In vivo regulation of protein-serine kinases by insulin in skeletal muscle of fructose-hypertensive rats

Sanjay Bhanot1, Baljinder S. Salh1, Subodh Verma2, John H. McNeill2, and Steven L. Pelech1

1 Department of Medicine, Faculty of Medicine, and 2 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effects of tail-vein insulin injection (2 U/kg) on the regulation of protein-serine kinases in hindlimb skeletal muscle were investigated in hyperinsulinemic hypertensive fructose-fed (FF) animals that had been fasted overnight. Basal protein kinase B (PKB) activity was elevated about twofold in FF rats and was not further stimulated by insulin. Phosphatidylinositol 3-kinase (PI3K), which lies upstream of PKB, was increased ~3.5-fold within 2-5 min by insulin in control rats. Basal and insulin-activated PI3K activities were further enhanced up to 2-fold and 1.3-fold, respectively, in FF rats. The 70-kDa S6 kinase (S6K) was stimulated about twofold by insulin in control rats. Both basal and insulin-stimulated S6K activity was further enhanced up to 1.5-fold and 3.5-fold, respectively, in FF rats. In control rats, insulin caused a 40-50% reduction of the phosphotransferase activity of the beta -isoform of glycogen synthase kinase 3 (GSK-3beta ), which is a PKB target in vitro. Basal GSK-3beta activity was decreased by ~40% in FF rats and remained unchanged after insulin treatment. In summary, 1) the PI3K right-arrow PKB right-arrow S6K pathway was upregulated under basal conditions, and 2) insulin stimulation of PI3K and S6K activities was enhanced, but both PKB and GSK-3 were refractory to the effects of insulin in FF rats.

insulin signaling; protein-serine/threonine kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE FRUCTOSE-HYPERTENSIVE RAT has been used extensively to examine the relationship among insulin resistance, hyperinsulinemia, and hypertension (6, 23-25). We and others have previously demonstrated that feeding normal rats a fructose-enriched diet results in marked insulin resistance in the animals (6, 23). To compensate for the decreased insulin-stimulated glucose disposal, the rats secrete more insulin, which results in compensatory hyperinsulinemia (6). This compensatory hyperinsulinemia offsets the insulin resistance and allows the animals to maintain normal plasma glucose levels despite the presence of severe insulin resistance. Results from several studies indicate that these metabolic defects may be intrinsically linked to the development of hypertension in the fructose-hypertensive rat (6, 23, 24). Although numerous studies have examined the association between insulin resistance and hypertension, the molecular mechanisms underlying insulin signaling in hypertensive states remain undetermined. In the present study, we have examined the regulation of several postreceptor protein-serine kinases (now thought to be critical to insulin-stimulated glycogen synthesis and glucose transport) in an insulin-resistant, hyperinsulinemic, hypertensive rat model, the fructose-fed rat.

Insulin-stimulated glucose utilization occurs primarily in the skeletal muscle, where most of the glucose is converted to glycogen (4, 27, 34). Insulin-stimulated glycogen synthesis has been shown to be markedly impaired in both animal and experimental hypertension (17, 18, 41). One of the protein kinases that has been implicated in the stimulation of glycogen synthesis by insulin is the seryl/threonyl protein kinase B (PKB) (12), also known as Rac-PK (26) or c-Akt (5). This enzyme, which is the cellular homolog of the viral oncogene v-Akt, has been shown to be activated by insulin in NIH-3T3 and Swiss 3T3 cells, rat adipocytes, and L6 myotubes (1, 13, 28). Furthermore, it was demonstrated that PKB inhibits glycogen synthase kinase-3 (GSK-3), an enzyme that phosphorylates and inhibits glycogen synthase in vitro (13). This led to the hypothesis that insulin activation of PKB and the subsequent inhibition of GSK-3 may be one of the mechanisms that enhance glycogen synthesis in vivo (13). PKB appears to lie downstream of the enzyme phosphatidylinositol 3-kinase (PI3K) in the insulin signaling pathway, because inhibitors of PI3K such as wortmannin inhibit the activation of PKB (8, 13).

