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

Yenshou Lin1,3, Samar I. Itani1, Theodore G. Kurowski1, David J. Dean1, Zhijun Luo1, Gordon C. Yaney1,2, and Neil B. Ruderman1,3

1 Diabetes and Metabolism Unit and 2 Obesity Research Center, Department of Medicine, and 3 Department of Physiology, Boston University Medical Center, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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

protein kinase C; phorbol dibutyrate; Akt/protein kinase B; glycogen synthase kinase-3; soleus muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN SUGGESTED THAT protein kinase C (PKC) activation plays a role in the downregulation of insulin signaling and the development of insulin resistance in skeletal muscle (see review in Refs. 8 and 28). Previous studies have demonstrated alterations in the activity and/or distribution of PKC isoforms in skeletal muscle in a variety of insulin-resistant states, including those induced by fat feeding (29), fructose ingestion (10), and glucose infusion (25). In addition, alterations in PKC distribution have been observed in insulin-resistant muscle of fa/fa and Goto-Kakizaki (GK) rats (2).

Studies employing phorbol esters (i.e., PKC activators) to investigate the relationship between PKC and insulin action in skeletal muscle have primarily focused on glucose transport. Thus it has been reported that, in the absence of insulin, phorbol 12-myristate 13-acetate (PMA), also referred to as 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulates glucose transport in rat epitrochlearis (16, 17) and extensor digitorum longus muscles (14) but has only a minimal effect on glucose transport in rat soleus muscle (33). Variable effects of phorbol esters on insulin-stimulated glucose transport have been reported (14, 16, 33). Glycogen synthesis has been less studied, although PMA has been shown to inhibit glycogen synthase activity (17) and glycogen synthesis (33) in muscles incubated in the absence of added insulin. The use of phorbol esters to evaluate the role of PKC as a central factor in the pathogenesis of insulin resistance in muscle (6, 7, 31) has been limited.

In the present study, we investigated the effects of preincubation with the phorbol ester phorbol 12,13-dibutyrate (PDBu) on insulin-stimulated glucose uptake and metabolism and on PKC distribution in rat soleus muscle. In addition, the effects of PDBu on key steps in the insulin-signaling cascade, including insulin receptor (IR), phosphatidylinositol 3'-kinase (PI3K), Akt/protein kinase B (PKB), and glycogen synthase kinase-3 (GSK3), were examined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals and muscle incubation. Male Sprague-Dawley rats weighing 50-65 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. All rats were fasted but had free access to water during the 18-20 h before they were killed. On the experimental day, rats were anesthetized with pentobarbital sodium (4-6 mg/100 g body wt ip), and soleus muscles from both limbs were isolated. Muscles were initially preincubated in Krebs-Henseleit solution (KHS) supplemented with 6 mM glucose and were continuously gassed with 95% O2-5% CO2 for 20 min. After this, they were incubated for 1 h with vehicle (0.1% DMSO) or various concentrations of calphostin C and then for 0.5-2 h with various concentrations of PDBu (or another phorbol ester) as described in RESULTS. Because the inhibitory activity of calphostin C is light dependent, these incubations were performed in clear glass tubes under the laboratory (cool-white fluorescent) light source. Subsequently, muscles were washed in KHS containing 6 mM glucose for 2 min and then incubated in KHS containing 6 mM glucose with or without 10 mU/ml insulin for 30 min. At the end of these incubations, muscles were blotted with a piece of gauze and then quick-frozen in liquid nitrogen. They were stored at -80°C before subsequent measurements were performed.

Measurement of glucose uptake and glucose disposition. Deoxy-[3H]glucose uptake and [U-14C]glucose incorporation into glycogen and lipid and oxidation to CO2 and lactate release were measured as described previously (18).

Tissue extraction for PKC studies. The method used for tissue extraction and PKC solubilization is modified from Schmitz-Peiffer et al. (29). Muscles (25-35 mg) were homogenized and extracted in 500 µl 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 µg/ml leupeptin, 4 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 2 mM diisopropyl fluorophosphate, 2.5 µg/ml pepstatin A, 10 mM sodium fluoride (NaF), and 1 mM beta -glycerophosphate. A portion of the homogenate (200 µl) was centrifuged at 140,000 g for 1 h at 4°C, and the supernatant (cytosolic fraction) was removed and stored in liquid nitrogen. The pellet was resuspended in 200 µl of homogenizing buffer containing 0.2% (wt/wt) of the detergent decanoyl-N-methyl-glucamide, known as MEGA-10. After sitting for 1 h at 4°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.

