulation Of

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DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.280.2.e229 ◽

2001 ◽

Vol 280 (2) ◽

pp. E229-E237 ◽

Cited By ~ 162

Author(s):

Eulàlia Montell ◽

Marco Turini ◽

Mario Marotta ◽

Matthew Roberts ◽

Véronique Noé ◽

...

Keyword(s):

Insulin Resistance ◽

Fatty Acids ◽

Protein Kinase C ◽

Protein Kinase ◽

Glucose Uptake ◽

Insulin Response ◽

Insulin Stimulation ◽

Kinase C ◽

Basal Glucose Uptake ◽

Stimulation Of

The increased availability of saturated lipids has been correlated with development of insulin resistance, although the basis for this impairment is not defined. This work examined the interaction of saturated and unsaturated fatty acids (FA) with insulin stimulation of glucose uptake and its relation to the FA incorporation into different lipid pools in cultured human muscle. It is shown that basal or insulin-stimulated 2-deoxyglucose uptake was unaltered in cells preincubated with oleate, whereas basal glucose uptake was increased and insulin response was impaired in palmitate- and stearate-loaded cells. Analysis of the incorporation of FA into different lipid pools showed that palmitate, stearate, and oleate were similarly incorporated into phospholipids (PL) and did not modify the FA profile. In contrast, differences were observed in the total incorporation of FA into triacylglycerides (TAG): unsaturated FA were readily diverted toward TAG, whereas saturated FA could accumulate as diacylglycerol (DAG). Treatment with palmitate increased the activity of membrane-associated protein kinase C, whereas oleate had no effect. Mixture of palmitate with oleate diverted the saturated FA toward TAG and abolished its effect on glucose uptake. In conclusion, our data indicate that saturated FA-promoted changes in basal glucose uptake and insulin response were not correlated to a modification of the FA profile in PL or TAG accumulation. In contrast, these changes were related to saturated FA being accumulated as DAG and activating protein kinase C. Therefore, our results suggest that accumulation of DAG may be a molecular link between an increased availability of saturated FA and the induction of insulin resistance.

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