We previously demonstrated that an intravenous injection of insulin in rats resulted in the activation of several seryl/threonyl protein kinases that were resolved as multiple peaks of myelin basic protein (MBP) phosphotransferase activity after fractionation by anion exchange chromatography (21). Furthermore, these peaks of MBP phosphotransferase activity remained unchanged in rats in which plasma glucose was clamped at basal levels after insulin administration (21), indicating that activation of these peaks was the direct result of insulin stimulation and was not secondary to the hypoglycemia that occurs after insulin injection. In the present study, we employed this model system to examine in vivo basal and insulin-stimulated activities of PI3K, PKB-alpha , p70 S6 kinase (p70 S6K), and GSK-3beta in the insulin-resistant, hypertensive, fructose-fed rats.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials. Regular insulin for intravenous injections was from Lilly; beta -glycerophosphate, EGTA, EDTA, MOPS, beta -methylaspartic acid, sodium orthovanadate, [gamma -32P]ATP, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, benzamidine, dithiothreitol (DTT), soybean trypsin inhibitor, pepstatin A, and the peptide inhibitor of cAMP-dependent protein kinase (PKI) were from Sigma. MBP was purified from bovine brain (15). The anti-PKB-alpha -PH, anti-PKB-alpha -CT, and anti-p70 S6K-NT (all polyclonal antibodies), as well as the monoclonal antibody raised against the 85-kDa subunit of rat PI3K, were purchased from Upstate Biotechnology (Lake Placid, NY). The anti-p70 S6K-CT antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-GSK-3beta antiserum was a kind gift from Dr. J. Woodgett (Ontario Cancer Institute, Toronto, ON, Canada); the GSK-3 substrate phosphopeptide and the control GSK-3 (Ala21) peptide were from Upstate Biotechnology. Alkaline phosphatase-conjugated goat anti-rabbit and goat anti-mouse IgGs and horseradish peroxidase-conjugated goat anti-mouse IgG were from Bio-Rad. Protein A-Sepharose, HR5/5 MonoQ, and MonoS columns were purchased from Pharmacia. The fructose diet was bought from Teklad Labs (Madison, WI). P-81 phosphocellulose filter paper was from Whatman. Ribosomal 40S subunits were prepared from rat liver by a procedure modified from that of Krieg et al. (29). All other chemicals and reagents were of the highest grade commercially available.

Experimental protocol. Sprague-Dawley rats were procured locally (body wt 130-150 g, age 5 wk). The animals were randomly assigned to either of the two experimental groups: control (n = 28) and fructose (n = 28). At 6 wk of age, the animals in the fructose group were started on a 66% fructose diet (66% fructose, 12% fat, and 22% protein), which had an electrolyte, protein, and fat content very comparable to the standard rat chow. The only difference was that the 60% vegetable starch present in normal rat chow was replaced by 66% fructose in the fructose diet. The fructose-induced metabolic changes become fully manifest 3-4 wk after initiation of the fructose diet (6); therefore, 6 wk after the initiation of the fructose diet, the rats were fasted overnight and crude muscle extracts were prepared, as we will describe in the next section. One week before termination, indirect systolic blood pressure (BP) was measured in conscious rats by use of the indirect tail cuff method without external preheating, as previously described (6). All experimental procedures were approved by the University of British Columbia Animal Care Committee.

Preparation of tissue extracts. The procedure described by Gregory et al. (20) and Pelech and Krebs (40) was modified and used to prepare tissue extracts. After pentobarbital anesthesia, 20 rats in each of the control and fructose groups were injected intravenously with insulin (2 U/kg dissolved in saline given into the tail vein), whereas the remaining animals (n = 8 in each group) were injected with saline alone. In each of the control and fructose-injected groups, the tissues were harvested 2 min (n = 7), 5 min (n = 7), or 15 min (n = 6) after the injection, and the skeletal muscles from the hind legs were removed (white muscle, primarily the gastrocnemius). The skeletal muscles were excised when the animals were anesthetized completely (surgical anesthesia) but were not dead. The muscles were removed rapidly, and the entire process (which took <1 min) was timed very accurately so that each animal was subjected to exactly the same procedure. The muscles were immediately homogenized in ice-cold MOPS buffer (25 mM, pH 7.2) containing (in mM) 5 EGTA, 2 EDTA, 75 beta -glycerophosphate, 1 sodium orthovanadate, 2 DTT, and various protease inhibitors (1 mM PMSF, 3 mM benzamidine, 5 µM pepstatin A, 10 µM leupeptin, and 200 µg/ml trypsin inhibitor). The homogenate was centrifuged at 10,000 g for 15 min at 4°C (Beckman J2-21), and the pellet was discarded. The supernatant was centrifuged at 100,000 g for 60 min (Beckman L8-60M), and the resultant supernatant was stored at -70°C until further analysis. At the time animals were killed, plasma samples were collected for subsequent insulin and glucose assays, which were performed as described previously (6).

