Alterations of npkc distribution, but normal Akt/PKB activation in denervated rat soleus muscle

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1 Am J Physiol Endocrinol Metab 283: E318 E325, First published April 16, 2002; /ajpendo Alterations of npkc distribution, but normal Akt/PKB activation in denervated rat soleus muscle YENSHOU LIN, 1,2 MATTHEW J. BRADY, 3 KRISTEN WOLANSKE, 1 RICHARD HOLBERT, 1 NEIL B. RUDERMAN, 1,2 AND GORDON C. YANEY 1 1 Diabetes and Metabolism Unit and 2 Department of Physiology, Boston University Medical Center, Boston, Massachusettes 02118; and 3 Department of Medicine, University of Chicago, Chicago, Illinois Received 29 August 2001; accepted in final form 12 April 2002 Lin, Yenshou, Matthew J. Brady, Kristen Wolanske, Richard Holbert, Neil B. Ruderman, and Gordon C. Yaney. Alterations of npkc distribution, but normal Akt/ PKB activation in denervated rat soleus muscle. Am J Physiol Endocrinol Metab 283: E318 E325, First published April 16, 2002; /ajpendo Denervation has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport in rat skeletal muscle. Insulin binding to its receptor, activation of the receptor tyrosine kinase and phosphatidylinositol 3 -kinase do not appear to be involved. On the other hand, it has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity. In this study, we further characterize these changes in PKC and assess other possible signaling abnormalities that might be related to the decrease of glycogen synthesis. The results reveal that PKC- and -, but not - or -, are increased in the membrane fraction 24 h after denervation and that the timing of these changes parallels the impaired ability of insulin to stimulate glycogen synthesis. At 24 h, these changes were associated with a 65% decrease in glycogen synthase (GS) activity ratio and decreased electrophoretic mobility, indicative of phosphorylation in GS in muscles incubated in the absence of insulin. Incubation of the denervated soleus with insulin for 30 min minimally increased glucose incorporation into glycogen; however, it increased GS activity threefold, to a value still less than that of control muscle, and it eliminated the gel shift. In addition, insulin increased the apparent abundance of GS kinase (GSK)-3 and protein phosphatase (PP)1 in the supernatant fraction of muscle homogenate to control values, and it caused the same increases in GSK-3 and Akt/protein kinase B (PKB) phosphorylation and Akt/PKB activity that it did in nondenervated muscle. No alterations in hexokinase I or II activity were observed after denervation; however, in agreement with a previous report, glucose 6-phosphate levels were diminished in 24-h-denervated soleus, and they did not increase after insulin stimulation. These results indicate that alterations in the distribution of PKC- and - accompany the impairment of glycogen synthesis in the 24-h-denervated soleus. They also indicate that the basal rate of glycogen synthesis and its stimulation by insulin in these muscles are diminished despite a normal activation of Akt/PKB and phosphorylation of GSK-3. The significance of the observed alterations to GSK-3 and PP1 distribution remain to be determined. denervation; soleus muscle; novel protein kinase C; Akt/ protein kinase B; glycogen synthase kinase 3; protein phosphatase-1; glycogen synthase DENERVATION OF SKELETAL MUSCLE for as few as 6 24 h has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport (8, 10, 20, 35). Although defects in glycogen synthase (GS) activation have been reported (8), the mechanism by which insulin action is impaired is not clear. Thus binding of insulin to its receptor and activation of the receptor tyrosine-kinase (9) and phosphatidylinositol 3 -kinase (PI3K) (11) do not appear to be affected, suggesting that events distal to PI3K activation are involved. It has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity (20). Alterations in the activity and/or distribution of novel (PKC- and -, npkc) and occasionally conventional (c)pkc isoforms have been demonstrated in skeletal muscle in a variety of insulin-resistant states, including those induced by fat feeding (33), fructose ingestion (15), and glucose infusion (24). In addition, alterations in PKC distribution have been observed in insulin-resistant muscle of fa/fa and Goto Kakizaki rats (1). Which PKC isoform(s) is altered in the denervated muscle is not known. It has also been reported that GS activity is impaired in denervated rat muscle in both basal and insulinstimulated states (8). GS, the rate-limiting enzyme in glycogen synthesis, is regulated by covalent modification and allosterical regulation (23, 34). Covalent modification occurs by phosphorylation, as GS can be phosphorylated on at least six residues by a variety of kinases that cumulatively inhibit its activity. One of these kinases, GS kinase (GSK)-3, is phosphorylated and inactivated by insulin. It has been proposed that Address for reprint requests and other correspondence: N. B. Ruderman, Diabetes and Metabolism Research Unit, Boston Medical Center, 650 Albany St., EBRC Rm. 820, Boston, MA ( nruderman@medicine.bu.edu). 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. E /02 $5.00 Copyright 2002 the American Physiological Society

