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Endocrinology Vol. 144, No. 12 5259-5267
Copyright © 2003 by The Endocrine Society

Effect of Hyperglycemia on Signal Transduction in Skeletal Muscle from Diabetic Goto-Kakizaki Rats

Tatiana L. Steiler, Dana Galuska, Ying Leng, Alexander V. Chibalin, Marc Gilbert and Juleen R. Zierath

Departments of Physiology and Pharmacology (T.L.S., D.G.) and Surgical Sciences (Y.L., A.V.C., J.R.Z.), Section for Integrative Physiology, Karolinska Institutet, SE-17177 Stockholm, Sweden; and Laboratoire de Physiopathologie de la Nutrition (M.G.), Universite Paris 7, 75251 Paris, France

Address all correspondence and requests for reprints to: Juleen R. Zierath, Ph.D., Professor of Physiology, Department of Surgical Sciences, Section for Integrative Physiology, Karolinska Institutet, von Eulers väg 4, II, SE-171 77 Stockholm, Sweden. E-mail: Juleen.Zierath{at}fyfa.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We determined basal and insulin-stimulated responses on signaling intermediates in soleus skeletal muscle from male Wistar and diabetic Goto-Kakizaki (GK) rats. Rats were infused with glucose (5 or 20 mM) for 3 h, followed by a continuous infusion of saline or insulin (3 U/kg·h) for 20 min. Under euglycemic and hyperglycemic conditions, basal and insulin-stimulated action on phosphatidylinositol (PI) 3-kinase, protein kinase B/Akt, and ERK were reduced in GK rats, whereas insulin-stimulated protein kinase C (PKC){zeta} activity was not altered. Interestingly, basal PKC{zeta} activity was increased under hyperglycemic conditions in GK and Wistar rats. This finding of increased PKC{zeta} activity was confirmed in vitro in isolated soleus muscle exposed to high extracellular glucose, and occurred concomitant with an increase in PI-dependent kinase 1 (PDK-1) activity. The glucose effects were not specific to PKC{zeta}, because an increase in phosphorylation of PKC{alpha}/ß and PKC{delta}, but not PKC{theta}, in isolated soleus muscle exposed to 25 mM glucose was observed. In conclusion, insulin signaling defects in diabetic GK rats are not corrected by an acute normalization of glycemia. Interestingly, acute hyperglycemia leads to a parallel increase in PDK-1, PKC{alpha}/ß, PKC{delta}, and PKC{zeta} phosphorylation/activity via a PI 3-kinase-protein kinase B/Akt-independent mechanism. The long-term consequence of elevated PDK-1 and PKC phosphorylation/activity should be considered in the context of diabetes mellitus, as hyperglycemia is a clinical feature of this disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC HYPERGLYCEMIA HAS been highlighted as a major factor contributing to the development of peripheral insulin resistance in both type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus (1, 2). Restoration of glycemia normalizes insulin action on glucose transport in isolated skeletal muscle from type II diabetic subjects (3) and in animal models of the disease (4). Thus, hyperglycemia appears to have a profound impact on insulin action through a desensitization of the insulin signaling cascade. The precise mechanism by which hyperglycemia impairs insulin action is not fully known, but increased biosynthetic activity within the hexosamine pathway is partly involved (5, 6). Uridine diphosphate-N-acetylglucosamine, the terminal metabolite of the hexosamine pathway, is a substrate of the O-linked N-acetylglucosamine transferase and can modify serine and threonine residues of cytosolic and nuclear proteins, linking the hexosamine pathway to insulin resistance (7). Although the serine/threonine targets of the hexosamine pathway have not been completely elucidated, protein kinase B (PKB/Akt) and protein kinase C (PKC) isoforms, members of the AGC serine/threonine kinase superfamily [comprising the prototypes protein kinase A (PKA), G (PKG), and C (PKC)], have been implicated in glucose-induced insulin resistance (reviewed in Ref. 8).

In addition to PKB/Akt and PKC, the AGC superfamily includes ribosomal S6 kinase, p70 ribosomal S6 kinase, mitogen- and stress-activated protein kinase, and serum- and glucocorticoid-inducible kinase. Most of the AGC family members are phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK-1) (9, 10). However, emerging evidence suggests that PDK-1-independent kinases may also regulate these targets (11). PKB/Akt and PKC{zeta} are examples of AGC family members implicated in the regulation of glucose metabolism (12, 13). In the nonobese Goto-Kakizaki (GK) diabetic rat, insulin action on PKB/Akt is impaired (4, 14). Importantly, improved glucose tolerance through normalization of glycemia by phlorizin treatment fully restores insulin-stimulated PKB/Akt activity and glucose transport (4). Thus, hyperglycemia may directly interfere with insulin signaling at the level of PKB/Akt, because the improvement in insulin action on this target after restoration of glycemia was completely disassociated from phosphatidylinositol (PI) 3-kinase (14). Consistent with this, in isolated skeletal muscle from Wistar rats, hyperglycemia inhibited insulin action on PKB/Akt, without affecting PI 3-kinase activity (15). Whether this is a general feature of AGC kinases, or specific to PKB/Akt, is not known.