Anion-exchange chromatography. Because of the existence of a multitude of protein kinases and other proteins in crude skeletal muscle extracts that could interfere with the various enzyme assays, the muscle extracts were fractionated to partially purify the various MBP kinases before specific immunoprecipitation assays were conducted. A fast protein liquid chromatography system was used for all the chromatographic fractionations of muscle extracts, as previously described (21). Briefly, samples containing 5 mg of the protein were applied at a flow rate of 0.8 ml/min to a MonoQ anion exchange column equilibrated with buffer A (in mM: 10 MOPS, pH 7.2, 25 beta -glycerophosphate, 5 EGTA, 2 EDTA, 2 sodium orthovanadate, and 2 DTT). The column was developed at the same flow rate with a 15-ml linear NaCl gradient (0-800 mM) in buffer A. Fractions (0.25 ml) were collected for assaying protein kinase activities, for Western blots, and for specific immunoprecipitation assays. For determination of GSK-3beta activity, samples containing 5 mg of the protein were applied at a flow rate of 0.8 ml/min to a MonoS cation exchange column equilibrated with buffer C (20 mM HEPES, pH 7.0); the column was developed at the same flow rate with a 15-ml linear NaCl gradient (0-400 mM) in buffer C, and 0.5-ml fractions were collected for subsequent assays.

Determination of phosphotransferase activities. MBP phosphotransferase activity from the MonoQ fractions was measured by employing a filter paper assay. The reaction mixture was comprised of 25 µg of substrate, 10 µl MonoQ fraction, 0.5 mM PKI, 50 µM [gamma -32P]ATP (specific activity ~2,000 cpm/pmol) and assay dilution buffer, pH 7.2 (in mM: 20 MOPS, 25 beta -glycerophosphate, 20 MgCl2, 5 EGTA, 2 EDTA, 1 DTT, and 1 sodium vanadate). The reaction was allowed to proceed for 10 min at 30°C and then was terminated by spotting 25 µl of the reaction mixture onto P-81 phosphocellulose paper. The papers were washed 5 times with 1% phosphoric acid to remove the free [gamma -32P]ATP and were then counted for radioactivity.

Ribosomal S6 kinase activity was determined by incubation of 10 µl of the MonoQ fractions for 30 min at 30°C with 40S ribosomal units as substrate, as described previously (21). GSK-3beta phosphotransferase activity was determined by incubation of 10 µl of the MonoS fractions with assay buffer containing 8 mM MOPS, 0.2 mM EDTA, 10 mM magnesium acetate, 0.1 mM ATP, and 125 µM of either the GSK-3 substrate phosphopeptide or the GSK-3 (Ala21) control peptide. The GSK-3 substrate phosphopeptide (GSK-3PP) contains serine residues at sites 3b, 3c, and phosphorylated site 4 from skeletal muscle glycogen synthase and is an excellent substrate for GSK-3. In contrast, the GSK-3 (Ala21) control peptide does not contain the site 4 serine residue, which is replaced with alanine; therefore, this peptide is not a substrate for GSK-3, because GSK-3 requires a prephosphorylated serine at site 4 to optimally phosphorylate sites 3b and 3c in the enzyme glycogen synthase. The reaction was initiated by the addition of 50 µM [gamma -32P]ATP (specific activity ~2,000 cpm/pmol) and allowed to proceed at 30°C for 30 min, after which it was terminated by spotting 25 µl of the reaction mixture onto P-81 phosphocellulose paper. The papers were washed 5 times with 1% phosphoric acid and counted for radioactivity.

Electrophoresis and immunoblotting. SDS polyacrylamide gel electrophoresis was performed (with 12.5% slab gels) as described by Laemmli (32). Gel electrophoresis was performed at 10 mA/gel overnight, and subsequently the proteins were transferred onto nitrocellulose membranes at 300 mA for 3 h. The membranes were blocked for 2 h with buffer containing 5% skim milk and sodium azide in 20 mM Tris · HCl, pH 7.4, and 0.25 M NaCl (TBS) and incubated with primary antibodies for 2 h or overnight. The membranes were then washed with TBS and Tween 20 (TTBS) and incubated for another hour with the secondary antibodies. After this, the membranes were washed again with TTBS and rinsed with TBS (without Tween 20), and the color reaction was performed for 5-30 min depending on the intensity of the bands. For procedures in which enhanced chemiluminescence was employed as the detection procedure, the secondary antibody was either goat anti-rabbit or the goat anti-mouse antibodies conjugated with horseradish peroxidase, and the incubation time was 45 min to 1 h.

