AJP - Endo Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E1169-E1177, 2008. First published April 22, 2008; doi:10.1152/ajpendo.00050.2008
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/6/E1169    most recent
00050.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liberman, Z.
Right arrow Articles by Eldar-Finkelman, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liberman, Z.
Right arrow Articles by Eldar-Finkelman, H.

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase CβII in the diabetic fat tissue

Ziva Liberman,1 Batya Plotkin,1 Tamar Tennenbaum,2 and Hagit Eldar-Finkelman1

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv; and 2Faculty of Life Science, Bar Ilan University, Ramat-Gan, Israel

Submitted 24 January 2008 ; accepted in final form 21 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser332. However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser336 on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser336 and Ser332 in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser336. Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKC{alpha} or PKCβII isoforms in cells enhanced IRS-1 phosphorylation at Ser336 and Ser332, and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser336. The expression level and activation state of PKCβII, but not PKC{alpha}, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCβII were also associated with enhanced phosphorylation of IRS-1 at Ser336/332 and elevated activity of GSK-3β. Finally, adenoviral mediated expression of PKCβII in adipocytes enhancedphosphorylation of IRS-1 at Ser336. Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCβII and GSK-3 at Ser336 and Ser332. Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.

insulin signaling; insulin resistance; obesity

INSULIN RESISTANCE is the principal predictor for the development of type 2 diabetes and is associated with many complications, including obesity, polycystic ovary syndrome, hypertension, and cardiovascular disease (2, 34). Insulin resistance is a pathological state in which target tissues, particularly muscle fat and liver, fail to respond properly to normal concentrations of insulin (53, 61, 71). The immediate targets of the insulin receptor, the insulin receptor substrate proteins, in particular insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), are key elements in mediating insulin signaling. The inappropriate regulation of these targets is believed to play a central role in insulin resistance. The IRS proteins become tyrosyl-phosphorylated by the insulin receptor tyrosine kinase, which in turn, initiates the recruitment of SH2 domain-containing molecules, such as the p85 regulatory subunit of PI 3-kinase, Grb-2, SHP2, Nck, and Crk (47, 59, 75). The tyrosine-phosphorylated IRSs then activate downstream effectors that mediate the metabolic and growth-stimulatory effects of insulin (47, 59, 75).

Recent studies indicate that phosphorylation of IRS-1 on serine/threonine residues produces inhibitory effects on insulin signaling (19, 25, 69). Some studies have correlated increased in vivo serine phosphorylation of IRS-1 with insulin resistance in muscle and liver (10, 11, 56, 58, 74). The protein kinases responsible for phosphorylation of IRS-1 include NH2-terminal c-Jun kinase (JNK) protein kinase C (PKC), mammalian target of rapamycin (mTOR), MAP kinase, inhibitor of {kappa}B kinase (IKKβ), and 5'-AMP-activated protein kinase (AMPK) (1, 15, 23, 37, 42, 46, 57, 74). It is suggested that serine phosphorylation may sterically inhibit the interactions between IRS-1 and the NPEY motif of the insulin receptor, making IRS-1 a poorer substrate for the insulin receptor (16, 46); alternatively phosphorylation could initiate IRS-1 degradation (52).

Previously, we (19, 41) identified the serine/threonine kinase glycogen synthase kinase-3, (GSK-3) as an IRS-1 kinase and mapped its phosphorylation site on IRS-1 to the highly conserved serine residue Ser332. Phosphorylation at Ser332 produced inhibitory effects on IRS-1-mediated signaling (41). It is noteworthy that GSK-3 has recently been targeted in the drug discovery program for insulin resistance and type 2 diabetes. Indeed, treatment with selective GSK-3 inhibitors produced antidiabetic effects reflected by improved glucose homeostasis and glucose tolerance in rodent models (13, 24, 43, 49, 54). Enhanced GSK-3 phosphorylation of IRS-1 (at Ser332) may represent a molecular basis for the contribution of GSK-3 to insulin resistance and type 2 diabetes; nevertheless, this mechanism is not fully understood. The requirement of GSK-3 for prephosphorylation of its substrates at the +4 position (21, 55) implicated IRS-1-serine336 as the "priming" site. This requirement for ordered phosphorylation means that the GSK-3 inhibitory effect is dependent on the function of this unknown priming kinase. In addition, the physiological relevance of IRS-1 phosphorylation to the diabetic condition has not been determined. The present study addressed these two issues. We show that PKCβII primes IRS-1 for GSK-3 phosphorylation. We further provide evidence for a physiological role of serine-phosphorylated IRS-1, along with elevated levels of GSK-3 activity and PKCβII, in insulin-resistant fat tissue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Purified PKC proteins, PKC inhibitors GF-109203X and GÖ-6976, and MEK inhibitor U-0126 were purchased from Calbiochem (San Diego, CA). LY-333531 was synthesized by custom synthesis. Antibodies against the N-terminus of IRS-1, PKC{alpha}, PKCβII, PKCβI, anti-phospho-tyrosine antibody, PY-99, were from Santa Cruz Biotechnology (Santa Cruz, CA). GSK-3 (Ser9) antibody was from Cell Signaling Technology (Beverly, MA), and GSK-3 (Tyr216) antibody was from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies directed against phosphorylated IRS-1 (at either Ser332 or Ser336) were generated by Bethyl Laboratories (Montgomery, TX). Synthetic IRS-1 peptide, based on the IRS-1 sequence RREGGMS332RPAS336VDG, was synthesized by Genemed Synthesis (San Francisco, CA) and described previously (41). Radioactive materials were purchased from Amersham Pharmacia Biotech (Pittsburg, PA). All other materials were from Sigma (Rehovot, Israel).

Animals. The background strain of all animals used was C57Bl/6J mice. Lepob/ob mice (referred to here as ob/ob) were purchased from Harlan (Israel). High-fat diet-induced diabetic (HF) mice were generated in the laboratory as described previously (65, 68). In brief, 4-wk-old mice received either standard laboratory food or a high-fat diet containing 35% lard (Bioserve, Frenchtown, NJ), in which 55% of the calories came from fat. The animals were housed in individual cages with free access to water in a temperature-controlled facility with a 12:12-h light-dark cycle. Animals were used after 16 wk of high-fat-diet feeding. Animals developed obesity, hyperglycemia, and insulin resistance (68). Animal care followed the protocol specified by the Institutional Animal Care and Use Committee.