PKC isoforms can be classified into subfamilies based on amino acid similarity and mode of activation. Conventional PKCs (cPKCs: {alpha}, ß1, ß2, and {gamma}) are dependent on both Ca2+ and diacylglycerol (DAG) for stimulation, novel PKCs (nPKCs: {delta}, {epsilon}, {theta}, and {eta}) are dependent on DAG, and atypical PKCs (aPKCs; {zeta} and {lambda}/{iota}) are independent of Ca2+ and DAG. PKC isoform distribution is altered in skeletal muscle from various diabetic animals, and this may impair insulin-stimulated glucose transport (16). In GK rats, reduced insulin-stimulated PKC{zeta} activity in skeletal muscle is restored after rosiglitazone treatment (17). Whether this defect is primarily a consequence of the hyperglycemic state is presently not known. An acute exposure of skeletal muscle to high glucose promotes the translocation and activation of PKC isoforms (18, 19, 20), and this may provide a mechanism for insulin resistance (21). Furthermore, high extracellular concentrations of glucose may activate PKC{zeta} via ERK (20).

To gain further insight into the molecular mechanisms underlying insulin action in skeletal muscle, we determined the effect of hyperglycemia on PDK-1 and two members of the AGC kinase superfamily, PKB/Akt and PKC{zeta}, which have been implicated to play a role in metabolic effects of insulin. Our aim was to determine whether these AGC kinase superfamily members are directly regulated by hyperglycemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male GK rats (200–250 g) were obtained from the Department of Endocrinology, Karolinska Hospital (Stockholm, Sweden). Age- and weight-matched male Wistar rats were used as controls (B&K Universal AB, Sollentuna, Sweden). All animals were housed under a 12-h light, 12-h dark cycle and had free access to water and standard rodent chow. All rats were fasted overnight (15–18 h) before each experiment. The animal ethical committee of the Stockholm North Animal Ethical Committee approved all studies.

In vivo studies
Rats undergoing the infusion studies were anesthetized with ketamine (125 mg/100 g body weight ip) before surgical placement of a catheter in the jugular vein for systemic infusions. Catheters were externalized through an incision behind the head. Animals were used for experimentation 4 d after surgery and were studied under euglycemic (glucose levels maintained at ~5 mM) or hyperglycemic (glucose levels maintained ~20 mM) conditions, in the absence or presence of insulin (Table 1Go). Thus, four groups of animals were studied. For all experimental conditions, endogenous insulin secretion was suppressed by a constant infusion of somatostatin (2 µg/kg·min). Sixty minutes after the onset of the somatostatin infusion, animals received either saline (euglycemic) or a bolus of glucose (0.5 ml of 30% glucose), followed by continuous glucose infusion at a flow rate of either 20.2 ± 0.9 or 16.5 ± 0.9 µl/100 g·min for Wistar or GK rats, respectively, to raise blood glucose concentration to approximately 20 mM (hyperglycemic). Three hours thereafter, animals were either killed (basal conditions) or administered a bolus of insulin (20 mU; Actrapid, Novo Nordisk, Bagsvaerd, Denmark), followed by a continuous insulin infusion (3 U/min·kg body weight) over a 20-min period (insulin-stimulated). The aim of the glucose infusion protocol was to reach two different steady-state glucose concentrations. The purpose of the 20-min insulin infusion at the end of the protocol was to obtain insulin-stimulated samples, during which the glucose infusion rate was kept constant. Thus, the glucose infusion rate does not reflect insulin sensitivity or resistance with this type of protocol. Blood samples for plasma glucose and insulin concentration were collected from the tail vein at 60, 240, or 260 min. Immediately after infusions were terminated, rats were anesthetized with sodium pentobarbital and hind-limb skeletal muscles were removed, directly frozen, and stored at -80 C for analysis.


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  		TABLE 1. Animal characteristics and blood chemistry during in vivo studies in Wistar and GK rats

 
In vitro studies
Rats were anesthetized with an ip injection of sodium pentobarbital (5 mg/100 g body weight ip). Isolated soleus muscles were used for in vitro incubation as described (14). Each soleus was split into two equal longitudinal portions before incubation. All incubation media were prepared from pregassed (95% O2/5% CO2) Krebs-Henseleit buffer containing 5 mM HEPES and 0.1% BSA (RIA grade). The gas phase in the vials was maintained at 95% O2/5% CO2. Muscles were incubated (30 C) for 3 h with either 5 or 25 mM glucose. Thereafter muscles were incubated in the absence or presence of insulin (60 nM) for 20 min. Incubations were terminated by immediately freezing the soleus muscle in liquid nitrogen and samples were stored at -80 C for analysis.