Immunoprecipitation studies. For PKB-alpha immunoprecipitation assays, 200 µl of the particular MonoQ fractions were incubated with an equal volume of 3% NETF (100 mM NaCl, 5 mM EDTA, 50 mM Tris · HCl pH 7.4, 50 mM NaF, 5% glycerol, and 3% Nonidet P-40) and 35 µl of protein A-Sepharose beads and a combination of 5 µl of the anti-PKB-alpha -PH (Rac1-PH) and 5 µl of the anti-PKB-alpha -CT (Rac1-CT) antibodies. After a 3-h incubation period at 4°C, the protein A-Sepharose beads were pelleted by centrifugation at 10,000 rpm for 2 min. The beads were washed twice with 3% NETF and then twice with KII buffer (in mM: 12.5 MOPS, 12.5 beta -glycerophosphate, 20 MgCl2, 5 EGTA, 0.25 DTT, and 50 sodium fluoride), after which kinase assays were performed. The reactions were initiated by the addition of 35 µl of KII buffer, 5 µl of 200 mM MgCl2, and 50 µM of [gamma -32P]ATP (specific activity ~2,000 cpm/pmol). The reaction was allowed to proceed for 20 min at 30°C, after which it was stopped by the addition of 20 µl of 5× sample buffer. The reaction contents were boiled and loaded onto 12.5% SDS polyacrylamide gels, and after transfer, the MBP bands were Ponceau stained, cut, and counted. The membrane was then probed with specific antibodies to visualize the protein of interest by use of immunoblotting procedures described in Electrophoresis and Immunoblotting. For the p70 S6K immunoprecipitation assays, 200 µl of the particular MonoQ fractions were incubated for 3 h with 10 µl of the anti-p70-CT antibody. After this, the kinase reaction was performed in a manner identical to that just described, except that S6 peptide was used as the substrate. For the GSK-3beta immunoprecipitation assays, 200 µl of the particular MonoS fractions were incubated for 3 h with 3 µl of the anti-GSK-3beta antiserum. The beads were washed twice with 3% NETF, twice with 100 mM Tris, pH 7.4, and then once with 10 mM Tris, pH 7.4. After this, the kinase reaction was performed as previously described for the GSK-3 MonoS fractions.

The following two controls were included for each immunoprecipitation experiment. 1) An "antibody" control consisted of the antibody, the protein A-Sepharose beads, the immunoprecipitation buffer, but no sample extract. This allowed us to exclude any antibody-specific bands in the immunoprecipitate. 2) A "beads" control consisted of the protein A-Sepharose beads, the sample extract, the immunoprecipitation buffer, but no antibody. This allowed us to correct for the background by subtracting the activity pulled down by proteins sticking to the beads alone from each sample.

For the PI3K assays, 4 µl of the monoclonal anti-p85 antibody were incubated with aliquots of the crude homogenates (1 mg protein), 40 µl of the protein A-Sepharose beads, and an equal volume of lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 10% glycerol). After overnight incubation, the beads were washed two times with lysis buffer and then twice with 10 mM Tris, pH 7.4. Subsequently, kinase assays were performed by the addition of phosphatidylinositol as the substrate (10 µg/sample). The reaction was initiated by the addition of 10 µCi of [gamma -32P]ATP in a reaction buffer containing 30 mM HEPES (pH 7.4), 30 mM MgCl2, 50 µM ATP, and 200 µM adenosine. Samples were incubated at room temperature for 15 min, after which the reaction was stopped by the addition of 0.1 ml of 1 M HCl. Each sample was subjected to chloroform-methanol (1:1, vol/vol) extraction, and 25 µl of the extracted lipid products were spotted onto oxalate-treated silica gel plates and chromatographed by thin-layer chromatography with ammonium hydroxide-water-methanol-chloroform (1:3:14:18, vol/vol/vol/vol). The lipid products were visualized by autoradiography, and then the PI 3-phosphate spot was cut and quantified in a scintillation counter.

Statistical analysis. Data are presented as means ± SE unless indicated otherwise. Results were analyzed by an analysis of variance (ANOVA) procedure followed by a Newman-Keuls test. A probability of P < 0.05 was taken to indicate a significant difference between means.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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General characteristics of the animals. The fructose-fed rats were hypertensive compared with the control group (BP, 138 ± 3 mmHg vs. control 123 ± 2 mmHg, P < 0.05). The fructose-fed rats were also hyperinsulinemic compared with the Sprague-Dawley controls (fasted plasma insulin, 2.3 ± 0.6 ng/ml vs. control 0.9 ± 0.1 ng/ml, P < 0.05 by ANOVA). Basal plasma glucose levels were similar between the control and fructose-fed rats (control, 6.6 ± 0.2 vs. fructose, 7.1 ± 0.3 mmol/l). The fructose-fed rats gained weight at a similar rate compared with the control group (body weight at the time of euthanasia: control, 372 ± 20 g vs. fructose, 373 ± 8 g). These results are in accordance with earlier results (6), indicating that despite being severely resistant to the glucoregulatory effects of insulin, these animals were able to maintain normal glucose levels by secreting increased amounts of plasma insulin.