2 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE E319 this results in the disinhibition of GS by diminishing its phosphorylation (14, 17, 41). It has also been suggested that GS dephosphorylation and activation can result from the insulin-stimulated activation of glycogen-targeted protein phosphatase-1 (PP1) (29). In the present study, we have attempted to address the mechanism of GS inhibition after sciatic nerve section by asking the following questions. 1) What are the PKC isoforms responsible for the observed alteration in membrane-associated PKC activity? 2) Is the timing of changes in PKC distribution consistent with the development of insulin resistance and documented elevation of malonyl-coa? 3) Do alterations in distal events in the insulin-signaling cascade or in the distribution of GS, GSK-3, and PP1 occur after denervation? Finally, when initial data confirmed an earlier report that the concentration of glucose 6-phosphate was diminished in the denervated soleus, we asked whether this might be related to a decrease in hexokinase activity. MATERIALS AND METHODS Experimental animals and denervation. Male Sprague- Dawley rats weighing g, purchased from Charles River Breeding Laboratories (Wilmington, MA), were maintained on a 12:12-h light-dark cycle in a temperature-controlled (19 21 C) animal room. Sciatic nerve section was performed under anesthesia with pentobarbital sodium (4 6 mg/100 g body wt ip) at various times before an experiment, as detailed in RESULTS. A sham operation was performed on the contralateral limb. All rats were fasted but had free access to water during the h before they were killed. In all instances, rats were killed at 12:00 noon. Incubation protocol. On the experimental day, rats were reanesthetized, and soleus muscles from both limbs were isolated. Muscles were initially incubated for 30 min at 37 C in Krebs-Henseleit solution (KHS) continuously gassed with 95% O 2-5% CO 2 and supplemented with 6 mm glucose and then for 2 min in glucose-free KHS. After that, they were transferred to KHS containing 6 mm glucose (with [U- 14 C]glucose when the measurements of glucose disposition were performed) and 10 mu/ml insulin as indicated. At the end of a 30-min incubation in this medium, muscles were quickly blotted with a piece of gauze and quick-frozen in liquid nitrogen. The muscles were stored at 80 C until the measurement of glucose disposition and other assays were performed. Measurement of glucose disposition. [U- 14 C]glucose incorporation in glycogen and lipid and its oxidation to CO 2 were measured as described previously (20). PKC immunoblotting. The processing of muscles used for tissue extraction and PKC solubilization was as described previously (26). Briefly, muscles (25 35 mg) were homogenized and extracted in 500 l of an ice-cold homogenizing buffer containing 250 mm sucrose, 50 mm Tris (ph 7.5), 2 mm EDTA, 0.4 mm EGTA, 10 mm dithiothreitol (DTT), 5 g/ml leupeptin, 4 g/ml aprotinin, 2 mm phenylmethylsulfonyl fluoride (PMSF), 2 mm diisopropyl fluorophosphate, 2.5 g/ml pepstatin A, 10 mm sodium fluoride (NaF), and 1 mm -glycerophosphate. A portion of the homogenate (200 l) was centrifuged at 140,000 g for 1 h at 4 C and the supernatant (cytosolic fraction) removed and stored in liquid nitrogen. The pellet was resuspended in 200 l of homogenizing buffer containing 0.2% (wt/vol) of the detergent decanoyl-nmethylglucamide (MEGA-10). After sitting for1hat4 C, the extract was centrifuged again at 140,000 g for 1 h, and the supernatant that contained solubilized protein (membrane fraction) was removed. All fractions were stored at 80 C before assay. Protein was determined with a detergent-tolerant protein assay kit from Bio-Rad (Hercules, CA). Equal amounts (20 30 g of protein) of the cytosol and membrane fractions from each muscle were subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (22). Protein was transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) using a semidry transfer apparatus as outlined by the manufacturer (Owl Scientific, Cambridge, MA). After transfer, the nitrocellulose membranes were blocked with 5% nonfat dried milk in TBS-T buffer (1.5 M NaCl and 50 mm Tris, ph 7.4, containing 0.05% Tween 20) overnight at 4 C. The membranes were probed with isozyme-specific antibodies against PKC (polyclonal Ab:, I, II,,,,,,, and ; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. After four 5-min washes in TBS-T buffer, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Boehringer Mannheim, Mannheim, Germany) for 45 min at room temperature. Visualization of bands was obtained by chemiluminescence as outlined by the manufacturer (Pierce Chemical, Rockford, IL). The optimal band intensity was determined to be linear with increasing time of exposure and amount of sample loaded (data not shown), as determined by the NIH image analysis software (free distribution by the National Institutes of Health, Bethesda, MD). Akt/PKB immunoprecipitation and activity assay. The processing of muscles used to assay Akt/PKB activity was as described previously (21). In brief, immunoprecipitates of Akt/PKB were obtained by incubating the muscle extract with an Akt/PKB-specific antibody (provided by Dr. P. Tsichlis, Jefferson Medical College) that had been preincubated with Sepharose A beads for 1 h. The beads were then washed and activity assessed by the phosphorylation of the substrate peptide crosstide (Upstate Biotechnology, Lake Placid, NY). In the Western blot, an equal amount of protein was loaded, separated by SDS-PAGE, and blotted with anti-phospho- Ser 473 antibody (New England Biolabs, Beverly, MA). Assay of GS activity and Western blot. Muscles were homogenized in 50 mm HEPES (ph 7.8), 10 mm EDTA, and 100 mm NaF plus protease inhibitors and then subjected to centrifugation for 10 min at 2,500 g. GS activity in the supernatant was measured as described previously (6, 25). Briefly, l of supernatant were assayed in a final volume of 100 l of GS buffer containing 5 mm UDP-glucose and 1 Ci/ml UDP-[U- 14 C]glucose, in both the presence and absence of 10 mm glucose 6-phosphate (G-6-P). Samples were incubated at 37 C and then placed on ice for 15 min. Ninety microliters of the reaction mixture were spotted on GF/A filters, dried for 3 s, and then placed in 70% ethanol on ice. Filters were washed for 20 min at 4 C and then washed two more times in 70% ethanol at room temperature. Filters were air-dried, and UDP-[U- 14 C]glucose incorporation into glycogen was measured by liquid scintillation counting. In the Western blot, an equal amount of protein was loaded, separated by SDS-PAGE, and blotted with anti-gs antibody (a generous gift from Dr. J. Lawrence Jr., University of Virginia). GS blots were visualized using HRP-conjugated secondary antibody followed by chemiluminescence as outlined by the manufacturer (Amersham). GSK-3 Western blot. Muscles were weighed (average mg) and homogenized in 1 ml of ice-cold buffer containing

3 E320 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE (in mm) 50 HEPES (ph 7.4), 1 EGTA, 1 EDTA, 10 -glycerolphosphate, 5 sodium pyrophosphate, 100 KCl 1 DTT, and 0.5 Na 3VO 4 and 10 l/ml protease inhibitor cocktail P8340 added just before use. Triton X-100 was added to each sample to achieve a final concentration of 0.5% (vol/vol) and the sample solubilized for 1 h at 4 C. The resultant lysate was centrifuged at 15,000 g for 10 min, and the supernatant was used in either Western blot assay for GSK-3 or the activity measurement. PP1 Western blot. For PP1 assay, the processing was as described previously (7). Briefly, muscle was homogenized in PP1 homogenization buffer [50 mm HEPES (ph 7.2), 2 mm EDTA, 0.2% 2-mercaptoethanol, and 2 mg/ml glycogen] plus 10 g/ml aprotinin, 1 mm benzamidine, and 0.1 mm PMSF added just before use. The homogenate was centrifuged at 10,000 g to yield supernatant for Western blot assay. In Western blot, an equal amount of protein was loaded, separated by SDS-PAGE, and blotted with anti-pp1 antibody (a generous gift from Dr. J. Lawrence, Jr.). PP1 blots were visualized using HRP-conjugated secondary antibody followed by chemiluminescence as outlined by the manufacturer (Amersham Pharmacia). G-6-P measurement. Muscles (25 35 mg) were homogenized in 1 ml of ice-cold 6% perchloric acid for 1 min. The precipitated protein was pelleted by centrifugation at 5,000 g for 15 min. The supernatant was neutralized with KHCO 3, and G-6-P was assayed spectrophotometrically as described by Lowry and Passoneau (27). Hexokinase activity measurement. Muscles (25 30 mg) were homogenized in 10-fold volume of homogenizing buffer (in mm: 20 Tris HCl, 900 KCl, 10 MgCl 2, 2 EDTA, and 10 glucose and 0.5% Tween 20). The homogenate was centrifuged at 10,000 g for 30 min, and the supernatant was split in half to measure total hexokinase activity and heat-stable hexokinase (hexokinase I). Hexokinase activity was assayed as described by Postic et al. (31). Hexokinase II activity was subsequently calculated by subtracting hexokinase I from total hexokinase activity. Statistical analysis. Data are presented as means SE. Treatment effects were