PKC immunoblotting and activity assay. Protein was determined with the detergent-tolerant protein assay kit Bio-Rad DC (Hercules, CA). Equal amounts (20-30 µg protein) of the cytosol and membrane fractions from each muscle were subjected to 10% SDS-PAGE by the method of Laemmli (24). Protein was transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) with a semi-dry transfer apparatus following a method outlined by the manufacturer (Owl Scientific, Cambridge, MA). After transfer, the nitrocellulose membranes were blocked with 5% nonfat dried milk in a buffer of 1.5 M NaCl and 50 mM Tris, pH 7.4, containing 0.05% Tween-20 (TBS-T) overnight at 4°C. The membranes were probed with isozyme-specific antibodies against PKC (polyclonal Ab: alpha , beta I, beta II, gamma , epsilon , theta , delta , eta , zeta , and iota ; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. After 4× 5-min washes in TBS-T buffer, membranes were incubated with horseradish peroxidase-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).

In some studies, muscles incubated to determine the effects of PDBu and calphostin C on glycogen synthesis were homogenized as described above and fractionated into cytosol and total particulate fractions for measurement of PKC activity. Activity was measured using the Biotrak System from Amersham. Given that we wished to measure only intrinsic PKC activity, peptide substrate, but no PKC activators (phosphatidylserine and phorbol ester), was added to the assay samples. After phosphorylation, substrate peptides were spotted on filter disks, washed, and counted as previously outlined (19).

Akt/PKB immunoprecipitation and activity assay. The processing of muscles used to assay Akt/PKB activity was as described previously (23). 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 2 h. The beads were then washed, and activity was assessed by the phosphorylation of the substrate peptide "crosstide" (Upstate Biotechnology, Lake Placid, NY).

IR, Akt, and GSK3 Western blot. Muscles were weighed (average 25-35 mg) and homogenized in 1 ml of ice-cold buffer containing 250 mM sucrose, 20 mM Tris (pH 7.4), 10 mM NaF, 5 mM EDTA, 2 mM Na3VO4, 2 mM NH4MbO4, 1 mM beta -glycerophosphate, 1 mM DTT, 1 µM microcystin-LR, and 10 µl/ml Protease Inhibitor Cocktail P8340. Igepal CA630 was added to each sample to achieve a final concentration of 1% (vol/vol), and the sample was solubilized for 1.5 h at 4°C. The resultant lysate was centrifuged at 15,000 g for 5 min, and the supernatant was used in Western blot assay of IR, Akt, GSK3, and various anti-phosphorylated residue antibodies.

PI3K assay. Muscle lysates were incubated with an antibody against IRS-1 and the immunocomplex precipitated by adding trisacryl protein A beads (Pierce). PI3K was assayed according to Kelly et. al. (20).

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preincubation with phorbol ester impairs insulin-stimulated glycogen synthesis. To assess the effect of PKC activation on insulin action, rat soleus muscles were incubated with phorbol esters. As shown in Fig. 1, preincubation with the PKC activators PMA and PDBu, but not the inactive phorbol ester 4-alpha phorbol, significantly inhibited the ability of insulin to stimulate glycogen synthesis. Figure 2 shows that maximal inhibition of the response to insulin by 1 µM PDBu was evident after 1 h of preincubation, whereas a lower concentration of 0.1 µM required 2 h to demonstrate any effect. At these same time points, the phorbol ester did not have an effect on this parameter in the absence of insulin.


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Fig. 1.   Effect of preincubation with phorbol esters on insulin-stimulated glycogen synthesis. Soleus muscles were preincubated for 60 min in a control medium (0.1% DMSO) or a medium containing a phorbol ester. Incorporation of [14C]glucose into glycogen was then measured over 20 min in the presence of 10 mU/ml of insulin and 6 mM glucose. PMA, phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate. Values are means ± SE; n = 4. *P < 0.05.



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Fig. 2.   Time course and dose response of PDBu incubation of insulin (Ins)-stimulated glycogen synthesis. Soleus muscles were preincubated with different concentrations of PDBu for the indicated time periods. The incorporation of [14C]glucose into glycogen in the presence and absence of insulin was determined as described in Fig. 1. Values are means ± SE; n = 4. *P < 0.05.