Insulin injection into the control animals caused a decrease in their plasma glucose concentration to 5.9 ± 0.3 mmol/l by 5 min postinjection and 2.5 ± 0.2 mmol/l by 15 min postinjection. Likewise, plasma glucose concentration dropped to 5.7 ± 0.1 mmol/l by 5 min and 3.2 ± 0.6 mmol/l by 15 min in the fructose-fed rats.

Increased basal MBP kinase activities in hyperinsulinemic rats. MonoQ fractionation on cytosolic extracts from the skeletal muscle extracts revealed multiple peaks of MBP phosphotransferase activity in the column fractions. Of the different peaks, peaks II, III, IV, and V were activated about twofold in control rats (Fig. 1A). These findings are in accordance with our previous studies, in which the kinetics and time course of each of these MBP peaks were reported (21). Interestingly, the basal activities of peaks II, III, and V were elevated about twofold in the hyperinsulinemic fructose-fed rats (Fig. 1B). Furthermore, insulin injection did not cause any increase in the MBP kinase peaks II, III, and V in fructose-fed rats, whereas peak IV was further activated by about twofold.


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Fig. 1.   MonoQ column chromatography of insulin-activated myelin basic protein (MBP) kinases in control and fructose-fed rats. Nos. I-V, peaks of MBP phosphotransferase activity. Skeletal muscle homogenates (5 mg) from control (0 min, open circle , A) or fructose-fed (0 min, triangle , B) rats and rats treated with iv insulin injection (15 min postinjection, control , A, and fructose-fed black-triangle, B) were fractionated over a MonoQ anion-exchange column (with a 0-800 mM linear NaCl gradient) and assayed for MBP kinase activity as described in EXPERIMENTAL PROCEDURES. Results are representative of >= 3 independent experiments.

PKB is an insulin-activated kinase in MonoQ peaks II and III. To determine the identity of the kinases that may contribute to MBP peaks II and III, we immunoblotted with antibodies specific for PKB-alpha (also known as Rac or c-Akt). Initial experiments demonstrated that the alpha -isoform of PKB was one of the MBP kinases that eluted in MBP peak fractions II and III. Therefore, we performed immunoprecipitation studies from the pooled MonoQ fractions 25-34 using antibodies directed against the carboxy terminus and the pleckstrin homology domain of PKB-alpha . These experiments revealed that there was a time-dependent increase in the activity of immunoprecipitated PKB-alpha in response to insulin in control rats (Fig. 2A). An increase in PKB-alpha activity was evident as early as 2 min after the insulin injection and was not accompanied by any change in the amount of immunoprecipitated protein (Fig. 2B). In the fructose-fed hyperinsulinemic rats, basal PKB-alpha activity was already increased about twofold, which corresponded with the basal increase in the MBP peaks II and III. Insulin administration did not cause any further increase in PKB-alpha activity in fructose-fed rats at any of the time points studied.


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Fig. 2.   Protein kinase B (PKB) is an insulin-activated kinase in MonoQ column peaks II and III. A: skeletal muscle homogenates from untreated control (C-I) and fructose-fed (F-I) as well as insulin-treated rats were collected at the various times indicated and fractionated over a MonoQ anion-exchange column. Eluted fractions 25-34 (that immunoblotted for PKB) were pooled, and immunoprecipitation assays were performed with a combination of anti-PKB-alpha -PH (Rac1-PH) and anti-PKB-alpha -CT (Rac1-CT) antibodies. Immunoprecipitates were then assayed for MBP kinase activity as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of 3 independent experiments. * P < 0.05 vs. C-I, ANOVA. B: representative Western blot from PKB-alpha immunoprecipitates (IP), showing similar amounts of immunoprecipitated protein among all treatment groups.

Activation of insulin-modulated ribosomal S6 kinases in vivo. To determine the activation of ribosomal S6 kinases in response to insulin, muscle extracts were fractionated by MonoQ chromatography and assayed using the 40S ribosomal protein as the substrate. Two major peaks of S6 phosphotransferase activity were detected in normal rats, which eluted at NaCl concentrations of ~100 and 350 mM, respectively (Fig. 3A). In fructose-fed rats, both peaks of S6 phosphotransferase activity were elevated basally by about twofold. However, insulin activation of peak I was decreased, whereas that of peak II was markedly increased when compared with control rats (Fig. 3B). Immunoblotting studies revealed that the 70-kDa ribosomal S6K, which lies downstream of PKB, coeluted with the second peak of S6 phosphotransferase activity (Fig. 3A, inset), which also corresponded with the fourth peak of MBP phosphotransferase activity (Fig. 1A). Immunoprecipitation studies from the second peak of S6 phosphotransferase activity demonstrated that the p70 S6K was activated about twofold by insulin in control rats within 15 min after insulin injection (Fig. 4A). This was accompanied by a gel mobility shift of the protein, which was maximal at 15 min postinjection (Fig. 4B). Basal p70 S6K activity was already elevated ~1.5-fold in the insulin-resistant rats (Fig. 4A). Surprisingly, insulin-stimulated p70 S6K activity could be further enhanced by another twofold after insulin treatment, which was accompanied by a corresponding gel mobility band-shift of the protein (Fig. 4B).