evaluated using a two-tailed Student s t-test. A P value 0.05 was considered to be statistically significant. RESULTS Impairment of insulin-stimulated GS at 24 h, but not 6 h, after denervation in the soleus muscle. In keeping with previous data (8, 20), glucose incorporation into glycogen in both the presence and absence of insulin was significantly decreased in the 24-h-denervated muscle, whereas glucose incorporation into lipid was enhanced. Six hours after denervation, neither of these alterations was evident (Fig. 1). Alteration of npkc distribution in muscle 24 h, but not 2 12 h, after denervation. As noted previously (20), an increase in membrane-associated PKC activity has been observed in muscle 24 h after denervation; however, the PKC isoforms responsible for this are not known. To answer this question, the cellular distribution of various PKC isoforms was compared in shamoperated and denervated muscle. Twenty-four hours after denervation, the abundance of PKC- and - (P 0.05), but not PKC- or -, was increased in the total homogenate, suggesting an increase in net synthesis. As shown in Fig. 2B, this was due to an increase in the Fig. 1. Glucose disposition in denervated rat soleus muscle. Soleus muscles from sham-operated control and contralaterally denervated rats were incubated in the presence of 6 mm [U- 14 C]glucose with or without 10 mu/ml insulin. Glucose incorporation into glycogen (top), total lipid (middle), and CO 2 (bottom) was examined 6 and 24 h after denervation. Data are means SE; n 5 6. *P 0.05, and **P abundance of these isoforms in the membrane fraction (P 0.05) without a corresponding decrease in the cytosolic fraction. To evaluate the time course of these changes in PKC distribution, similar studies were performed in muscle taken 2, 6, and 12 h after denervation (Fig. 3). In contrast to the finding at 24 h, no changes in the membrane abundance of PKC- or - were found at these times. Interestingly, at 12 h, a decrease in the mass of PKC- in the membrane fraction was found in both control and denervated muscle, suggesting it could be subject to diurnal variation. Activation of Akt/PKB by insulin is unaffected by denervation. The ability of insulin to activate its receptor tyrosine kinase (9) and PI3K (11) is not diminished in 24-h-denervated muscle. This plus the differential effect of insulin on glucose incorporation into glycogen and lipid synthesis in these muscles suggest that more distal events in the insulin-signaling pathway are involved. As shown in Fig. 4, this event did not appear to involve Akt/PKB. Thus both basal Akt/PKB activity and insulin-stimulated activation of this kinase were the same in denervated and control soleus muscles. Likewise, the phosphorylation of Ser 473 on Akt/PKB, an indicator of its activation by insulin, was unaffected by denervation. GS activity is impaired by denervation. The abundance and activity of GS were measured in control and

4 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE E321 Fig. 2. Effect of 24 h of denervation on protein kinase C (PKC) isoform distribution in rat soleus muscle. Immunoblots of shamoperated and denervated leg samples from the same rats were quantified by densitometry, as described in MATERIALS AND METHODS. A: Total H., total homogenate; Ctrl, control; Den, denervation. The blots shown are representative of 1 experiment repeated 4 times. B: values for the cytosolic and membrane fractions of denervated muscle are normalized to comparable values for sham-operated muscle. Abundance of PKC isoforms in denervated muscles is expressed as %difference from abundance in cytosolic or membrane fraction from control, nondenervated muscles. Data are expressed as means SE; n *P h-denervated muscles incubated in the presence and absence of insulin. Figure 5A shows that the activity of the active form of the enzyme (expressed as the activity ratio G-6-P/ G-6-P) was decreased by 60% in denervated muscle incubated with a medium devoid of insulin. Surprisingly, insulin caused a greater percentage increase in the activity of GS in the denervated soleus than it did in control muscles, although activity was still significantly higher in the control muscle. Total GS activity (Fig. 5B) was also slightly lower in denervated than in control muscle, although in the presence of insulin this difference was not statistically significant. None of these changes was associated with an alteration in the abundance of GS (Fig. 5C). On the other hand, an immunoblot of GS showed a gel shift in the denervated soleus that was not observed after incubation with insulin (Fig. 5C). Effects of denervation and insulin on GSK-3 and PP1 distribution and GSK-3 phosphorylation. The abundance of GSK-3 and, to an even greater extent, its phosphorylation on Ser 9 were markedly decreased