Incubation with PDBu does not inhibit glucose uptake. In contrast to glycogen synthesis, PDBu had no effect on insulin-stimulated 2-deoxyglucose uptake, glucose incorporation into total lipid, or oxidation to CO2. PDBu treatment also did not alter these parameters in the absence of insulin (Fig. 3).


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Fig. 3.   Effect of preincubation with PDBu on glucose disposition. Soleus muscles were preincubated, and [14C]glucose incorporation into glycogen and total lipid and its oxidation into CO2 were determined subsequently, as described in legend to Fig. 1. Uptake of 2-deoxy-D-[1,2-3H]glucose was determined under similar conditions in a separate experiment (see MATERIALS AND METHODS). Values are means ± SE; n = 6. *P < 0.01.

Impairment of insulin-stimulated glycogen synthesis by PDBu is partially reversed by preincubation with calphostin C. Preincubation with calphostin C, a PKC inhibitor that competes with phorbol ester at the diacylglycerol binding site (IC50 of 50 nM) (5, 21), had no effect on glycogen synthesis in either the presence or the absence of insulin (Fig. 4). On the other hand, it partially (30~40%) reversed the inhibitory effect of 1 µM PDBu on insulin-stimulated glycogen synthesis.


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Fig. 4.   Effect of preincubation with calphostin C on the impairment of insulin-stimulated glycogen synthesis induced by PDBu. Soleus muscles were preincubated for 20 min in 6 mM glucose and then for 60 min in 6 mM glucose supplemented with either DMSO (final concentration 0.1%) or calphostin C. They were then incubated for 1 h in a medium containing PDBu and 6 mM glucose, washed for 2 min in a glucose-free medium, and then incubated for 30 min with a medium containing 6 mM glucose and [14C]glucose. Values are means ± SE; n = 4-5. *P < 0.05.

Distribution and activity of PKC between cytosolic and membrane fractions. As shown in Fig. 5, decreases in PKC-alpha , -beta I, -beta II, -epsilon , and -theta , but not -zeta , were observed in the cytosol after preincubation with PDBu in both the presence and the absence of insulin. In the case of PKC-alpha , -beta I, -epsilon , and -theta (but not -beta II or -zeta ), this was associated with an increase in membrane-associated isoforms. PKC-theta also showed an additional band in the membrane fraction after incubation with PDBu. Calphostin C competes with phorbol ester at the C1 binding site of PKC. As is also shown in Fig. 5, the distribution of PKC isoforms was not affected by calphostin C alone, and surprisingly, calphostin C did not reverse the translocation of PKCs caused by PDBu.


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Fig. 5.   Protein kinase C (PKC) distribution in soleus muscles preincubated with PDBu and/or calphostin C. Muscles were incubated as described in MATERIALS AND METHODS. After the incubation, muscles were quickly frozen in liquid nitrogen and stored at -80°C. At a later time, they were homogenized and fractionated. Equal amounts of protein from each fraction were loaded and separated by 8% SDS-PAGE. After transfer to polyvinylidene difluoride (PVDF), membranes were immunoblotted with antibodies to specific PKC isoforms. Actin was used as a control. Each blot shown is representative of 1 experiment that was repeated 4 times.

PKC activity in the membrane and cytosol fractions from these muscles was assayed without the addition of activators (see METHODS). As shown in Fig. 6, kinase activity in the cytosol was not altered by incubation with either PDBu or PDBu plus calphostin in muscles incubated with insulin (Fig. 6A). In contrast, activity in the membrane fraction was increased by incubation with PDBu, and this increase in activity was totally inhibited by calphostin C (Fig. 6B).


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Fig. 6.   PKC activity in cytosol (A) and membrane (B) fractions of soleus muscle preincubated with PDBu and/or calphostin C. The intrinsic activity of PKC in the 2 fractions was assayed in the absence of activators (phosphatidylserine and phorbol ester), as described in MATERIALS AND METHODS and legend to Fig. 4. Values are means ± SE; n = 4. *P < 0.05.

Alterations in the insulin-signaling cascade induced by PDBu. To identify a defect in insulin signaling caused by PDBu, several signaling molecules thought to be involved in insulin action were examined. As shown in Fig. 7A, the abundance of Akt/PKB was not altered by PDBu; however, the gel shift of Akt/PKB caused by insulin, as well as its phosphorylation on threonine 308 and serine 473 and the insulin-stimulated increase in its activity, was substantially depressed by PDBu (Fig. 7, A-D).