Cell transfections. PTB-2 constructs that code residues 1–350 of IRS-1 were described previously (41). PKC constructs coding for rabbit PKC{alpha} and PKCβII, cloned in SRD plasmids were kindly provided by Nathan Daskal (Sackler School of Medicine, Tel Aviv University). HEK 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were transfected transiently with IRS-1 or PTB-2 constructs (5 µg each), using the calcium phosphate method as described (18). Expression of IRS-1 was verified by Western blot analysis using IRS-1-specific antibody. Cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min), indicated PKC inhibitor (5 µM for 4 h), lithium (20 mM), or U-0126 (10 µM), as described in the text. CHO/IR/IRS-1 cells were transiently transfected with PKCβII plasmid using jetPEI transfection reagent (Polyplus-Transfection, J1/Kirch Cedex, France), following the manufacturer's protocol. The cells were treated with insulin (20 nM) and lysates prepared as described below.

Cell extraction and Western blot analysis. Cells were lysed in ice-cold buffer G (20 mM Tris, pH 7.5, 10 mM β-glycerophosphate, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 0.5 mM orthovanadate, 1 mM benzamidine, 5 µg/ml leupeptin, 25 µg/ml aprotinin, 500 nM microcystine LR, and 1% Triton X-100). Cell extracts were centrifuged for 20 min at 15,000 g. Supernatants were collected, and equal amounts of proteins (30 µg) were boiled with SDS sample buffer and subjected to gel electrophoresis (7.5–12% polyacrylamide gels). After transfer to nitrocellulose membranes, specific antibodies were used to detect relevant proteins as described in text.

Phosphorylation of IRS-1 peptide by PKC and PKA. Purified PKC isoforms were incubated with 200 µM IRS-1 synthetic peptide in a reaction mixture of 20 mM Tris·HCl (pH 7.3), 10 mM MgCl2, 100 µM [{gamma}-32P]ATP, DAG (3.7 ng/ml), and 40 µM phosphatidylserine (PS) at 30°C for 20 min. CaCl2 (1 mM) was added to PKC{alpha} and PKCβ mixtures. The reactions were spotted on p81 paper (Whatman, Kent, UK), washed with phosphoric acid, and counted for radioactivity. PKC activity was confirmed using myelin basic protein (MBP) as a substrate.

PKC activity in CHO/IR/IRS-1 cells. Cells expressing PKCβII were treated with insulin (20 nM) for 15 min. cell lystaes were prepared as described above. The resulting supernatants were passed through DE-52 (Whatman, Maidstone, UK) minicolumns that were equilibrated with column buffer (20 mM Tris, pH 7.5, 5 mM EGTA, 5 mM EDTA, 10% glycerol). PKCβII was eluted with column buffer including 0.25M NaCl. The partially purified PKC activity was determined by in vitro kinase assays as described above, except that MBP was used as a substrate, and some assays included the PKCβ inhibitor LY-333531 (100 nM).

Tissue extraction. Epididymal fat pads were homogenized with ice-cold buffer H (25 µg/ml each of 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 50 mM NaF, 5% glycerol, 1% Triton X-100, leupeptin, aprotinin, and pepstatin A). Homogenates were centrifuged at 10,000 g, and supernatants were collected. Western blot analyses with indicated antibodies were performed. IRS-1 was immunoprecipitated from the supernatants of fat extracts with specific {alpha}-IRS-1 antibody. The immunoprecipitates were subjected to gel electrophoresis followed by immunoblot analysis with indicated antibodies. In another set of experiments, fat homogenates were centrifuged at 1,000 g, and supernatants were collected and subjected to another centrifugation at 100,000 g for 1 h at 4°C. The supernatants were collected as soluble fractions and referred to as the cytosol fraction. The pellets were dissolved in with ice-cold buffer H in a volume equal to that of soluble fractions; these are termed the membrane fraction. In additional experiments, the epididymal fat pads were incubated with PMA for indicated time points and or with insulin (10 nM) for 15 min. Tissue homogenates were prepared as described, and IRS-1 was immunoprecipitated from tissue extracts as described.

Overexpression of PKCβII in fat tissue. Epidydimal fat pads from C57bL/6J mice were excised into small pieces and incubated with recombinant adenovirus coding for PKCβ2 or with a "control" virus encoding β-galactosidase (generated by Dr. Tamar Tennenbaum, Bar-Ilan University, Israel) together with lipofectamine (10 µg/ml; Invitrogen Life Technologies, Carlsbad, CA) in low glucose (5 mM)-DMEM supplemented with 1% BSA, as previously described (67). Tissues were processed as described above.

Statistics and densitometry analyses. Gel band quantitation was performed by densitometry analysis, and statistics were calculated using Origin 6.0 Professional software (OriginLab, Northampton, MA) and Student's t-test. For all analyses, P < 0.05 was considered statistically significant. Results are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IRS-1 Ser336 phosphorylation is primed by a classical PKC. The phosphorylation of IRS-1 at Ser332 by GSK-3 is dependent on its prior phosphorylation at Ser336 as is typically required in GSK-3 substrates (41). The experiment presented in Fig. 1A is in agreement with this. The IRS-1 mutant, S336A, in which Ser336 was mutated to alanine, was expressed in HEK 293 cells, and the isolated protein was subjected to immunoblot analysis using an antibody, {alpha}pIRS-1Ser332, that recognizes only the Ser332-phosphorylated form of IRS-1 (41). The antibody recognized wild-type IRS-1 (WT-IRS-1), but it showed a very weak signal with the mutant (Fig. 1A). This indicated that Ser332 was not phosphorylated in the absence of prior phosphorylation at Ser336.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. Role of PKC in IRS-1 phosphorylation at Ser336. A: wild-type (WT) IRS-1 and S336A mutants were expressed transiently in HEK 293 cells. Proteins were analyzed by Western blot analysis using anti-phospho- ({alpha}p)IRS-1Ser332 antibody. B: PTB-2, containing IRS-1 residues 1–350, was expressed transiently in HEK 293 cells. Cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min), forskolin (10 µM, 30 min), and 20% FCS (30 min). Equal amounts of protein from cell extracts were subjected to Western blot analysis using specific {alpha}pIRS-1Ser336 or {alpha}IRS-1 antibody as indicated. C: PTB-2 was expressed transiently in HEK 293 cells. Cells were treated with GF-109203X and GÖ-6976 (5 µM each for 6 h) prior to stimulation with PMA. Equal amounts of protein from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser336, {alpha}pIRS-1Ser332, and {alpha}IRS-1 antibodies as indicated. D: PTB-2 was expressed transiently in HEK 293 cells. Cells were treated with LiCl (20 mM for 4 h) or U-0126 (10 µM for 2 h) prior to stimulation with PMA. Equal amounts of protein from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser336, {alpha}pIRS-1332, and {alpha}IRS-1 antibodies as indicated. E: WT-IRS-1 was transiently expressed in HEK 293 cells. Cells were treated with PMA (100 nM) for 30 min or 24 h. Equal amounts of protein prepared from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser332, {alpha}pIRS-1Ser336, and {alpha}IRS-1 antibodies. IRS-1 proteins are indicated. A–C and E: NT, nontransfected cells. For each panel, a gel representative of 3 independent experiments is shown.