in the 15,000-g supernatant of 24-h-denervated soleus incubated in the absence of insulin (Fig. 6). Incubation Fig. 3. Time course of changes in PKC distribution in the membrane fraction after denervation. At the indicated times, rats were killed, and soleus muscles from the denervated ( ) and sham-operated (}) limb were taken and processed as described in Fig. 2. Data comparing the abundance of individual isoforms in the membrane fraction are compared with those in the cytosolic fraction of the shamoperated limb. Results are means SE; n *P with insulin reversed both of these abnormalities. As shown in Fig. 7, denervation caused a similar alteration in PP1 distribution, which was also reversed by insulin. The concentration of G-6-P was diminished in denervated muscle in the presence and absence of insulin, whereas hexokinase I and II activities were unchanged. The failure of insulin to stimulate glycogen synthesis in denervated muscle, despite its ability to phosphorylate GSK-3 and activate GS, caused us to examine the effect of denervation on the concentration of G-6-P. In keeping with the findings of Burant et al. (8), the concentration of G-6-P was diminished by 50% in the 24-h-denervated soleus, and insulin failed to increase its concentration as it did in a control muscle (Fig. 8A). Thus, after insulin stimulation, the concentration of Fig. 4. Activation of Akt/protein kinase B (PKB) by insulin in 6- and 24-h-control and -denervated soleus muscles. Soleus muscles from sham-operated control leg and contralaterally denervated leg 6 and 24 h after sciatic nerve section were incubated in medium containing 6 mm glucose with or without 10 mu/ml insulin. Data are means SE; n 5. The Western blot of phospho-ser 473 Akt/PKB (P-S473 Akt, inset) is representative of 1 experiment repeated 8 times.

5 E322 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE Fig. 5. Effect of denervation on activity and abundance of glycogen synthase (GS). Soleus muscles from sham-operated and 24-h-denervated leg were processed as described in MATERIALS AND METHODS. Muscle homogenates were centrifuged at 2,500 g for 10 min, and equal amounts of supernatant protein were loaded onto SDS-polyacrylamide gel or used to measure the GS activity and abundance. A: glucose 6-phosphate (G-6-P) activity ratio ( G-6-P/ G-6-P)ofGS.B: total GS activity. C: abundance of GS in control and denervated muscle incubated with or without insulin. Western blot is representative of 4 experiments. Data are means SE; n 4. *P G-6-P was 70% lower in the denervated soleus. As shown in Fig. 8B, these differences were not paralleled by differences in the activity of hexokinase I or II between control and denervated muscle. DISCUSSION The increases in the concentration of DAG and PKC activity have been associated with insulin resistance in skeletal muscle in several model systems, including the denervated muscle (20). Denervation causes a dichotomy in insulin action: the ability of insulin to stimulate glucose incorporation into glycogen is markedly inhibited, whereas its ability to direct glucose into DAG and lipids is enhanced. These findings plus the observation that membrane-associated PKC activity is increased 24 h after denervation (20) led to the hypothesis that an increased formation of complex lipids, such as DAG, could initiate the events leading to insulin resistance (impaired glycogen synthesis) in denervated muscle by causing a sustained activation of one or more Fig. 6. Effect of denervation on abundance and serine phosphorylation of GS kinase (GSK)-3. After treatment, muscle was homogenated and centrifuged at 15,000 g for 10 min, and equal amounts of supernatant protein were loaded onto SDS-polyacrylamide gel or used to measure GSK-3 activity. A: abundance of GSK-3 blot. B: blot of Ser 9 phosphoryation (P-S9) in GSK-3 in control and denervated muscle incubated with or without insulin. Each blot is representative of 8 experiments. Results summarized by densitometry are shown in histograms, with the value in control muscle (neither denervation nor insulin) set as 100%. Data are means SE; n 8. *P conventional or novel PKC isoforms (12, 20). Thus, at various time points after sciatic nerve sectioning, we examined changes in glucose disposition, PKC isoform distribution, and the activity of metabolic enzymes and Fig. 7. Effect of denervation on abundance of protein phosphatase (PP) 1. Muscles were incubated and processed as described in legend to Fig. 6. Top: abundance of PP1 in Western blot of control and denervated muscle incubated with or without insulin. Each blot is representative of 7 experiments. Bottom: results summarized by densitometry are shown in histogram, with the value in control muscle (neither denervation nor insulin) set as 100%. Data are means SE; n 7. *P 0.05.