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Fig. 7.   Effect of preincubation with PDBU on the abundance, phosphorylation, and activity of Akt/PKB in soleus muscle. After incubation, as described in legend to Fig. 4 (except no calphostin C), preincubated muscles were quickly frozen and stored at -80°C. At a later time, they were homogenized in Akt/PKB buffer, and the 15,000-g supernatant was collected (see MATERIALS AND METHODS). A: equal amounts of protein were separated on 8% SDS-polyacrylamide gels, and the gels were transferred to a PVDF membrane. The membrane was blotted with Akt/PKB antibody. B and C: after stripping, the membrane was probed with anti-phospho-Thr308 Akt/PKB and anti-phospho-Ser473 Akt/PKB antibody. D: supernatant was immunoprecipitated (IP) with Akt/PKB antibody and conjugated to beads, and Akt/PKB activity was determined using crosstide as substrate (see MATERIALS AND METHODS). Each point represents means ± SE of 5 muscles. *P < 0.05.

As shown in Fig. 8, preincubation with PDBu did not alter the abundance of the insulin receptor, but it significantly decreased its tyrosine phosphorylation (P < 0.05). The phosphorylation of GSK3-alpha and -beta , downstream molecules of Akt/PKB, was also depressed by PDBu. As with the insulin receptor and Akt/PKB, no change in GSK3 abundance was observed (Fig. 9). PI3K activity tended to be diminished; however, this was not statistically significant (Fig. 10).


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Fig. 8.   Effect of preincubation with PDBu on tyrosine phosphorylation of the insulin receptor (IR). Muscles incubated as described in Fig. 7 were quickly frozen and stored in -80°C. Muscles were homogenized, and the 15,000-g supernatant was collected. A: equal amounts of protein were separated on 6% SDS-polyacrylamide gels, and the gel was transferred to a PVDF membrane, which was blotted with anti-IR antibody. At left: molecular mass markers. B: after membrane was stripped, it was probed with anti-phosphotyrosine antibody (representative blot in insert). Bars represent means ± SE of 5 muscles. *P < 0.05.



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Fig. 9.   Effect of preincubation with PDBu on glycogen synthase kinase-3 (GSK3) phosphorylation. Muscles were incubated and processed as described in legend to Fig. 7. They were homogenized in GSK3 buffer, and the 15,000-g supernatant was collected. A and B: immunoblots of GSK3-alpha and GSK3-beta , respectively, after separation on 10% SDS-polyacrylamide gels. At left, molecular mass markers. C: immunoblots of GSK3 phosphorylated on Ser9 and Ser21 (top). Each bar (middle and bottom) represents mean ± SE of 4 muscles. *P < 0.05.



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Fig. 10.   Effect of preincubation with PDBu on phosphatidylinositol 3-kinase (PI3K) activity. Muscles were incubated and processed as described in legend to Fig. 7. PI3K activity was assayed in an insulin receptor substrate-1 (IRS-1) immunoprecipitate from the muscle lysate. Results are means ± SE; n = 5.

Effect of PDBu on glucose uptake is dose dependent. Because there was no change in insulin-stimulated glucose uptake and PI3K activity in muscles incubated with 1 µM PDBu, we next examined the effect of incubating muscle with a higher concentration of PDBu. As shown in Fig. 11, a small but significant decrease in insulin-stimulated 2-deoxyglucose uptake (20%, P < 0.01) was observed in soleus incubated with 3 µM, but not 1 µM, PDBu. In contrast, insulin-stimulated glycogen synthesis was similarly depressed at the two PDBu concentrations.


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Fig. 11.   Effect of different concentrations of PDBu on glucose uptake and glucose disposition. Muscles were incubated and processed as described in legend to Fig. 3. A: glucose uptake; B: incorporation of [14C]glucose into glycogen; C: conversion of [14C]glucose into lactate. See MATERIALS AND METHODS for details. Values are means ± SE; n = 5. *P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The key findings in this study are as follows. First, insulin action was selectively inhibited in muscles preincubated with PDBu. Insulin-stimulated glycogen synthesis was diminished, but 2-deoxyglucose uptake and glucose incorporation into total lipid and its oxidation to CO2 were unaffected. Second, the impairment of insulin-stimulated glycogen synthesis induced by PDBu was associated with changes in the distribution of PKC-alpha , -beta I, -beta II, -theta , and -epsilon and with decreases in the ability of insulin to phosphorylate the insulin receptor, Akt/PKB, and GSK3 and to increase Akt/PKB activity.