 
The amino acid sequence upstream of Ser336 (S332RPAS336) contains the motif RXXS, typical for recognition by the AGC family of protein kinases such as cAMP-dependent protein kinase (PKA), PKC, or S6 ribosomal protein kinases (44, 62). Thus, we sought to examine the effect of these kinases on the phosphorylation of IRS-1 at Ser336. We generated a polyclonal antibody, {alpha}pIRS-1Ser336, that recognizes only the Ser336-phosphorylated form of IRS-1. The specificity of this new antibody was validated (data not shown). Next, HEK 293 cells were transiently transfected with the PTB-2 construct that codes residues 1–350 of IRS-1 (41). We chose to use this short construct initially, as it allowed us to focus the study on the specific region phosphorylated by GSK-3. Cells were treated with PMA (a potent PKC activator), forskolin (a potent PKA activator), or a "general" stimulator serum (20% FCS). Phosphorylation of PTB-2 at Ser336 was evaluated by Western blot analysis using the {alpha}pIRS-1Ser336 antibody. Results indicated that, of these stimulators, only PMA enhanced Ser336 phosphorylation (Fig. 1B). Hence, it appeared that a PKC family member was likely the priming kinase(s).

PKC represents a large family of isoforms that are categorized into classical, novel, and atypical PKCs (45, 48). The amino acids surrounding Ser336 fulfill the substrate specificity requirement for PKC isoforms that include a basic residue (arginine) at position –3 and a hydrophobic residue (valine) at position +1 (36, 50). As atypical PKCs are insensitive to PMA, these isoforms are likely not the priming kinase. To further narrow the list of candidate PKC isoforms, the cells were treated with a broad-spectrum PKC inhibitor, GF-109203X, or with a selective inhibitor of the classical PKC isoforms ({alpha}, β, and {gamma}), GÖ-6976, prior to stimulation by PMA. Treatment with either PKC inhibitor abolished PMA-induced phosphorylation at Ser336 (Fig. 1C). Inhibition of PKC by these inhibitors was verified by reduced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein (data not shown). Treatment with PMA enhanced GSK-3 phosphorylation of IRS-1 at Ser332, and the PKC inhibitors reduced this phosphorylation (Fig. 1C). This further suggested that PMA can prime IRS-1 for GSK-3 phosphorylation.

When cells were treated with a selective GSK-3 inhibitor, lithium (38), PMA-induced phosphorylation of Ser332 was abolished (Fig. 1D). This indicated that PMA-induced phosphorylation of Ser332 is mediated by GSK-3. We then examined whether other targets activated by PMA, such as the MAP kinase pathway, are involved. Cells were treated with the MEK inhibitor U-0126 prior to the stimulation with PMA. This treatment did not affect PMA-enhanced phosphorylation of Ser336 or Ser332 (Fig. 1D). Finally, we confirmed our results using "full length" IRS-1. Cells that expressed WT-IRS-1 were treated with PMA for 30 min and 24 h. A 30-min PMA treatment enhanced IRS-1 phosphorylation at Ser336 and Ser332 (Fig. 1E), whereas downregulation of PKC by the longer PMA treatment (24 h) reduced phosphorylation below basal levels (Fig. 1E). All together, the results suggest that a classical PKC isoform phosphorylate IRS-1 at Ser336 and facilitates GSK-3-phosphorylation at Ser332.

PKC{alpha} and PKCβII phosphorylate cellular IRS-1 at Ser336. Our results suggested that a classical PKC (i.e., PKC{alpha} and/or PKCβ) is likely involved in the phosphorylation of IRS-1 at Ser336. Thus, we decided to focus our studies on PKC{alpha} and PKCβ. First, we examined whether PKC{alpha} or PKCβ can directly phosphorylate IRS-1 at serine336. An IRS-1 peptide (RREGGMSRPAS336VDG) (41) was used as the substrate for in vitro kinase assays with purified PKC isoforms. Both PKC{alpha} and PKCβII phosphorylated the IRS-1 peptide, but PKCβII was a "more" effective kinase (Fig. 2A). Interestingly, other PKC isoforms, such as PKC{delta}, PKC{varepsilon}, and PKC{eta}, weakly phosphorylated the IRS-1 peptide (not shown). This further indicated that conventional PKCs can phosphorylate IRS-1 at Ser336.


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 2. PKC{alpha} and PKCβII phosphorylate cellular IRS-1 at Ser336. A: PKC{alpha} and PKCβII isoforms were incubated with IRS-1 peptide substrate, lipid coactivators, calcium, and [{gamma}-32P]ATP, as described in MATERIALS AND METHODS, at 30°C for 20 min. Phosphate incorporation into peptide was determined and is depicted as fold change relative to control. Results are means of 3 independent experiments ±SE. Control (c) was reaction incubated without kinase. B: HEK 293 cells were transiently transfected with IRS-1 construct and either PKC{alpha} or PKCβII constructs. Cells were treated with PMA (100 nM, 30 min). Equal amounts of protein prepared from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser332, {alpha}pIRS-1Ser336, and {alpha}IRS-1 antibodies as indicated (bottom). Expression of PKC{alpha} or PKCβII was also determined using a specific antibody (bottom). Densitometry analysis was used to determine level of phosphorylation of IRS-1 at Ser336 (top). Results represent means of 3 independent experiments ±SE, *P < 0.05. C: cells expressing PTB-2 protein were treated with PKCβII-selective inhibitor LY-333531 (10 µM, 2 h) prior to stimulation with PMA as described. Equal amounts of protein prepared from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser336, {alpha}pIRS-1Ser332, and {alpha}IRS-1 antibodies. D: CHO/IR/IRS-1 cells were treated with 20 nM insulin at indicated time points. Equal amounts of protein prepared from cell extracts were subjected to Western blot analysis using {alpha}pIRS-1Ser336, {alpha}pIRS-1Ser332, and {alpha}IRS-1 antibodies. Phosphorylation of PKC{alpha} at Ser657 by insulin (30-min treatment) is shown at bottom. E: CHO/IR/IRS-1 cells were transiently transfected with PKCβII plasmid as described in MATERIALS AND METHODS. Cells were treated with insulin (20 nM) for 15 min. Activity of partially purified PKCβII was determined by in vitro kinase assays as described in MATERIALS AND METHODS. Results present fold activation of kinase activity that was determined in control nontransfected cells and are means of 2 independent experiments. Ectopic expression of PKCβII is shown at bottom. B–D: a gel representative of 3 independent experiments is shown.