PI 3-kinase activity
A portion of frozen soleus muscle (25–30 mg) was homogenized in 500 µl of ice-cold buffer containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.5 mM Na3VO4, 1% Triton X-100, 10% vol/vol glycerol, 20 mM Tris (pH 8.0), 10 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, and 10 µg/ml aprotinin. Samples were solubilized by continuous stirring for 1 h at 4 C and centrifuged at 12,000 x g for 10 min (4 C). Protein was determined using a commercial kit (Bio-Rad Laboratories, Richmond, CA). The supernatant was immunoprecipitated overnight (4 C) with antiphosphotyrosine antibody (Signal Transduction Laboratories, Lexington, KY) coupled to protein A Sepharose (Sigma, St. Louis, MO). The immune complex was washed, and PI 3-kinase activity was determined as described (22). Data were quantitated using a phosphoimager (Bio-Rad Laboratories).

PDK 1 activity
A portion of frozen soleus muscle (25–30 mg) was homogenized in 500 µl of ice-cold buffer containing 50 mM Tris (pH 8.0), 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na4P2O7, 10 mM ß-glycerophosphate, 1 mM sodium vanadate, 1 µM microcystin, and 20 mM ß-mercaptoethanol. Samples were solubilized by continuous stirring for 40 min at 4 C and centrifuged at 12,000 x g for 10 min (4 C). Protein was determined as described above. The supernatant was immunoprecipitated 2 h (4 C) with PDK-1 antibody (gift from Dr. Dario Alessi, MRC Protein Phosphorylation Unit, Dundee, UK) coupled to protein A/G Sepharose (Sigma). The immune complex was washed, and PDK-1 activity was determined as described (23). Radioactivity was counted using a liquid scintillation counter (LKB Wallac, Stockholm, Sweden).

PKC{zeta} activity
The remaining portion of soleus muscle (25–30 mg) was homogenized in 600 µl of ice-cold buffer containing 20 mM Tris/HCl (pH 7.5), 250 mM sucrose, 1.2 mM EGTA, 20 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM NaF, 1% Triton X-100, 0.5% Nonidet P-40, and 2 µg/ml pepstatin. Samples were solubilized by continuous stirring for 30 min and centrifuged at 3000 x g for 15 min (4 C). Protein was determined as described above. The supernatant was immunoprecipitated overnight (4 C) with anti-PKC{zeta} antibody (Upstate Biotechnologies, Lake Placid, NY) coupled to protein A/G Sepharose (Sigma). The immune complex was washed, and PKC{zeta} activity was determined as described (24). Radioactivity was counted using a liquid scintillation counter.

Protein kinase phosphorylation and expression
Aliquots (30-50 µg) of homogenate prepared for the PI 3-kinase assay were solubilized in Laemmli buffer containing 100 mM diothiothreitol, separated by SDS-PAGE (7.5% resolving gel) and transferred to polyvinylidenedifluoride membranes (Millipore Corp., Bedford, MA). Protein expression and phosphorylation was assessed by immunoblot analysis using commercially available antibodies. PKB/Akt phosphorylation was assessed using a polyclonal rabbit antibody that recognizes Ser473 (New England BioLabs, Inc., Boston, MA). ERK protein expression was assessed using a pan-ERK antibody, and phosphorylation was assessed using a phosphospecific antibody that recognizes Thr202/Tyr204 (New England BioLabs). PDK-1 expression was assessed using a polyclonal antibody that recognizes the C-terminal region of human PDK-1, and phosphorylation was assessed using phosphospecific antibodies that recognize either Tyr373/376 or Ser241 (Cell Signaling Technology, Beverly, MA). PKC{alpha}/ß phosphorylation was assessed using a phosphospecific antibody that recognizes Thr638/641 (Cell Signaling Technology). PKC{delta} phosphorylation was assessed using a phosphospecific antibody that recognizes Ser643/676 (Cell Signaling Technology). PKC{theta} phosphorylation was assessed using a phosphospecific antibody that recognizes Thr538 (Cell Signaling Technology). PKC{zeta} protein expression was assessed using a monoclonal antibody that recognizes the C-terminal region of human PKC{zeta} (Transduction Laboratories), and phosphorylation was assessed using a polyclonal antibody that recognizes Thr410/403 (Cell Signaling Technology). After blocking with 7.5% fat-free dry milk and washing (TBST: 10 mM Tris, 100 mM NaCl, 0.02% Tween 20) polyvinylidenedifluoride membranes were incubated with appropriate secondary antibody (1:25,000, goat antirabbit IgG HRP conjugate, Bio-Rad Laboratories). Phosphorylated proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and quantified by densitometry (Bio-Rad Laboratories).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animal characteristics
Wistar and GK rats were matched for body weight (Table 1Go). Basal blood glucose levels were significantly elevated in diabetic GK rats compared with Wistar rats. Serum insulin concentration was similar between GK and Wistar rats. Euglycemic (glucose levels ~5 mM) or hyperglycemic (glucose levels ~20 mM) conditions were maintained for 3 h using a modified glucose clamp technique (Fig. 1Go). Thereafter rats were killed (basal) or further exposed to insulin for 20 min (insulin-stimulated). Plasma glucose levels under the euglycemic and hyperglycemic conditions were similar between Wistar and GK rats (Table 1Go). Plasma insulin levels at the end of the 20-min infusion were markedly increased in Wistar and GK rats. Glucose use during the 20-min insulin infusion was similar between Wistar and GK rats under euglycemic conditions (244 ± 16 vs. 240 ± 17 µmol glucose/kg·min, respectively) and 20% lower in GK vs. Wistar rats under hyperglycemic conditions (453 ± 40 vs. 584 ± 20 µmol/kg·min-1, respectively; P < 0.05).