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Fig. 3.   MonoQ column chromatography of insulin-activated S6 kinases in control and fructose-fed rats. Skeletal muscle homogenates (5 mg) from control (0 min, open circle , A) or fructose-fed (0 min, triangle , B) rats and rats treated with intravenous insulin injection (15 min postinjection, control , A, and fructose-fed black-triangle, B) were fractionated over a MonoQ anion-exchange column (with a 0-800 mM linear NaCl gradient) and assayed for S6 kinase activity as described in EXPERIMENTAL PROCEDURES. A, inset: eluted fractions 30-44 were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-p70 S6K-CT antibody. Only fractions showing immunologically identifiable protein bands are shown. Results are representative of >= 3 independent experiments.



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Fig. 4.   Activation of p70 S6 kinase (p70 S6K) in vivo. A: skeletal muscle homogenates from untreated control (C-I) and fructose-fed (F-I) as well as insulin-treated rats were collected at the various times indicated and fractionated over a MonoQ anion-exchange column. Eluted fractions 32-40 (that immunoblotted for p70 S6K) were pooled, and immunoprecipitation assays were performed with the anti-S6K-CT antibody. Immunoprecipitates were then assayed for S6 peptide (RRLSSLRA) kinase activity as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of 3 independent experiments. * P < 0.05 vs. C-I, ANOVA. B: representative Western blot from p70 S6K immunoprecipitates showing similar amounts of immunoprecipitated protein among all treatment groups.

Increased PI3K activation in fructose-fed rats. Because both PKB and p70 S6K activities exhibited intriguing changes in the fructose-fed rats, we next examined the effects of insulin on PI3K, which has been shown to function upstream of PKB in a signaling pathway. Immunoprecipitation studies with the monoclonal anti-p85 PI3K antibody demonstrated that PI3K was maximally stimulated within 5 min after insulin injection by ~3.5-fold in control animals (Fig. 5, A and B). An increase in enzyme activity could be observed as early as 2 min postinjection, and the activity returned to near basal levels by ~15 min postinjection. Basal PI3K activity was elevated about twofold in the hyperinsulinemic rats, which was similar to the changes observed with PKB and p70 S6K. Insulin-stimulated PI3K activity was increased ~4.7-fold in the insulin-resistant animals (when compared with control basal levels) and peaked at 5 min after insulin injection (Fig. 5, A and B).


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Fig. 5.   Increased phospatidylinositol 3-kinase (PI3K) activation in fructose-fed rats. A: crude skeletal muscle homogenates (1 mg) from untreated control (C-I) and fructose-fed (F-I) as well as insulin-treated rats were collected at the various times indicated and subjected to immunoprecipitation assays with the monoclonal anti-p85-PI3K antibody as described in EXPERIMENTAL PROCEDURES. Autoradiogram of thin-layer chromatograph is shown. B: radioactivity in spots corresponding to PI 3-phosphate (PI3P) was measured; results are means ± SE of 3 independent experiments after normalization for amount of immunoprecipitated protein. * P < 0.05 vs. C-I, ANOVA.

Regulation of glycogen synthase kinase-3beta in fructose-fed rats. Recent reports have demonstrated that the enzyme glycogen synthase kinase-3beta (GSK-3beta ) is phosphorylated and inhibited by PKB in vitro and that this enzyme may be a physiological substrate for PKB (13). Crude muscle homogenates were subjected to MonoS chromatography and assayed for phosphotransferase activity directed against GSK-3PP. Results from these experiments revealed two basal peaks of GSK-3PP phosphotransferase activity, which eluted at a NaCl concentration of ~150-225 mM (Fig. 6A). When the same fractions were assayed using the control (Ala21) peptide as the substrate (not a substrate for GSK-3), no detectable phosphotransferase activity was observed. Immunoblotting studies identified GSK-3beta only within MonoS fractions 26-29 (Fig. 6C). Insulin administration in control rats caused a decrease in the activity of both the peaks, the first of which has been previously shown to contain the alpha -isoform of GSK-3 (44). In fructose-fed rats, basal activity in the second GSK-3PP peak was depressed, although the first GSK-3PP peak remained unchanged (Fig. 6B). Insulin injection appeared to cause a slight increase in both the GSK-3PP peaks, although the change did not attain statistical significance. To further confirm that the changes in the second peak of GSK-3PP phosphotransferase activity were due to GSK-3beta , immunoprecipitation studies were performed on the specific MonoS fractions by use of antiserum against this isoform of GSK-3. These experiments confirmed that insulin caused a >= 50% inhibition of GSK-3beta in control rats and that basal GSK-3beta activity was already depressed in fructose-fed rats (Figs. 6D and 7). No change was observed after insulin administration in fructose-fed rats at any of the time points studied.