Previous studies of the effects of preincubation with phorbol esters on insulin-stimulated glucose transport in rat skeletal muscle have yielded variable results (Table 1). Thus it was shown that PMA stimulates 3-methylglucose transport in rat epitrochlearis muscle both in the absence of insulin (17) and in the presence of insulin at a submaximal concentration (16), whereas at a supraphysiological insulin concentration it had no effect (16). Similar findings were observed in the extensor digitorum longus muscle (14). In contrast, TPA (33) (the same compound as PMA) and PDBu (this study) had minimal if any effect on 2-deoxyglucose uptake in rat soleus muscle in either the presence or the absence of insulin. Incubation with another phorbol ester, 12-deoxyphorbol 13-phenylacetate 20-acetate, caused a three- to fourfold increase in 3-methylglucose uptake both in the presence of insulin at submaximal and maximal concentrations and in its absence (16). Collectively, these results suggest that the disparate findings in the literature may be related to differences in the type of muscle and phorbol ester used and in experimental design. Also, in general they suggest that phorbol ester effects on glucose transport are most evident in muscles incubated either in the absence of insulin or when insulin was present at a physiological concentration. To our knowledge, the effects of phorbol esters on insulin-stimulated glycogen synthesis in rat muscle were not examined in any of these studies.

                              
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Table 1.   A comparison: effect of preincubation with phorbol esters on glucose metabolism in skeletal muscle

Our results indicate that preincubation with PDBu leads to a selective inhibition of insulin-stimulated glycogen synthesis. Prior studies bear only indirectly on this finding. Sowell et al. (33) also observed an inhibition of glycogen synthesis, but not glucose transport, in rat soleus muscle; however, these experiments were carried out only in the absence of insulin. Likewise, Avignon et al. (2) reported that RO 31-8220, a PKC inhibitor, increases glycogen synthesis, but not glucose uptake, in both the presence and the absence of insulin in incubated rat soleus muscle. In addition, they found that RO 31-8220 reversed the impairment of insulin-stimulated glycogen synthesis in soleus muscle of the GK rat but had no effect on glycogen synthesis in the absence of insulin (2). In general, these observations suggest that PKC is a negative regulator of the glycogen synthetic pathway, even when it does not affect glucose transport.

The molecular mechanism by which PKCs inhibit insulin-stimulated glycogen synthesis remains to be determined. One possibility is that activation of PKC (e.g., by a phorbol ester) directly leads to phosphorylation and inhibition of glycogen synthase, as has been reported to occur in other tissues (1, 3). Against this notion, however, we found that PDBu caused no inhibition of glycogen synthesis in soleus muscle incubated in the absence of added insulin (Fig. 3), although, as noted in the preceding paragraph, others have observed such an effect. Another possibility is that PDBu acts by inhibiting one or more steps in the insulin-signaling cascade. In keeping with such a mechanism, preincubation with this phorbol ester decreased the ability of insulin to phosphorylate Akt/PKB (90%) and GSK3 (35%) and to increase Akt/PKB activity (inhibited by 50%). In contrast, it failed to diminish the activation of PI3K by insulin and diminished insulin receptor tyrosine phosphorylation by only 30%. Thus, at the high concentration of insulin used, activation of the insulin receptor tyrosine kinase, although somewhat diminished in muscles incubated with PDBu, was sufficient to allow a normal activation of PI3K. The normal activation of PI3K by insulin in solei incubated with PDBu was surprising, because Akt/PKB phosphorylation and activity were diminished, and PI3K is generally thought to be one of its upstream activators. A similar dissociation between PI3K and Akt activation by insulin in muscle has been observed in both EDL of the GK rat (32) and EDL of control rats preincubated in a hyperglycemic medium (not containing insulin) for 2-4 h (23). A noteworthy finding in both of these studies was that the ability of insulin to stimulate glucose incorporation into glycogen was impaired; however, as in the present study, insulin-stimulated glucose transport was maintained. What accounts for the dissociation of PI3K and Akt/PKB in these situations remains to be determined, as does the reason inhibition of Akt/PKB is associated with inhibition of glucose incorporation into glycogen but not glucose transport into the muscle cell. This could occur if Akt/PKB is involved in the regulation by insulin of glycogen synthesis but not glucose transport. Early studies with cultured cells (15, 22, 34) suggested that Akt/PKB plays a key role in mediating insulin-stimulated glucose transport; however, this has recently been questioned (13). Alternatively, insulin-stimulated glycogen synthesis could simply be more sensitive to inhibition of the insulin receptor tyrosine kinase and Akt than are glucose transport and lipid synthesis, and/or it may simply be an earlier event in the evolution of insulin resistance in the muscle cell. The fact that both glucose transport and glycogen synthesis are inhibited concurrently in a wide variety of in vivo models in which insulin signaling at the level of its receptor and Akt is impaired (30, 35) is consistent with this notion.