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  		FIG. 1. In vivo study protocol. Horizontal arrows represent infusion of somatostatin, glucose, and saline or insulin during the in vivo study. Vertical arrows represent time at which animals were killed to harvest soleus skeletal muscle for signal transduction analysis.

 
In vivo effects of hyperglycemia on phosphotyrosine-associated PI 3-kinase activity
Under euglycemic conditions, insulin significantly increased PI 3-kinase activity 1.9- and 2.9-fold in Wistar and GK rats, respectively (Fig. 2Go). However, basal and insulin-stimulated PI 3-kinase activity was significantly lower in GK vs. Wistar rats. Hyperglycemia did not alter basal or insulin-stimulated PI 3-kinase activity in either Wistar or GK rats.



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  		FIG. 2. In vivo effects of hyperglycemia on phosphotyrosine-associated PI 3-kinase activity. Awake rats were infused with 5 (euglycemia) or 20 (hyperglycemia) mM glucose for 3 h followed by a 20-min infusion with saline (basal; open bar) or insulin (insulin-stimulated; closed bar). Phosphotyrosine-associated PI 3-kinase activity was determined in soleus muscle. A, Representative autoradiogram of thin layer chromatographic separation of reaction products. B, Graph is mean ± SE for five to six rats. Values are percentage of Wistar insulin-stimulated phosphotyrosine-associated PI 3-kinase activity. *, P < 0.05 basal vs. insulin-stimulated conditions; , P < 0.05 GK vs. Wistar rats.

 
In vivo effects of hyperglycemia on PKB/Akt phosphorylation
Under euglycemic conditions, insulin significantly increased PKB/Akt phosphorylation 4- and 9-fold in Wistar and GK rats, respectively (Fig. 3Go). Similar to the results for PI 3-kinase activity, basal and insulin-stimulated PKB/Akt phosphorylation was significantly reduced in the GK vs. Wistar rats. Hyperglycemia did not alter basal PKB phosphorylation in either GK or Wistar rats. Insulin-stimulated PKB phosphorylation tended to be lower in Wistar but not GK rats; however, this difference was not significant.



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  		FIG. 3. In vivo effects of hyperglycemia on PKB/Akt phosphorylation. Awake rats were infused with 5 (euglycemia) or 20 (hyperglycemia) mM glucose for 3 h followed by a 20-min infusion with saline (basal; open bar) or insulin (insulin-stimulated; closed bar). PKB/Akt phosphorylation was determined in soleus muscle. A, Representative immunoblot analysis showing PKB/Akt phosphorylation. B, Graph is mean ± SE for five to six rats. Values are percentage of Wistar insulin-stimulated PKB/Akt phosphorylation. *, P < 0.05 basal vs. insulin-stimulated conditions; , P < 0.05 GK vs. Wistar rats.

 
In vivo effects of hyperglycemia on ERK phosphorylation
Under euglycemic conditions, insulin significantly increased ERK phosphorylation 2.3-fold and 1.9-fold in Wistar and GK rats, respectively (Fig. 4Go). Insulin-stimulated ERK phosphorylation was significantly decreased in GK rats. Hyperglycemia did not alter basal or insulin-stimulated ERK phosphorylation is either Wistar or GK rats.



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  		FIG. 4. In vivo effects of hyperglycemia on ERK phosphorylation. Awake rats were infused with 5 (euglycemia) or 20 (hyperglycemia) mM glucose for 3 h followed by a 20-min infusion with saline (basal; open bar) or insulin (insulin-stimulated; closed bar). ERK phosphorylation was determined in soleus muscle. A, Representative immunoblot analysis showing ERK phosphorylation. B, Graph is mean ± SE for five to six rats. Values are percentage of Wistar insulin-stimulated ERK phosphorylation. *, P < 0.05 basal vs. insulin-stimulated conditions; , P < 0.001 GK vs. Wistar.