6 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE E323 Fig. 8. G-6-P content and hexokinase (HK) I and II activity in denervated soleus muscle. A: soleus muscle was homogenized, and the G-6-P content was measured by glucose-6-phosphate dehydrogenase with a spectrophotometer. B: total, type I, and type II HK activity were measured on muscle homogenates from control and denervated muscles. Data are means SE; n 7 8. *P signaling molecules to better understand how insulinstimulated glycogen synthesis is inhibited. The distribution of individual PKC isoforms in denervated soleus muscle was examined by Western blot. We focused on PKC-, -, and -, because they are the predominant isoforms expressed in skeletal muscle and changes in their distribution have been found in a variety of insulin-resistant states (1, 15, 24, 33), and on the atypical isoform PKC-, which is thought to play an important role in the regulation of glucose transport (38) and protein synthesis (28) by insulin. The results revealed that only the DAG-sensitive npkc isoforms PKC- and - were affected by denervation; their abundance in both the whole homogenate and the membrane fraction was increased at 24 h (Fig. 2B). In contrast, no changes in the mass of PKC- and PKC- were observed. Given that PKC- is also DAG sensitive, it is unclear why it did not respond in similar fashion. One possibility is that changes in DAG did not colocalize with PKC- ; alternatively, changes in intracellular calcium as well as in DAG might be required to alter its membrane targeting. Irrespective of the explanation, the observation that the changes in PKC- and PKC- abundance and distribution are not observed at 2 and 12 h after denervation, when insulin-stimulated glycogen synthesis is still normal, suggests that these alterations in PKC could play a causal role. The increase in total lipid synthesis also temporally correlates with a repartitioning of free fatty acids into complex lipids such as phosphatidic acid and DAG, as might be expected after a rise in malonyl-coa (32). Although this increase in malonyl-coa peaked between 6 and 8 h after denervation, it persisted at twice the control levels for more than 24 h. Activation of npkcs might impair insulin action by a number of mechanisms. Thus increased PKC activity has been shown in certain settings to serine-phosphorylate the insulin receptor, leading to decreases in the ability of insulin to stimulate glucose transport and various of its metabolic effects (2, 5, 18, 39). Against such a mechanism being operative in 24-h-denervated muscle, inhibition of early steps in the insulin-signaling cascade (receptor tyrosine kinase, PI3K, Akt/PKB) was not observed in these muscles (Refs. 9 and 11 and this study). In addition, insulin-stimulated glycogen synthesis was severely impaired after denervation, whereas the ability of insulin to stimulate glucose transport and overall glucose utilization was only minimally diminished and its ability to stimulate glucose incorporation into lipid was enhanced (Ref. 20 and this study). Collectively, these findings suggest that the decrease in glycogen synthesis in denervated muscle 1) is not secondary to decreased glucose transport and, 2) if it is related to impaired signaling, either a distal event in the cascade mediated by insulin or changes in an early event are involved. Whether the observed alteration in PKC- and - contributed to these changes remains to be determined, although it is noteworthy that incubation of the soleus with 1 M of phorbol dibutyrate for 1 h, which activates both cpkc and npkc isoforms, inhibits insulin-stimulated glycogen synthesis but not glucose transport (26, 37). Furthermore, inhibition of the insulin receptor tyrosine kinase and PI3K were minimal in these muscles, whereas a clear-cut decrease in Akt/PKB phosphorylation was observed (26). In 24-h-denervated solei incubated in a medium devoid of insulin, the activity of the active form of GS is diminished by 60% and electrophoretic mobility of GS is altered, consistent with phosphorylation. Unexpectedly, these changes were associated with a decrease in the abundance of both PP1 and GSK-3 in the 15,000-g supernatant of the muscle homogenate and with a decreased phosphorylation of GSK-3 in this fraction. Presumably, the net effect of these changes was to increase GS phosphorylation. Although insulin stimulated glycogen synthesis only minimally in the 24-h-denervated soleus, it activated Akt/PKB and GS to the same extent that