 
Next, HEK 293 cells were transiently cotransfected with IRS-1 and PKC{alpha} or PKCβII constructs. The cells were treated with PMA or left unstimulated, and the phosphorylation of IRS-1 at Ser336 and Ser332 was determined. Overexpression of PKC{alpha} or PKCβII enhanced IRS-1-phosphorylation of Ser336 and Ser332 under both basal and PMA-stimulated conditions (Fig. 2B). This indicated that cellular PKC{alpha}/β mediate the phosphorylation of IRS-1. To further explore the potential role of PKCβ, the cells were treated with a selective PKCβ inhibitor, LY-333531 (33). Treatment with LY-333531 reduced PMA-induced phosphorylation of Ser336 to nearly basal levels (Fig. 2C). This suggested that PKCβ is the most important priming kinase of IRS-1 in cellular conditions.

Finally, we examined how a physiological activator of PKC, insulin, (6), affects IRS-1 phosphorylation. Insulin treatment of CHO cells overexpressing both the insulin receptor and IRS-1 indicated that insulin enhanced IRS-1 Ser336 phosphorylation after 2 min and persisted at longer time points (15 and 30 min; Fig. 2D). There was a slight increase in Ser332 phosphorylation after 2 min of insulin treatment, and phosphorylation substantially increased after 15 min. At longer time points (15–30 min), we could detect coordinated elevation in the phosphorylation of Ser336 and Ser332. To further examine whether PKC was activated by insulin, we examined phosphorylation levels of PKC{alpha} at Ser657, a phosphorylation associated with enhanced PKC{alpha} activity (8) (Fig. 2D). We could not detect PKCβ in these cells. However, ectopically expressed PKCβII was activated by insulin (Fig. 2D). All together, insulin can activate both PKC{alpha} and PKCβ in these cells.

Elevated activity of PKCβII and GSK-3 in diabetic fat tissue. The results presented thus far indicate that PKC{alpha} and/or PKCβ phosphorylate IRS-1 at Ser336. However, the relevance of this phosphorylation to in vivo regulation in a diabetic system was unknown. Therefore, we focused on diabetic fat tissue in which GSK-3 is known to be hyperactive (20). Two model systems were used: obese, insulin resistant, ob/ob mice (76) and nutritionally-induced diabetic mice [HF mice (65, 68)]. Elevation in GSK-3β activity in the fat tissue from ob/ob and HF mice is demonstrated in Fig. 3A. Phosphorylation of GSK-3β at a key inhibitory site, Ser9 (66), is reduced, whereas phosphorylation at Tyr216, a site important for GSK-3 activation (26), is increased. There was no change in total GSK-3 protein levels between control and diabetic samples (Fig. 3A). Remarkably, expression levels of PKCβII, but not PKCβI or PKC{alpha}, were elevated in the ob/ob and HF fat tissues (Fig. 3B). Phosphorylation of PKC{alpha} at Ser657 (8) was somewhat lower in the diabetic samples (Fig. 3B), indicating that PKC{alpha} activity was not elevated in this tissue. To further verify that PKCβII was active, we examined its localization in cell membranes (40). Indeed, levels of PKCβII associated with the fat cell membranes were specifically elevated in the ob/ob and HF samples (Fig. 3C). There was no elevation in levels of other PKC isoforms, including {theta}, {varepsilon}, {delta}, and {zeta}, in the diabetic fat tissue (data not shown).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Glycogen synthase kinase (GSK)-3β and PKCβII are hyperactive in fat tissue of obese diabetic animals. Epididymal fat pad tissue extracts were prepared from ob/ob, high-fat diet-fed (HF), and control lean (C) animals. A: phosphorylation of GSK-3 at Ser9 and Tyr216 were determined by Western blot analysis as described in MATERIALS AND METHODS. B: abundances of PKCβII, PKCβI, and PKC{alpha} were determined by Western blot analysis as described in MATERIALS AND METHODS. Phosphorylation of PKC{alpha} at Ser657 was detected with specific {alpha}PKC{alpha} (Ser657) antibody. Densitometry analysis of PKC{alpha} and PKCβII bands is shown in bottom. Results are means of data from 9–12 animals ±SE, **P < 0.01. A and B: a gel representative of analysis of samples from 2 animals from groups of 9–12 is shown. C: fat tissue was subjected to subcellular fractionation as described in MATERIALS AND METHODS. Equal amounts of protein from cytosolic and membrane fractions were subjected to immunoblot analysis using antibodies against PKCβII. Insulin receptor (IR, bottom) served as a marker for validity of the membrane fraction. MAPK expression levels confirmed an equal load in each fraction. Densitometry analysis of the PKCβII bands is shown in bottom as fold of control. Shown is a representative gel with analysis of 2 of 6 samples.