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Fig. 6.   MonoS column chromatography of glycogen synthase kinase-3beta (GSK-3beta ) in control and fructose-fed rats. Skeletal muscle homogenates (5 mg) from control (0 min, open circle , A) and fructose-fed (0 min, triangle , B) rats and rats treated with intravenous insulin injection (15 min postinjection, control , A, and fructose-fed black-triangle, B) were fractionated over a MonoS cation-exchange column (with a 0-400 mM linear NaCl gradient) and assayed with GSK-3 substrate phosphopeptide (GSK-3PP) as substrate. The same fractions were also assayed with GSK-3 (Ala21) control peptide (control, 0 min  and 15 min , fructose-fed, 0 min down-triangle and 15 min black-down-triangle ) for activity as described in EXPERIMENTAL PROCEDURES. C: eluent fractions 21-34 were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-GSK-3beta antibody. Only fractions showing immunologically identifiable GSK-3beta bands are shown. Results are representative of >= 3 independent experiments. D: eluent fractions 26-29 were pooled, and immunoprecipitation assays were performed with anti-GSK-3beta antiserum. Immunoprecipitates were then assayed for GSK-3PP activity as described in Experimental Procedures. Results are means ± SE of 3 independent experiments. * P < 0.05 vs. C-I, ANOVA.



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Fig. 7.   Insulin regulation of GSK-3beta activity in control and fructose-fed rats. Crude skeletal muscle homogenates (1 mg) from untreated control (C-I) and fructose-fed (F-I) as well as insulin-treated rats were collected at the various times indicated and subjected to immunoprecipitation assays with anti-GSK-3beta antiserum. Immunoprecipitates were then assayed for GSK-3PP activity, as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of 3 independent experiments. * P < 0.05 vs. C-I, ANOVA.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In vivo insulin injection in normal rats activated PI3K, PKB-alpha , and p70 S6K in muscle lysates in a time-dependent fashion. There was a temporal correlation between the activation of these kinases such that the maximal activation of PI3K preceded that of PKB-alpha , the latter being activated earlier than the p70 S6K. Furthermore, activation of PKB-alpha was accompanied by a concurrent inhibition of GSK-3beta , supporting the notion that GSK-3beta may be a physiological substrate for PKB. One of the primary findings of the study was that basal activities of PI3K, PKB-alpha , and p70 S6K were increased in fructose-fed rats, indicating that this pathway was chronically upregulated in these hyperinsulinemic rats. This is in marked contrast to results in insulin-resistant, hyperinsulinemic models of experimental non-insulin-dependent diabetes mellitus (NIDDM), where basal PI3K activity is markedly decreased (2, 19). Similarly, PKB activation has been reported to be reduced in muscle samples obtained from insulin-resistant diabetic rats, which is in contrast to the present findings (30, 48).

An important difference between the fructose-fed model and several other rodent models of diabetes is that the fructose-fed rats are not hyperglycemic. Hyperglycemia per se could lead to changes in the regulation of signal transduction pathways, because it leads to glucose insensitivity. Therefore, some of the differences in particular kinase activities between fructose-fed rats and those studied previously (19, 31) could be explained by the differences in circulating glucose concentrations between the different animal models. However, models such as the fa/fa Zucker rat are also either normoglycemic or mildly hyperglycemic, and yet they demonstrate very different changes in the activities of the enzymes studied (for example, insulin-stimulated PI3K activity is impaired in muscle from fa/fa rats, in contrast to the fructose-fed rats). Taken together, these data raise the possibility that the mechanisms underlying the insulin resistance of hypertension may differ from those seen in NIDDM. However, it is necessary to study the regulation of these enzymes in other insulin-resistant hypertensive models before any conclusions can be drawn.