 
In vivo effects of hyperglycemia on PKC{zeta} activity
Under euglycemic conditions, insulin significantly increased PKC{zeta} activity in Wistar and GK rats 1.6-fold and 2.4-fold, respectively (Fig. 5Go). Somewhat consistent with the results for PI 3-kinase and PKB/Akt, basal PKC{zeta} activity tended to be reduced in GK rats; however, this difference was not significant. In contrast to our results for PI 3-kinase, PKB/Akt, and ERK, insulin-stimulated PKC{zeta} was similar between Wistar and GK rats. Under hyperglycemic conditions, basal PKC{zeta} activity was significantly increased 1.8-fold in Wistar rats, with a similar trend noted in GK rats. Interestingly, insulin did not have a synergistic effect on the elevated basal PKC{zeta} activity in either Wistar or GK rats. Despite this apparent insulin resistance, the absolute insulin-stimulated PKC{zeta} activity in Wistar and GK rats was similar between euglycemic and hyperglycemic conditions.



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  		FIG. 5. In vivo effects of hyperglycemia on PKC{zeta} activity. Awake rats were infused with 5 (euglycemia) or 20 (hyperglycemia) mM glucose for 3 h followed by a 20-min infusion with saline (basal; open bar) or insulin (insulin-stimulated; closed bar). PKC{zeta} activity was determined in soleus muscle. Graph is mean ± SE for five to six rats. Values are percentage of Wistar insulin-stimulated PKC{zeta} activity. *, P < 0.05 basal vs. insulin-stimulated conditions; , P < 0.01 GK vs. Wistar under euglycemic conditions.

 
Protein expression.
In previous studies we have assessed protein expression of IR, IRS-1, p85{alpha} regulatory subunit of PI 3-kinase, Akt, GLUT4, and glycogen synthase in skeletal muscle from GK vs. Wistar rats and were unable to detect any significant change between the strains (4, 14). PDK-1 protein expression was similar between Wistar and GK rats (1.63 ± 0.16 vs. 1.53 ± 0.15 arbitrary units, respectively; not significant, NS). ERK protein expression was similar between Wistar and GK rats (2.14 ± 0.17 vs. 2.52 ± 0.18 arbitrary units, respectively; NS). PKC{zeta} protein expression was similar between Wistar and GK rats (0.91 ± 0.17 vs. 0.86 ± 0.15 arbitrary units, respectively; NS). We interpret these data to suggest that signaling defects in skeletal muscle from GK rats arise largely from functional defects rather than from a reduced level of a particular signal transducer.

In vitro effects of high extracellular glucose concentration on PKC{zeta} activity and phosphorylation
To determine whether the elevation in basal PKC{zeta} activity under hyperglycemic conditions was a glucose-specific response, isolated soleus muscle from Wistar rats were incubated in vitro for 3 h with either 5 mM or 25 mM glucose. Thereafter, soleus muscle was incubated for an additional 20 min in the absence or presence of 60 nM insulin. Insulin significantly increased PKC{zeta} activity 2-fold in soleus muscle incubated in the presence of 5 mM glucose (Fig. 6AGo). Interestingly, exposure of soleus muscle to 25 mM glucose led to a significant 1.7-fold increase in PKC{zeta} activity over the response observed in the presence of 5 mM glucose. Insulin did not further increase PKC{zeta} activity in the presence of 25 mM glucose. However, insulin-stimulated PKC{zeta} activity was similar in soleus muscle exposed to 5 vs. 25 mM glucose. Similar results were obtained for PKC{zeta} phosphorylation (Fig. 6BGo). Insulin significantly increased PKC{zeta} phosphorylation 1.5-fold in soleus muscle incubated in the presence of 5 mM glucose. Exposure of soleus muscle to 25 mM glucose led to a significant 1.3-fold increase in PKC{zeta} phosphorylation over the response observed in the presence of 5 mM glucose. Insulin did not further increase PKC{zeta} phosphorylation in the presence of 25 mM glucose.



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  		FIG. 6. In vitro effects of high extracellular glucose concentration on PKC{zeta} activity and phosphorylation. Soleus muscle from Wistar rats was incubated for 3 h in the presence or 5 or 25 mM glucose. Thereafter muscles were exposed to the same concentration of glucose and incubated for an additional 20 min in the absence (basal) or presence (insulin-stimulated) of 60 nM insulin. PKC{zeta} activity (A) and phosphorylation (B) were determined. Graphs are mean ± SE for 5–10 muscles. Values are percentage of insulin-stimulated PKC{zeta} activity/phosphorylation in soleus muscle exposed to 5 mM glucose. Inset on B is representative immunoblot showing PKC{zeta} phosphorylation. *, P < 0.01; , P < 0.005 vs. muscle exposed to 5 mM glucose.