On the basis of immunoblots with specific antibodies, preincubation with PDBu caused a translocation of PKC-alpha , -beta I, -theta , -epsilon , and possibly -beta II, from the cytosol to a membrane fraction (Fig. 5). Whether one, some, or all of these isoforms mediated the observed changes in insulin signaling and action or whether the alterations in PKC distribution are an epiphenomenon remains to be determined. Studies in other insulin-resistant muscles do not answer this question. Changes in the distribution of PKC-epsilon and -theta , but not other isoforms, have been observed in skeletal muscle of intact rats in a number of insulin-resistant states, including those induced by fat feeding (29), fructose ingestion (10), and glucose infusion (25). In contrast, alterations in PKC-alpha , -beta , -epsilon , and -delta have been described in muscle of fa/fa and GK rats (2). In rats made insulin resistant by raising plasma free fatty acid levels during a euglycemic hyperglycemic clamp, altered PKC-theta distribution has been reported (12). In denervated muscle, in which, as in PDBu-treated muscle, the ability of insulin to stimulate glycogen synthesis but not glucose transport is impaired, PKC-theta and, to a lesser extent, PKC-epsilon , were altered (26). For the most part, these reports suggest that changes in the distribution of novel PKC isoforms correlate most closely with the development of insulin resistance in skeletal muscle.

Incubation with calphostin C partially prevented the impairment of insulin-stimulated glycogen synthesis caused by PDBu (Fig. 4). Interestingly, calphostin C did so without reversing the effect of the phorbol ester on PKC isoform intracellular translocation (Fig. 5), but it totally inhibited the activation of membrane-associated PKC activity caused by PDBu (Fig. 6). Furthermore, it did so without affecting PKC activity in the cytosol. Given its lipophilic nature, these findings suggest that calphostin C taken up by the muscle was predominantly restricted to the membrane compartment during the period of incubation. The apparent absence of calphostin C in the cytosol could account for its failure to affect PKC translocation. It could also explain why it only partially reversed the effect of PDBu on insulin-stimulated glycogen synthesis.

That translocation/activation of one or more PKC isoforms can lead to insulin resistance has been suggested by studies in tissues other than skeletal muscle, including murine adipocytes (11), fibroblasts (4), vascular cells, and heart (27). In addition, multiple insulin-resistant states associated with altered PKC distribution or activity have been observed in muscles of both experimental animals (see review in Ref. 28) and humans (9). How these findings relate to the observations of PKC in the present study remains to be determined.

In conclusion, our results show that insulin-stimulated glycogen synthesis, but not glucose uptake, is impaired in soleus muscle preincubated with 1 µM PDBu. This impairment of insulin action was associated with alterations in the distribution of PKC-alpha , -beta I, -beta II, -theta , and -epsilon , but not -zeta , and with decreased tyrosine phosphorylation of the insulin receptor. In addition, insulin-induced phosphorylation of Akt/PKB and GSK3 was depressed. The data strongly suggest that translocation and/or activation of one or more PKC isoforms causes defects in insulin signaling that lead to a selective impairment of insulin-stimulated glycogen synthesis.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-49147 and DK- 19514.


    FOOTNOTES

Address for reprint requests and other correspondence: N. B. Ruderman, Chief, Diabetes and Metabolism Research Unit, Boston Medical Center, 650 Albany St., EBRC Rm. 820, Boston, MA 02118 (E-mail: nruderman{at}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.

Received 17 August 2000; accepted in final form 15 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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