Insulin injection further enhanced PI3K activity in fructose-fed rats, indicating that the defect in insulin signaling in these rats resided distal to the insulin receptor. Similarly, there was a marked increase in p70 S6K activity after insulin administration in these rats, although PKB-alpha activity could not be further stimulated above basal levels. This observation indicates that inputs other than the PI3K-PKB pathway are responsible for the activation of p70 S6K in fructose-fed rats. Several enzymes, including the classical PKC isoforms and the p21-activated kinases (also called PAKs) have been demonstrated to indirectly activate p70 S6K (10, 11). Which, if any, of these activate p70 S6K in insulin-resistant animals remains to be determined. It has been reported that activation of p70 S6K is not essential for glucose utilization, as rapamycin fails to inhibit glucose transport in response to insulin (9). Our findings are consistent with these results, because activation of p70 S6K was not impaired in animals that were refractory to insulin's glucoregulatory effects.

GSK-3 is an enzyme that has been reported to regulate several of insulin's physiological effects, including glycogen and protein synthesis (13, 47). Recent studies have demonstrated that GSK-3 is a substrate of the enzyme PKB in vitro and in vivo (13). Intriguingly, although basal PKB-alpha activity was increased, basal GSK-3 activity was chronically inhibited in fructose-fed rats, which is consistent with the notion that the PKB- GSK-3 pathway is upregulated in these hyperinsulinemic rats. What is perhaps more interesting is the observation that, although both PKB-alpha and GSK-3beta activities remained unchanged after insulin injection in fructose-fed rats, the animals still displayed a fall in plasma glucose concentration after insulin injection. This indicates that activation of enzymes other than the PKB right-arrow GSK-3 pathway is involved in mediating insulin's glucoregulatory effects in fructose-fed rats. It should be noted that PKB-alpha is one of the three isoforms of PKB and that insulin has been shown to have differential effects on the activation of different PKB isoforms (46). Only the PKB-alpha isoform was examined in the present study, which is the primary isoform that is activated by insulin in skeletal muscle (46). The beta -isoform of PKB has been shown to be minimally activated by insulin in muscle, although in adipocytes, both the alpha - and beta -isoforms are stimulated to a similar extent.

These data support recent observations that inhibition of GSK-3 by insulin may not be sufficient to explain insulin-induced activation of glycogen synthase. For example, the beta 3-receptor agonists BRL-37344 and isoproterenol decreased the GSK-3 activity in epididymal fat cells without having any effect on the activity ratio of glycogen synthase (38). Furthermore, the mechanisms underlying the glucoregulatory effects of insulin may be distinct in different insulin-target tissues. For example, in rat skeletal muscle, the stimulation of glycogen synthase by insulin was shown to be partly sensitive to inhibition by rapamycin (3), whereas in rat adipocytes rapamycin had no effect on the glycogen synthase activity ratio (37).

The observation that, despite being insulin resistant, the fructose-fed rats demonstrated an apparently normal response to an acute insulin injection, deserves mention. When the fructose-fed rats are subjected to an acute insulin injection (2 U/kg), they attain plasma insulin concentrations that are at least two- or threefold higher than those seen during the normal postabsorptive state in these hyperinsulinemic rats. This amount of insulin, combined with the endogenous insulin already present in the animals, is sufficient to offset any insulin resistance and, hence, these animals show a normal decrease in plasma glucose levels.

In conclusion, we have demonstrated that the PI3K right-arrow PKB right-arrow p70 S6K pathway is chronically upregulated in insulin-resistant, hyperinsulinemic, fructose-hypertensive rats. In addition, although insulin stimulation of PI3K and p70 S6K activities is further enhanced in fructose-fed rats, both PKB and GSK-3beta are resistant to insulin's effects. To our knowledge, this is the first report that has examined the regulation of the PI3K pathway in hyperinsulinemic hypertensive rats. Further studies are required to elucidate insulin signal transduction in other models of experimental hypertension and to link the activation of the various enzymes to the final biological effects of insulin.


    ACKNOWLEDGEMENTS

We thank Violet Yuen for expert technical assistance during preparation of the tissue extracts, Dr. J. Woodgett for the GSK-3beta antiserum, and Dr. Jasbinder Sanghera for valuable advice during the course of this study.


    FOOTNOTES

This study was supported in part by grants from the Medical Research Council of Canada (MRCC) (to J. H. McNeill and S. L. Pelech). S. Bhanot was the recipient of a Heart and Stroke Foundation of Canada Fellowship, S. Verma was the recipient of a MRCC Fellowship, and S. L. Pelech was the recipient of a MRCC Industrial Scientist Award.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. L. Pelech, Dept. of Medicine, Rm. S125, 2nd floor, Koerner Pavilion, 2211 Wesbrook Mall, Univ. of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada (E-mail: spelech{at}home.com).

Received 29 October 1998; accepted in final form 20 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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