 
PKC phosphorylates IRS-1 at Ser336 in fat tissue. The results presented in Fig. 3 prompted us to focus our study on the potential role of PKCβII in the phosphorylation of IRS-1 in fat tissue. Epidydimal fat tissues isolated from C57Bl/6J mice were treated with PMA for various times, and the extent of IRS-1 phosphorylation was determined. Treatment with PMA led to rapid activation of Ser336 phosphorylation of IRS-1 after 5 min of exposure; this phosphorylation was consistently elevated during the 30-min treatment (Fig. 4A) and associated with enhanced phosphorylation at Ser332 (not shown). We next examined how this phosphorylation would affect insulin signaling. The tissue was treated with PMA (30 min) prior to stimulation with insulin (15 min). IRS-1 protein was immunoprecipitated from cell lysates, and tyrosine phosphorylation levels were determined (Fig. 4B). Indeed, pretreatment with PMA reduced insulin-induced tyrosine phosphorylation of IRS-1, supporting our notion that phosphorylation of Ser336/332 inhibits insulin signaling. We next sought to examine the role of PKCβII in the phosphorylation of IRS-1 in this tissue. The isolated fat tissues were exposed to recombinant adenovirus encoding PKCβII or to a control adenoviral vector encoding β-galactosidase. Levels of expression of PKCβII and phosphorylation of IRS-1 were determined in the infected tissues. As shown in Fig. 4C, IRS-1 phosphorylation at Ser336 was significantly enhanced in PKCβII-infected adipocytes. This suggested that IRS-1 phosphorylation at Ser336 is modulated by PKCβII in fat tissue.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 4. Modulation of IRS-1 serine phosphorylation by PKC in fat tissue. A: epidydimal fat tissues were incubated with PMA (100 nM) for indicated times as desribed in MATERIALS AND METHODS. Tissue extracts were prepared and subjected to gel electrophoresis followed by immunoblot analysis with {alpha}pIRS-1Ser336. Densitometry analysis of IRS-1-phosphorylated bands is shown in bottom. Results are means of data from 4 independent experiments ±SE, *P < 0.05. B: same as A, except that tissue was treated with PMA for 30 min prior to stimulation with insulin (10 nM) for an additional 15 min. IRS-1 was immunoprecipitated from tissue extracts, and samples were subjected to immunoblot analysis using anti-phospho-tyrosine and anti-IRS-1 antibody. C: epididymal fat tissues were infected with adenovirus coding for PKCβII or β-galactosidase (β-gal), as described in MATERIALS AND METHODS. Tissue extracts were prepared and subjected to gel electrophoresis followed by immunoblot analysis with {alpha}pIRS-1Ser336, {alpha}PKCβII, and {alpha}β-actin antibodies. Densitometry analysis of phosphorylated IRS-1 is shown at right, and results are means ± SE of 4 independent experiments, *P < 0.05. Representative gels from 2 of 4 experiments are shown.

 
IRS-1 phosphorylation at Ser332 and Ser336 is elevated in diabetic fat tissue. Our studies have indicated so far that PKCβII is elevated in diabetic fat tissue and is capable of phosphorylating IRS-1 specifically at Ser336. We next evaluated IRS-1 serine phosphorylation in the diabetic fat tissue. IRS-1 was immunoprecipitated from fat tissue extracts prepared from control, ob/ob, and HF animals. The immunoprecipitates were subjected to immunoblot analysis using the anti-phospho-IRS-1 antibody. A specific increase in IRS-1 phosphorylation at Ser336/332 was clearly detected in ob/ob and HF samples (Fig. 5A). Total amounts of IRS-1 were lower in the diabetic fat tissues than in the control tissue (Fig. 5A), indicating that, in fact, a larger pool of IRS-1 molecules were phosphorylated at Ser336/332 in these tissues. Results indicated 2- and 1.6-fold increases in phosphorylations at Ser332 and Ser336, respectively, in the diabetic fat tissues relative to control (Fig. 5B). Interestingly, the extent of IRS-1 phosphorylation was closely correlated with the degree of obesity developed in the models (Fig. 5C).


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. IRS-1 Ser332/336 phosphorylation is elevated in fat tissue of diabetic animals. A: IRS-1 proteins were immunoprecipitated from fat tissue extracts from ob/ob, HF, and control animals. Immunoprecipitates were subjected to Western blot analysis using {alpha}pIRS-1Ser332, {alpha}pIRS-1Ser336, and {alpha}IRS-1 antibodies. Total IRS-1 is shown at bottom. Gels representative of samples 2 animals from groups of 9–12 animals are shown. B: ratio of phosphorylated to total IRS-1 as calculated from densitometry analysis. Ratio obtained from control was designated 1, and results are presented as fold of control. Results are means of samples from 9–12 animals ±SE, **P < 0.01. C: blood glucose levels, plasma insulin, body weight, and fat pad mass of ob/ob, HF, and control animals are listed. Values are means ± SE for samples from 9–12 animals, *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Serine/threonine phosphorylation of IRS-1 is a negative feedback regulator in insulin signaling. Evidence that the kinase GSK-3 phosphorylated of IRS-1 at Ser332 increased our understanding the factors that contribute to insulin resistance (41). However, GSK-3 phosphorylation at Ser332 requires prior phosphorylation of IRS-1 at Ser336; the nature of the priming kinase was unknown. We realized that the priming kinase likely belonged to the AGC-serine/threonine protein kinase family, and by using a novel anti-phospho-IRS-1 (Ser336) antibody, we identified PMA-sensitive PKC isoforms as potential candidates for the priming kinase. The use of inhibitors with specificity for certain PKC isoforms and PKC-overexpressing cell systems implicated classical PKCs {alpha} or β. The expression level and kinase activity of PKCβII, but not PKC{alpha}, was significantly upregulated in the fat tissue of two diabetic animal models, ob/ob and HF mice. This was also associated with elevated levels of IRS-1 phosphorylation at Ser336 and Ser332 in samples from the diabetic animals compared with normal tissue. Using adenoviral-mediated gene delivery, we confirmed that PKCβII can phosphorylate IRS-1 at Ser336 in the fat tissue. Our results suggested that PKCβII is likely the in vivo priming kinase responsible for IRS-1 phosphorylation at Ser336 at least under diabetic conditions in the fat tissue.

We further noticed that levels of PKCβII, GSK-3 activity, and IRS-1 phosphorylation, were consistently higher in the fat tissue from ob/ob animals compared with HF animals, implying a link between these parameters and the severity of obesity and/or insulin resistance (Fig. 4C). Phosphorylation of IRS-1 at Ser336 and Ser332 was previously shown to constrain insulin signaling (41); consistently, prior treatment with PMA reduced insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 4B). We thus suggest that PKCβII/GSK-3-mediated phosphorylation of IRS-1 represents a mechanism that contributes to insulin resistance in fat tissue. This sequential phosphorylation can represent another important level of regulation of insulin signaling. Namely, as long as Ser336 is not phosphorylated by PKC, GSK-3 cannot promote the phosphorylation of Ser332. On the other hand, phosphorylation of both sites is expected to produce a stronger and effective impact on insulin signaling. Hence, the activity of both kinases is an important and essential factor in contributing to insulin resistance.