 
In vitro effects of high extracellular glucose concentration on phosphorylation of PKC{alpha}/ß, -{delta}, and -{theta}
To determine whether glucose alters phosphorylation of other PKC isoforms, we assessed phosphorylation of PKC{alpha}/ß, -{delta}, and -{theta} in isolated soleus muscle from Wistar rats incubated in vitro for 3 h with either 5 mM or 25 mM glucose, followed by an additional incubation for 20 min in the absence or presence of 60 nM insulin (Fig. 7Go). Insulin increased PKC{alpha}/ß phosphorylation 1.2-fold in soleus muscle incubated in the presence of 5 mM glucose; however, change did not reach significance (Fig. 7AGo). Exposure of soleus muscle to 25 mM glucose led to a significant 1.4-fold increase in PKC{alpha}/ß phosphorylation over the response observed in the presence of 5 mM glucose, with a further 1.2-fold increase in noted in the presence of insulin. Insulin significantly increased PKC{delta} phosphorylation 2.3-fold in soleus muscle incubated in the presence of 5 mM glucose (Fig. 7BGo). Exposure of soleus muscle to 25 mM glucose led to a significant 2.2-fold increase in PKC{alpha}/ß phosphorylation over the response observed in the presence of 5 mM glucose, with a further 50% increase noted in the presence of insulin. Neither insulin nor glucose altered PKC{theta} phosphorylation in the incubated skeletal muscle (Fig. 7CGo).



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  		FIG. 7. In vitro effects of high extracellular glucose concentration on phosphorylation of PKC{alpha}/ß, PKC{delta}, and PKC{theta}. Soleus muscle from Wistar rats was incubated for 3 h in the presence or 5 or 25 mM glucose. Thereafter muscles were exposed to the same concentration of glucose and incubated for an additional 20 min in the absence (basal) or presence (insulin-stimulated) of 60 nM insulin. Phosphorylation of PKC{alpha}/ß (A), PKC{delta} (B), and PKC{theta} (C) was determined. Inset is representative immunoblot showing isoform-specific phosphorylation. Graphs are mean ± SE for eight to 10 muscles. Values are percentage of insulin-stimulated phosphorylation in soleus muscle exposed to 5 mM glucose. *, P < 0.01 vs. muscle exposed to 5 mM glucose; , P < 0.01 vs. muscle exposed to 25 mM glucose.

 
In vitro effects of high extracellular glucose concentration on PKB/Akt phosphorylation
To determine whether the in vitro glucose effects on PKC{zeta} activity were specific, basal and insulin-stimulated PKB/Akt phosphorylation was determined in isolated soleus muscle from Wistar rats after in vitro exposure for 3 h to either 5 or 25 mM glucose (data not shown). Basal PKB/Akt phosphorylation in muscle exposed to either 5 or 25 mM glucose was below the detection level of the assay. Thus, glucose does not appear to have a direct measurable effect on PKB/Akt phosphorylation. Insulin induced a marked increase in PKB/Akt phosphorylation, with similar effects noted in soleus muscle exposed to either 5 or 25 mM glucose (100 ± 11 vs. 106 ± 10% Wistar insulin-stimulated response, respectively).

In vitro effects of high extracellular glucose concentration on PDK-1 activity
In an effort to delineate the mechanism by which changes in the extracellular glucose concentration directly increase PKC{zeta} activity, we determined PDK-1 activity under basal and insulin-stimulated conditions in isolated soleus muscle from Wistar rats after in vitro exposure for 3 h to either 5 or 25 mM glucose. Insulin did not affect PDK-1 activity (Fig. 8AGo). Regardless of whether insulin was absent or present, PDK-1 activity was significantly greater in soleus muscle exposed to 25 mM glucose (1.6-fold or 1.4-fold increase for soleus muscle incubated in the presence of 25 mM glucose without or with insulin, respectively). We also assessed phosphorylation of PDK-1 on Tyr373/376 and Ser241 by immunoblot analysis using phosphospecific antibodies. Insulin significantly increased phosphorylation of PDK-1 on Tyr373/376 2-fold (Fig. 8BGo). Exposure of soleus muscle to 25 mM glucose led to a significant 1.8-fold increase in PDK-1 phosphorylation on Tyr373/376, with a further 77% increase noted in the presence of insulin. PDK-1 phosphorylation on Ser241 was not altered by either insulin or glucose exposure (Fig. 8CGo). However, a decrease in the electrophoretic mobility of PDK-1 was noted in skeletal muscle exposed to insulin.



