Role of PDK1 in insulin-signaling pathway for glucose  metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (ΔPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas ΔPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or ΔPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3α/β, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither ΔPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3α/β pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or ΔPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.

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Insulin-Sensitive Phospholipid Signaling Systems and Glucose Transport. Update II

Experimental Biology and Medicine ◽

10.1177/153537020122600404 ◽

2001 ◽

Vol 226 (4) ◽

pp. 283-295 ◽

Cited By ~ 59

Author(s):

Robert V. Farese

Keyword(s):

Protein Kinase ◽

Glucose Metabolism ◽

Glucose Transport ◽

Intracellular Signaling ◽

Glucose Transporter ◽

De Novo ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Cell Types ◽

Biologically Active

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidyicholine with subsequent increases in phosphatidic acid (PA) and diacyiglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

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PTEN, but Not SHIP2, Suppresses Insulin Signaling through the Phosphatidylinositol 3-Kinase/Akt Pathway in 3T3-L1 Adipocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m501949200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22523-22529 ◽

Cited By ~ 81

Author(s):

Xiaoqing Tang ◽

Aimee M. Powelka ◽

Neil A. Soriano ◽

Michael P. Czech ◽

Adilson Guilherme

Keyword(s):

Signaling Pathway ◽

Glucose Transport ◽

Insulin Signaling ◽

Glycogen Synthase ◽

Sh2 Domain ◽

Insulin Signaling Pathway ◽

Dominant Negative ◽

Tensin Homolog ◽

Gene Ablation ◽

Mitogen Activated Protein

Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3′-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3α (GSK-3α) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes.

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Inhibition of insulin signaling and glycogen synthesis by phorbol dibutyrate in rat skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.1.e8 ◽

2001 ◽

Vol 281 (1) ◽

pp. E8-E15 ◽

Cited By ~ 17

Author(s):

Yenshou Lin ◽

Samar I. Itani ◽

Theodore G. Kurowski ◽

David J. Dean ◽

Zhijun Luo ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Glucose Transport ◽

Insulin Signaling ◽

Phorbol Esters ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Insulin Receptor Tyrosine Kinase ◽

Pkc Isoforms ◽

Glucose Incorporation

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 μM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-α, -βI, -θ, and -ε, and probably -βII, from the cytosol to membranes. Preincubation with 1 μM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3′-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2 was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2 are unaffected.

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Defective insulin signaling in skeletal muscle of the hypertensive TG(mREN2)27 rat

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00396.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. E1074-E1081 ◽

Cited By ~ 40

Author(s):

Julie A. Sloniger ◽

Vitoon Saengsirisuwan ◽

Cody J. Diehl ◽

Betsy B. Dokken ◽

Narissara Lailerd ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Angiotensin Ii ◽

Insulin Receptor ◽

Signaling Pathway ◽

Glucose Transport ◽

Insulin Signaling ◽

Glycogen Synthase ◽

Insulin Signaling Pathway ◽

Whole Body

Essential hypertension is frequently associated with insulin resistance of skeletal muscle glucose transport, with a potential role of angiotensin II in the pathogenesis of both conditions. The male heterozygous TG(mREN2)27 rat harbors the mouse transgene for renin, exhibits local elevations in angiotensin II, and is an excellent model of both hypertension and insulin resistance. The present study was designed to investigate the potential cellular mechanisms for insulin resistance in this hypertensive animal model, including an assessment of elements of the insulin-signaling pathway. Compared with nontransgenic, normotensive Sprague-Dawley control rats, male heterozygous TG(mREN2)27 rats displayed elevated ( P < 0.05) fasting plasma insulin (74%), an exaggerated insulin response (108%) during an oral glucose tolerance test, and reduced whole body insulin sensitivity. TG(mREN2)27 rats also exhibited decreased insulin-mediated glucose transport and glycogen synthase activation in both the type IIb epitrochlearis (30 and 46%) and type I soleus (22 and 64%) muscles. Importantly, there were significant reductions (∼30–50%) in insulin stimulation of tyrosine phosphorylation of the insulin receptor β-subunit and insulin receptor substrate-1 (IRS-1), IRS-1 associated with the p85 subunit of phosphatidylinositol 3-kinase, Akt Ser473 phosphorylation, and Ser9 phosphorylation of glycogen synthase kinase-3β in epitrochlearis and soleus muscles of TG(mREN2)27 rats. Soleus muscle triglyceride concentration was 25% greater in the transgenic group compared with nontransgenic animals. Collectively, these data provide the first evidence that the insulin resistance of the hypertensive male heterozygous TG(mREN2)27 rat can be attributed to specific defects in the insulin-signaling pathway in skeletal muscle.

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Vanadium and the cardiovascular functions

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-089 ◽

2004 ◽

Vol 82 (10) ◽

pp. 833-839 ◽

Cited By ~ 18

Author(s):

Lise Coderre ◽

Ashok K Srivastava

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Signaling Pathway ◽

Glucose Transport ◽

Insulin Signaling ◽

Insulin Signaling Pathway ◽

Vanadium Compounds ◽

Muscle Contractility ◽

Smooth Muscle Contractility ◽

Glut 4

Inorganic and organic compounds of vanadium have been shown to exhibit a large range of insulinomimetic effects in the cardiovascular system, including stimulation of glucose transporter 4 (GLUT-4) translocation and glucose transport in adult cardiomyocytes. Furthermore, administration of vanadium compounds improves cardiac performance and smooth muscle contractility, and modulates blood pressure in various models of hypertension and insulin resistance. Vanadium compounds are potent inhibitors of protein tyrosine phosphatases. As a result, they promote an increase in protein tyrosine phosphorylation of several key components of the insulin signaling pathway, leading to the upregulation of phosphatidylinositol 3-kinase and protein kinase B, two enzymes involved in mediating GLUT-4 trans location and glucose transport. In addition, vanadium has also been shown to activate p38 mitogen-activated protein kinase and increase Ca2+ levels in several cell types. The ability of vanadium compounds to activate these signaling events may be responsible for their ability to modulate cardiovascular functions.Key words: vanadium compounds, glucose transport, smooth muscle contractility, insulin signaling pathway.

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