The PKC family, and in particular DAG-PKC, were previously shown to have regulatory roles in insulin signaling, and their expression levels and/or activities were associated with insulin resistance in type 2 diabetes in patients and animal models (14, 27, 29, 64). PKC isoforms may be differentially regulated by high concentrations of glucose or free fatty acids in various cell types such as muscle and adipocytes (4, 35, 64). It is also apparent that the contribution of PKC isoforms to insulin signaling and/or insulin resistance is tissue specific. For example, PKC{theta} and PKC{varepsilon} are implicated in skeletal muscle insulin resistance (3, 22, 31, 60), PKC{alpha} and -{varepsilon} are elevated in diabetic liver (14), and PKC{zeta} has an essential role in insulin-induced glucose transport in adipocytes (5, 6). Our studies highlight the specific role of PKCβII in diabetic fat tissue, although we do not exclude the possibility that additional PKC isoforms may be involved.

The roles for GSK-3 and PKCβII as negative modulators in insulin signaling were previously described, and both protein kinases are promising drug targets for treatment of insulin resistance and type 2 diabetes (17, 39). PKCβ has been strongly implicated in the pathogenesis of diabetic vascular complications and has been shown to be a key regulator in hyperglycemia-induced retina, renal, and cardiovascular dysfunction (28, 39, 72). Elevated levels of PKCβII were detected in vascular smooth muscle and skeletal muscle of obese/diabetic patients (32, 39). Use of the selective PKCβ inhibitor LY-333531 restores insulin-stimulated glucose transport in rodent models (30, 51, 70). However, the roles of PKCβII and GSK-3 in insulin-resistant fat tissue are less clear. Evidence from knockout studies indicated that PKCβ plays important role in metabolic regulation of fat tissue and energy expenditure (5, 7, 9, 63). Loss of PKCβ improved insulin sensitivity, reduced fat mass, and protected against weight gain (7, 63). Elevation of GSK-3 activity was detected in diabetic fat tissues of rodents and humans and is correlated with adipogenesis and weight gain (12, 20). Here, we suggest new roles for PKCβII and GSK-3 in a concerted regulation of insulin action in fat tissue. Specifically, our view is that, under hyperglycemic/hyperlipidemic conditions, PKCβII and GSK-3 activities are elevated in fat tissue and ensure continuous phosphorylation of IRS-1 at Ser336 and Ser332. This, in turn, impairs insulin action in the fat tissue.

In summary, our work highlights the cooperative activities of PKCβII and GSK-3 in phosphorylation of IRS-1 and the role of this phosphorylation in mediating insulin resistance. We suggest that combined inhibition of PKCβII and GSK-3 may represent a new strategy for treatment of insulin resistance and type 2 diabetes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Ginsberg-Fay and Arthur Levenstein Foundation for Diabetes Research at Tel Aviv University.