it did in nondenervated muscles. In addition, it eliminated the gel shift of GS seen in the absence of insulin, and it restored the abundance of PP1 and GSK-3 in the muscle supernatant to control values. The net effect appeared to be activation of GS most likely related to its dephosphorylation. Studies to assess translocation between the supernatant and pellet fraction of these molecules are needed to explore the basis for these changes in subcellular distribution. In keeping with the earlier study by Burant et al. (8), insulin did not increase the concentration of G-6-P in denervated muscle as it did in control muscle (Fig. 8A). As a result, the concentration of G-6-P in the dener-

7 E324 REDISTRIBUTION OF PKC- AND - IN DENERVATED MUSCLE vated muscle incubated with insulin was only 25% of that of a control muscle. This persistently low concentration of G-6-P offers one explanation for the limited effect of insulin on the rate of glucose incorporation into glycogen in situ in the incubated soleus after denervation (Fig. 1) despite its pronounced effect on GS activity (Fig. 5). Interestingly, this result points to the fact that 24-h denervation did not affect the ability of insulin to increase the intrinsic activity of GS, as had been previously thought (7). The reason for this failure to increase the G-6-P concentration despite an apparently normal stimulation of glucose uptake and glycolysis (13, 20) in the denervated soleus is not known. The data in this study suggest that it is not attributable to a decrease in hexokinase activity, with the caveat that this reflects the intrinsic activity of the enzyme. The efficient phosphorylation of glucose by hexokinase requires its coupling to mitochondria, and this is increased by muscle contraction (3, 32, 42). Therefore, one might speculate that denervation results in a less efficient coupling and a decrease in the in situ activity of hexokinase. Another hypothetical possibility is that insulin-stimulated glucose transport directed to glycogen, vs. down the glycolytic pathway, is selectively depressed. There are no data in the literature relevant to this subject; however, it has been demonstrated that insulin and exercise recruit GLUT4 from different pools (30). Whether these transporters differentially direct glucose taken up by the muscle cell to different sites has not been studied. That denervation leads to an impairment of insulinstimulated glycogen synthesis, and sometimes glucose transport in muscle, has long been appreciated (8, 10, 35). The results of different studies are somewhat difficult to compare because of such variables as muscle type, duration of denervation, and nutritional state (8, 35, 36). In an experiment with rat diaphragm 1 day after denervation, Smith and Lawrence (35) observed a 50% decrease in glucose incorporation into glycogen and a decreased activity ratio for GS in both the presence and absence of insulin. In addition, they failed to observe a decrease in 2-deoxyglucose uptake at that time but did so 3 days after denervation. Later studies suggest that this secondary decrease in glucose transport is related to late-occurring decreases in the abundance of GLUT4 (4, 13, 19), as well as alterations in insulin signaling (16, 40). The same investigators demonstrated that, in an incubated epitrochlearis muscle prelabeled with 32 P, activation of GS by insulin is associated with the dephosphorylation of GS on at least two sites. In muscle denervated for 3 days, they found that the ability of insulin to do this was lost, as was its ability to activate GS (36). Studies in muscle denervated for 1 day were not reported. In conclusion, our results show that the inhibition of insulin-stimulated glycogen synthesis in rat soleus muscle 24 h after denervation correlates with altered distribution of PKC- and PKC- and an increase in glucose incorporation into lipids. The reason that the ability of insulin to stimulate glucose incorporation into glycogen is depressed in these muscles remains an enigma. Data from this study suggest that it is not due to an impaired ability of insulin to alter Akt/PKB and GSK-3 phosphorylation or to activate GS. The results also suggest that a redistribution of GSK-3 and PP1 occurs in denervated muscle; however, its relation to the pathophysiology of impaired glycogen synthesis is unclear, but it may relate to the phosphorylation state of GS. We thank T. Kurowski, Dr. D. Dean, and V. 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