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  		FIG. 8. In vitro effect of glucose and insulin on PDK-1 activity and phosphorylation. Soleus muscle from Wistar rats was incubated for 3 h in the presence or 5 or 25 mM glucose. Thereafter muscles were exposed to the same concentration of glucose and incubated for an additional 20 min in the absence (basal) or presence (insulin-stimulated) of 60 nM insulin. A, PDK-1 activity; B, PDK-1 phosphorylation on Tyr373/376; C, PDK-1 phosphorylation on Ser241. Graphs are mean ± SE for five to 10 muscles. Values are percentage of insulin-stimulated PDK-1 activity in soleus muscle exposed to 5 mM glucose. Insets on B and C are representative immunoblots showing PDK-1 phosphorylation. *, P < 0.05 vs. muscle exposed to 5 mM glucose; , P < 0.01 vs. insulin-stimulated muscle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hyperglycemia has deleterious effects on insulin signaling, but the underlying mechanism is not fully understood (4, 14, 25). To gain further insight into the molecular mechanisms underlying glucose-induced insulin resistance, basal and insulin-stimulated responses of PI 3-kinase, ERK, and two members of the AGC kinase super family, PKB/Akt and PKC{zeta}, were measured under euglycemic and hyperglycemic conditions. Several reports have implicated PKB/Akt as a major site of glucose-induced insulin resistance (4, 14, 25); however, the effects of hyperglycemia on PKC{zeta} and ERK are less clear. Insulin action on PI 3-kinase, PKB/Akt, and ERK was impaired in diabetic GK rats, whereas PKC{zeta} activity was normal. This impairment in insulin signaling cannot be explained by a reduction in protein expression of these signal transducers (present study and Refs. 4 and 14). We hypothesized that impaired insulin signaling in diabetic GK rats would be improved after acute restoration of glycemia, yet these signaling defects were evident, regardless of the level of glycemia. In earlier studies (4, 14), restoration of glycemia in GK rats by 4 wk phlorizin treatment was associated with improvements in insulin action. Thus, a more prolonged period of euglycemia seems to be required to achieve improvements in insulin signaling.

In Wistar and GK rats, basal PKC{zeta} activity was elevated under hyperglycemic conditions, suggesting glucose directly increases PKC{zeta} activity. This effect of hyperglycemia was specific for PKC{zeta}, as basal responses for PI 3-kinase, PKB/Akt, and ERK were not different between euglycemic and hyperglycemic conditions. However, under in vivo conditions, we cannot exclude the possibility that other systemic factors may increase PKC{zeta} in response to hyperglycemia. Thus, to determine whether glucose had a direct effect on PKC{zeta}, isolated skeletal muscle was exposed to 5 or 25 mM glucose in vitro. Consistent with our in vivo observation, PKC{zeta} activity and phosphorylation was increased in skeletal muscle exposed to 25 mM glucose. Furthermore, the effect of glucose on PKC{zeta} was independent of changes in PI 3-kinase activity, and occurred in parallel with an increase in PDK-1 activity. Our observations are consistent with earlier studies whereby exposure of isolated skeletal muscle (18) or mesangial cells (26) to high extracellular concentrations of glucose was associated with PKC{zeta} translocation. Taken together these data suggest that the level of glycemia may directly regulate PKC{zeta} activity/phosphorylation in skeletal muscle.

PDK-1 is a downstream target of PI 3-kinase that was first described as an activator of PKB/Akt (23, 27, 28) and later shown to activate members of the AGC kinase superfamily, including PKC{zeta} (29). Here we provide evidence that a 3-h exposure of skeletal muscle to 25 mM glucose was associated with an increase in PDK-1 activity, providing a potential mechanism for the observed glucose-mediated changes in PKC activity. However, glucose did not alter PKB/Akt phosphorylation, indicating a divergence in the effects of PDK-1 on these downstream substrates. Although the mechanism of PDK-1 activation is not completely resolved, PDK-1 requires phosphorylation on several tyrosine (30) and serine (31) residues for full activation. PDK-1 has been reported to be both constitutively active in resting cells (31) as well as activated in response to growth factor stimulation (30). Tyrosine phosphorylation of PDK-1 may occur as a consequence of PDK-1 translocation to the plasma membrane or in response to insulin stimulation (32). Although serine phosphorylation of PDK-1 has been proposed to result from autophosphorylation of the kinase (31), the possibility of a serine/threonine kinase that might phosphorylate PDK-1 cannot be excluded. The intracellular location of the temporary complex formed by PDK-1 and its substrates can determine substrate specificity. Studies in 3T3L1 adipocytes, in which wild-type or plasma membrane targeted PDK-1 was overexpressed, show that full activation of PKB/Akt requires PDK-1 translocation to the plasma membrane, whereas PKC{zeta} can be fully activated independently of insulin by overexpression of wild-type cytosolic PDK-1 (33). Thus, differences in the intracellular compartmentalization of PDK-1 may provide a mechanism for the divergent effects on PKB/Akt and PKC{zeta}.