   ACKNOWLEDGMENTS
 
This work is in partial fulfillment of the PhD requirements for Z. Liberman at the Sackler School of Medicine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Eldar-Finkelman, Dept. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel (e-mail: heldar{at}post.tau.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aguirre V, Uchida T, Yenush L, Davis R, White MF. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275: 9047–9054, 2000.[Abstract/Free Full Text]
  2. Amos AF, McCarty DJ, Zimmet P. The rising global burden of diabetes and its complications: estimates and projections to the year 2010. Diabet Med 14, Suppl 5: S1–S85, 1997.[Medline]
  3. Avignon A, Yamada K, Zhou X, Spencer B, Cardona O, Saba-Siddique S, Galloway L, Standaert ML, Farese RV. 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. Diabetes 45: 1396–1404, 1996.[Abstract]
  4. Bandyopadhyay G, Sajan MP, Kanoh Y, Standaert ML, Burke TR Jr, Quon MJ, Reed BC, Dikic I, Noel LE, Newgard CB, Farese R. Glucose activates mitogen-activated protein kinase (extracellular signal-regulated kinase) through proline-rich tyrosine kinase-2 and the Glut1 glucose transporter. J Biol Chem 275: 40817–40826, 2000.[Abstract/Free Full Text]
  5. Bandyopadhyay G, Standaert ML, Kikkawa U, Ono Y, Moscat J, Farese RV. Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda. Biochem J 337: 461–470, 1999.[CrossRef][Web of Science][Medline]
  6. Bandyopadhyay G, Standaert ML, Zhao L, Yu B, Avignon A, Galloway L, Karnam P, Moscat J, Farese RV. 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. J Biol Chem 272: 2551–2558, 1997.[Abstract/Free Full Text]
  7. Bansode RR, Huang W, Roy SK, Mehta M, Mehta KD. Protein kinase C deficiency increases fatty acid oxidation and reduces fat storage. J Biol Chem 283: 231–236, 2008.[Abstract/Free Full Text]
  8. Bornancin F, Parker PJ. Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J Biol Chem 272: 3544–3549, 1997.[Abstract/Free Full Text]
  9. Bosch RR, Bazuine M, Wake MM, Span PN, Olthaar AJ, Schurmann A, Maassen JA, Hermus AR, Willems PH, Sweep CG. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3–L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane. Mol Endocrinol 17: 1230–1239, 2003.[Abstract/Free Full Text]
  10. Bouzakri K, Karlsson HK, Vestergaard H, Madsbad S, Christiansen E, Zierath JR. IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas transplant recipients. Diabetes 55: 785–791, 2006.[Abstract/Free Full Text]
  11. Bouzakri K, Roques M, Gual P, Espinosa S, Guebre-Egziabher F, Riou JP, Laville M, Le Marchand-Brustel Y, Tanti JF, Vidal H. Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes 52: 1319–1325, 2003.[Abstract/Free Full Text]
  12. Ciaraldi TP, Oh DK, Christiansen L, Nikoulina SE, Kong AP, Baxi S, Mudaliar S, Henry RR. Tissue-specific expression and regulation of GSK-3 in human skeletal muscle and adipose tissue. Am J Physiol Endocrinol Metab 291: E891–E898, 2006.[Abstract/Free Full Text]
  13. Cline GW, Johnson K, Regittnig W, Perret P, Tozzo E, Xiano L, Damico C, Shulman GI. Effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in Zucker diabetic fatty (fa/fa) rats. Diabetes: 2903–2910, 2002.
  14. Considine RV, Nyce MR, Allen LE, Morales LM, Triester S, Serrano J, Colberg J, Lanza-Jacoby S, Caro JF. Protein kinase C is increased in the liver of humans and rats with non-insulin-dependent diabetes mellitus: an alteration not due to hyperglycemia. J Clin Invest 95: 2938–2944, 1995.[Web of Science][Medline]
  15. De Fea K, Roth RA. Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochemistry 36: 12939–12947, 1997.[CrossRef][Web of Science][Medline]
  16. Eck MJ, Dhe-Paganon S, Trub T, Nolte RT, Shoelson SE. Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor. Cell 85: 695–705, 1996.[CrossRef][Web of Science][Medline]
  17. Eldar-Finkelman H. Glycogen synthase kinase-3: an emerging therapeutic target. Trend Mol Med 8: 126–132, 2002.[CrossRef]
  18. Eldar-Finkelman H, Agrast GM, Foord O, Fischer EH, Krebs EG. Expression and characterization of GSK-3 mutants and their effect on glycogen synthase activity. Proc Natl Acad Sci USA 93: 10228–10233, 1996.[Abstract/Free Full Text]
  19. Eldar-Finkelman H, Krebs EG. Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. Proc Natl Acad Sci USA 94: 9660–9664, 1997.[Abstract/Free Full Text]
  20. Eldar-Finkelman H, Schreyer SA, Shinohara MM, LeBoeuf RC, Krebs EG. Increased glycogen synthase kinase-3 activity in diabetes- and obesity- prone C57BL/6J mice. Diabetes 48: 1662–1666, 1999.[Abstract]
  21. Fiol CJ, Mahrenholz AM, Wang Y, Roeske RW, Roach PJ. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem 262: 14042–14048, 1987.[Abstract/Free Full Text]
  22. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI. Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade. Diabetes 48: 1270–1274, 1999.[Abstract]
  23. Gual P, Gremeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti JF. MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612 and 632. Diabetologia 46: 1532–1542, 2003.[CrossRef][Web of Science][Medline]
  24. Henriksen EJ, Kinnick TR, Teachey MK, O'Keefe MP, Ring D, Johnson KW, Harrison SD. Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab 284: E892–E900, 2003.[Abstract/Free Full Text]
  25. Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM. IRS-1 mediated inhibition of insulin receptor tyrosine kinase activity in TNF-a and obesity induced insulin resistance. Science 271: 665–668, 1996.[Abstract]
  26. Hughes K, Nicolakaki E, Plyte SE, Totty NF, Woodgett JR. Modulation of glycogen synthase kinase-3 family by tyrosine kinase phosphorylation. EMBO J 12: 803–808, 1993.[Web of Science][Medline]
  27. Idris I, Gray S, Donnelly R. Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes. Diabetologia 44: 659–673, 2001.[CrossRef][Web of Science][Medline]
  28. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 89: 11059–11063, 1992.[Abstract/Free Full Text]
  29. Inoguchi T, Xia P, Kunisaki M, Higashi S, Feener EP, King GL. Insulin's effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. Am J Physiol Endocrinol Metab 267: E369–E379, 1994.[Abstract/Free Full Text]
  30. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 272: 728–731, 1996.[Abstract]
  31. Itani SI, Pories WJ, Macdonald KG, Dohm GL. Increased protein kinase C theta in skeletal muscle of diabetic patients. Metabolism 50: 553–557, 2001.[CrossRef][Web of Science][Medline]
  32. Itani SI, Zhou Q, Pories WJ, MacDonald KG, Dohm GL. Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes 49: 1353–1358, 2000.[Abstract]
  33. Jirousek MR, Gillig JR, Gonzalez CM, Heath WF, McDonald 3rd JH, Neel DA, Rito CJ, Singh U, Stramm LE, Melikian-Badalian A, Baevsky M, Ballas LM, Hall SE, Winneroski LL, Faul MM. (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta. J Med Chem 39: 2664–2671, 1996.[CrossRef][Web of Science][Medline]
  34. Kahn R. Dealing with complexity in clinical diabetes: the value of archimedes. Diabetes Care 26: 3168–3171, 2003.[Free Full Text]
  35. Kawano Y, Rincon J, Soler A, Ryder JW, Nolte LA, Zierath JR, Wallberg-Henriksson H. Changes in glucose transport and protein kinase Cbeta(2) in rat skeletal muscle induced by hyperglycaemia. Diabetologia 42: 1071–1079, 1999.[CrossRef][Web of Science][Medline]
  36. Kazanietz MG, Areces LB, Bahador A, Mischak H, Goodnight J, Mushinski JF, Blumberg PM. Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent protein kinase C isozymes. Mol Pharmacol 44: 298–307, 1993.[Abstract]
  37. Kim JA, Yeh DC, Ver M, Li Y, Carranza A, Conrads TP, Veenstra TD, Harrington MA, Quon MJ. Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by Mouse Pelle-like kinase/interleukin-1 receptor-associated kinase: cross-talk between inflammatory signaling and insulin signaling that may contribute to insulin resistance. J Biol Chem 280: 23173–23183, 2005.[Abstract/Free Full Text]
  38. Klein PS, Melton DA. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93: 8455–8459, 1996.[Abstract/Free Full Text]
  39. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 47: 859–866, 1998.[Abstract]
  40. Kraft AS, Anderson WB. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301: 621–623, 1983.[CrossRef][Medline]
  41. Liberman Z, Eldar-Finkelman H. Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling. J Biol Chem 280: 4422–4428, 2005.[Abstract/Free Full Text]
  42. Liu YF, Paz K, Herschkovitz A, Alt A, Tennenbaum T, Sampson SR, Ohba M, Kuroki T, LeRoith D, Zick Y. Insulin stimulates PKCzeta-mediated phosphorylation of insulin receptor substrate-1 (IRS-1). A self-attenuated mechanism to negatively regulate the function of IRS proteins. J Biol Chem 276: 14459–14465, 2001.[Abstract/Free Full Text]
  43. Lochhead PA, Coghlan M, Rice SQ, Sutherland C. Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes 50: 937–946, 2001.[Abstract/Free Full Text]
  44. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science 298: 1912–1934, 2002.[Abstract/Free Full Text]
  45. Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem J 332: 281–292, 1998.[Web of Science][Medline]
  46. Moeschel K, Beck A, Weigert C, Lammers R, Kalbacher H, Voelter W, Schleicher ED, Haring HU, Lehmann R. Protein kinase C-zeta-induced phosphorylation of Ser318 in insulin receptor substrate-1 (IRS-1) attenuates the interaction with the insulin receptor and the tyrosine phosphorylation of IRS-1. J Biol Chem 279: 25157–25163, 2004.[Abstract/Free Full Text]
  47. Myers MGJ, White MF. Insulin signal transduction and the IRS proteins. Annu Rev Pharmacol Toxicol 36: 615–658, 1996.[CrossRef][Web of Science][Medline]
  48. Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J 2002.
  49. Nikoulina SE, Ciaraldi TP, Mudaliar S, Carter L, Johnson K, Henry RR. Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle. Diabetes 51: 2190–2198, 2002.[Abstract/Free Full Text]
  50. Nishikawa K, Toker A, Johannes FJ, Songyang Z, Cantley LC. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem 272: 952–960, 1997.[Abstract/Free Full Text]
  51. Ohshiro Y, Ma RC, Yasuda Y, Hiraoka-Yamamoto J, Clermont AC, Isshiki K, Yagi K, Arikawa E, Kern TS, King GL. Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase Cbeta-null mice. Diabetes 55: 3112–3120, 2006.[Abstract/Free Full Text]
  52. Pederson TM, Kramer DL, Rondinone CM. Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes 50: 24–31, 2001.[Abstract/Free Full Text]
  53. Pessin JE, Saltiel AR. Signaling pathways in insulin action: molecular targets of insulin resistance. J Clin Invest 106: 165–169, 2000.[Web of Science][Medline]
  54. Plotkin B, Kaidanovich O, Talior I, Eldar-Finkelman H. Insulin mimetic action of synthetic phosphorylated peptide inhibitors of glycogen synthase kinase-3. J Pharmacol Exp Ther 974–980, 2003.
  55. Plyte SE, Hughes K, Nikolakaki E, Pulverer BJ, Woodgett JR. Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta 1114: 147–162, 1992.[Medline]
  56. Qiao LY, Zhande R, Jetton TL, Zhou G, Sun XJ. In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents. J Biol Chem 277: 26530–26539, 2002.[Abstract/Free Full Text]
  57. Ravichandran LV, Esposito DL, Chen J, Quon MJ. Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin. J Biol Chem 276: 3543–3549, 2001.[Abstract/Free Full Text]
  58. Rui L, Aguirre V, Kim JK, Shulman GI, Lee A, Corbould A, Dunaif A, White MF. Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107: 181–189, 2001.[CrossRef][Web of Science][Medline]
  59. Saltiel AR, Pessin JE. Insulin signaling pathways in time and space. Trends Cell Biol 12: 65–71, 2002.[CrossRef][Web of Science][Medline]
  60. Schmitz-Peiffer C, Browne CL, Oakes ND, Watkinson A, Chisholm DJ, Kraegen EW, Biden TJ. Alterations in the expression and cellular localization of protein kinase C isozymes epsilon and theta are associated with insulin resistance in skeletal muscle of the high-fat-fed rat. Diabetes 46: 169–178, 1997.[Abstract]
  61. Shulman GI. Cellular mechanisms of insulin resistance in humans. Am J Cardiol 84: 3J–10J, 1999.[Web of Science][Medline]
  62. Songyang Z, Blechner S, Hoagland N, Hoekstra MF, Piwnica-Worms H, Cantley LC. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol 4: 973–982, 1994.[CrossRef][Web of Science][Medline]
  63. Standaert ML, Bandyopadhyay G, Galloway L, Soto J, Ono Y, Kikkawa U, Farese RV, Leitges M. Effects of knockout of the protein kinase C beta gene on glucose transport and glucose homeostasis. Endocrinology 140: 4470–4477, 1999.[Abstract/Free Full Text]
  64. Steiler TL, Galuska D, Leng Y, Chibalin AV, Gilbert M, Zierath JR. Effect of hyperglycemia on signal transduction in skeletal muscle from diabetic Goto-Kakizaki rats. Endocrinology 144: 5259–5267, 2003.[Abstract/Free Full Text]
  65. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Diet-induced type II diabetes in C57BL/6J mice. Diabetes 37: 1163–1167, 1988.[Abstract]
  66. Sutherland C, Leighton IA, Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem J 295: 15–19, 1993.[Web of Science][Medline]
  67. Talior I, Tennenbaum T, Kuroki T, Eldar-Finkelman H. PKC{delta}-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase. Am J Physiol Endocrinol Metab 288: E405–E411, 2005.[Abstract/Free Full Text]
  68. Talior I, Yarkoni M, Bashan N, Eldar-Finkelman H. Increased glucose uptake promotes oxidative stress and PKC{delta} activation in adipocytes of obese, insulin-resistant mice. Am J Physiol Endocrinol Metab 285: E295–E302, 2003.[Abstract/Free Full Text]
  69. Tanti JF, Gremeaux T, van Obberghen E, Le Marchand-Brustel Y. Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. J Biol Chem 269: 6051–6057, 1994.[Abstract/Free Full Text]
  70. Tuttle KR, Bakris GL, Toto RD, McGill JB, Hu K, Anderson PW. The effect of ruboxistaurin on nephropathy in type 2 diabetes. Diabetes Care 28: 2686–2690, 2005.[Abstract/Free Full Text]
  71. Virkamaki A, Ueki K, Kahn R. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103: 931–943, 1999.[Web of Science][Medline]
  72. Way KJ, Katai N, King GL. Protein kinase C and the development of diabetic vascular complications. Diabet Med 18: 945–959, 2001.[CrossRef][Web of Science][Medline]
  73. Welsh GI, Proud CG. Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B. Biochem J 294: 625–629, 1993.[Web of Science][Medline]
  74. Werner ED, Lee J, Hansen L, Yuan M, Shoelson SE. Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302. J Biol Chem 279: 35298–35305, 2004.[Abstract/Free Full Text]
  75. White MF. The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res 53: 119–138, 1998.[Medline]
  76. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of mouse obese gene and its human homologue. Nature 372: 425–432, 1994.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/6/E1169    most recent
00050.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liberman, Z.
Right arrow Articles by Eldar-Finkelman, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liberman, Z.
Right arrow Articles by Eldar-Finkelman, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.