High concentrations of glucose or glucosamine are associated with modifications of PI 3-kinase, Akt, and glycogen synthase (33, 34, 35), suggesting a potential mechanism by which glucose can directly alter the kinetic properties of signaling intermediates and enzymes. Here we describe that activation of PDK-1 and PKC{zeta} can be regulated by changes in the extracellular glucose concentration. Furthermore, these effects are not specific to PKC{zeta}, because we also noted an increase in phosphorylation of PKC{alpha}/ß and PKC{delta}, but not PKC{theta}, in isolated soleus muscle exposed to 25 mM glucose. Our results are consistent with a previous study whereby glucose was reported to activate several PKC isoforms, including {alpha}, {delta}, {epsilon}, and {zeta} in Rat-1 fibroblasts (19). Furthermore, we provide in vivo evidence in diabetic GK rats for increased PKC{zeta} activity and phosphorylation under hyperglycemic conditions. Interestingly, basal responses for PI3-kinase, Akt, and ERK under euglycemic conditions were reduced in GK rats. Because protein expression of these targets was similar between GK and Wistar rats (present study and Refs. 4 and 14), the reduced basal responses may be a consequence of activated PDK-1 or PKC; however, this remains to be determined. The long-term consequence of elevated PDK-1 and PKC isoforms phosphorylation/activity should be considered in the context of diabetes mellitus, as hyperglycemia is a clinical feature of this disease.

PI 3-kinase activity and PKB/Akt phosphorylation were not directly increased in skeletal muscle in response to high extracellular glucose. This finding suggests that a phosphatidylinositol-3,4,5-triphosphate-independent mechanism can account for the glucose-induced changes in activity of PDK-1 and PKC{zeta}. Indeed, glucose has been reported to directly activate PKC{zeta} through a sequential activation of the dantrolene-sensitive, nonreceptor proline-rich tyrosine kinase-2 and components of the ERK pathway, including GRB2, Son of Sevenless, RAS, RAF, MAPK/ERK kinase 1, ERK, and phospholipase D (20). Through this signaling cascade, levels of phosphatidic acid (PA), a known activator of PKC{zeta}, are increased. However, our present and previous (36) results fail to provide evidence that ERK phosphorylation is increased in response to hyperglycemia. Although the involvement of ERK in this signaling cascade is less apparent, our studies do not exclude a role for phospholipase D/PA in the regulation of PKC{zeta} by hyperglycemia. In vitro exposure of isolated epitrochlearis muscle to exogenous PLC is associated with increased membrane-associated PKC activity (37). Furthermore, de novo synthesis of DAG from glucose can also lead to PKC activation (38, 39), providing a potential mechanism to account for the increase in PKC{alpha}/ß and -{delta} phosphorylation. Although PKC{zeta} is an atypical PKC isoform that is activated in a DAG-independent manner, DAG can be phosphorylated into PA by diacylglycerol kinase (40), providing an additional PA-mediated mechanism for the glucose effects on PKC{zeta}. Thus, glucose might selectively activate PKC{zeta} through a phosphatidylinositol-3,4,5-triphosphate-independent mechanism, whereby other phospholipids, such as PA, may serve as suitable lipid substrates to activate PKC{zeta}.

In summary, diabetic GK rats have defects in insulin signal transduction in skeletal muscle at the level of PI 3-kinase, Akt, and ERK. Despite a reduction in insulin-stimulated signaling, the fold changes appeared to be similar or even higher in GK rats under euglycemic conditions. This could be counterintuitive, as it may be viewed as increased insulin sensitivity. Nevertheless, the insulin signaling defects persist even after an acute in vivo normalization of glycemia. Importantly, we provide evidence that PDK-1 is a glucose-sensitive target that activates PKC isoforms ({alpha}/ß, {delta}, and {zeta}) through a mechanism that is independent of changes in PI 3-kinase, PKB/Akt, and ERK phosphorylation. The glucose effect on PKC does not appear to be a general feature of AGC kinases, as PKB/Akt activity was unaffected by hyperglycemia. Whether other PDK-1 substrates such as mitogen- and stress-activated protein kinase 1 and p90rsk are activated in response to hyperglycemia remains to be determined. Finally, the consequence of elevated activity/phosphorylation of PDK-1 and PKC isoforms in skeletal muscle from type 2 diabetic subjects remains to be determined.


   Footnotes
 
This work was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, Novo-Nordisk Foundation, Marcus and Amalia Wallenberg’s Foundation, Torsten and Ragnar Söderberg’s Foundation, and the Foundation for Scientific Studies of Diabetology.

Abbreviations: DAG, Diacylglycerol; GK, Goto-Kakizaki; NS, not significant; PDK, PI-dependent kinase 1; PI, phosphatidylinositol; PKB, protein kinase B.

Received April 9, 2003.

Accepted for publication August 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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