Effects of Insulin on DiacylglycerolProtein Kinase C Signaling in Rat Diaphragm and Soleus Muscles and Relationship to Glucose Transport TATSUO ISHIZUKA, DENISE R. COOPER, HERMAN HERNANDEZ, DONNA BUCKLEY, MARY STANDAERT, AND ROBERT V. FARESE

Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulininduced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3HI myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts

From the James A. Haley Veterans Administration Hospital and Departments of Internal Medicine and Biochemistry, University of South Florida College of Medicine, Tampa, Florida. Address correspondence and reprint requests to Robert V. Farese, MD, Research Service (151), J.A. Haley Veterans Administration Hospital, 13000 Bruce Downs Boulevard, Tampa, FL 33612. Received for publication 20 April 1989 and accepted in revised form 29 September 1989.

DIABETES, VOL. 39, FEBRUARY 1990

independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues. Diabetes 39:181-90,1990

I

nsulin has been found to increase diacylglycerol (DAG) production and provoke changes in protein kinase C (PKC) in BC3HI myocytes (1-3), rat adipose tissue (2,4,5), and rat hepatocytes (unpublished observations). In the BC3HI myocyte, insulin has been found to increase DAG, both by increased de novo synthesis of phosphatidic acid (PA; 6,7), which may be directly converted to DAG (6,7), and by hydrolysis of phospholipids, including a phosphatidylinositol (Pl)-polysaccharide complex (8,9) and phosphatidylcholine (PC; 7,10) but not phosphatidylinositol 4',5'-bisphosphate (2). In BC3H1 myocytes (11,12) and adipocytes (13-15), the effects of insulin on DAG and PKC may contribute to insulin effects on glucose transport. However, skeletal muscle is the major tissue in which insulin increases glucose utilization in vivo (16), and there are few reported studies of insulin effects on DAG-PKC signaling systems in this tissue. Walaas et al. (17) reported that insulin activates PKC in the rat diaphragm, but the cause of this activation is uncertain as is its importance for insulin effects on glucose transport. Indeed, Sowell et al. (18) observed little or no stimulatory effects of phorbol esters and exogenously added DAGs on 2-deoxyglucose (2-DOG) uptake in both rat diaphragm and soleus muscles and suggested that DAG-PKC signaling may not be important in insulin regulation of glucose transport in these tissues. In this study, we 1) examined the effects of insulin on DAG production as a cause for PKC activation in the rat diaphragm and soleus muscles, 2) examined effects of insulin on PKC activation in the rat soleus

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muscle, and 3) reassessed the effects of DAG-PKC activation on stimulation of 2-DOG uptake in the rat diaphragm and soleus muscles along with rat adipocytes and BC3H1 myocytes. RESEARCH DESIGN AND METHODS Skeletal muscle experiments. Male Holtzman rats, weighing -100-150 g, were fed ad lib and killed by decapitation. Diaphragms attached to adjacent rib cages were excised, and each diaphragm was cut into two hemidiaphragms, one serving as a control and one for treatment with insulin, 12O-tetradecanoylphorbol-13-acetate (TPA), or phospholipase C. Soleus muscles were excised, and tension was maintained by ligatures attached to the tendons: the two soleus muscles from each rat provided one control and one stimulated sample. Diaphragms and soleus muscles were incubated at 37°C in 25-ml Erlenmeyer flasks under 95% O2/5% CO2 in 5 ml of medium 199 (M-199), containing 117 mM NaCI, 1 mM NaH2PO4, 0.8 mM MgSO4, 26 mM NaHCO3, 4 mM KCI, and 1 mM CaCI2, or Krebs-Ringer bicarbonate buffer (KRBB), containing 119 mM NaCI, 4.8 mM KCI, 1 mM NaH2PO4,1.2 mM MgSO4,1 mM CaCI2, and 24 mM NaHCO3, each containing 12 mM HEPES (pH 7.4), 0.1 % bovine serum albumin (BSA), 5.8 mM glucose, and 2 mM sodium pyruvate, unless specified otherwise. For most lipid experiments, diaphragms or soleus muscles were incubated for 30 or 60 min in M-199 or KRBB with 10 or 20 ixCi [2-3H]glycerol (final concn 0.2 or 0.4 pM, respectively), insulin was then added, and incubation was continued for 1-10 min. In some lipid experiments, after a 30- to 60-min equilibration in M-199 or KRBB, [3H]glycerol was added simultaneously with insulin during the final treatment period (2-10 min). After incubation, the tissues were removed (hemidiaphragms were rapidly dissected from the rib cage), blotted, weighed, and homogenized (Brinkman Polytron) or sonicated in CHCI3/CH3OH/H2O (2:1:1 vol/vol/vol). Lipid extracts were washed three times with H2O, and aliquots were chromatographed on thin-layer chromatography plates to separate neutral lipids and phospholipids as described previously (2,6). DAG content of lipid extracts was determined by the DAG kinase method of Preiss et al. (19). For [1,2-3H]-2-DOG-uptake experiments, diaphragms and soleus muscles were first incubated for 30 min in M-199 or KRBB, each containing 5.8 mM glucose, and then incubated for 30 min in either glucose-containing M-199 or glucose-free KRBB, with or without (controls) insulin, TPA, or phospholipase C. [3H]-2-DOG (VCi), L-[1- 14 C]glucose (0.1 jxCi), and unlabeled 2-DOG (0.1 mM) were then added, and incubation was continued for 10 min (occasionally 30 min where specified). After incubation, tissues were removed, rapidly rinsed in isotope-free medium (diaphragms were dissected free of bone and connective tissue), blotted, weighed, homogenized in 5% trichloroacetic acid, and counted simultaneously for 3H and 14C at 55 and 90% efficiencies, respectively. Corrections for [3H]-2-DOG in tissue samples unrelated to specific transport were determined by measurement of radioactivity of L-['"C]glucose. Uptake of [3H]-2-DOG in both tissues was linear with respect to time (up to 30 min) and was inhibited by cytochalasin B. In PKC experiments, rat soleus muscles were incubated in glucose-free KRBB (as in [3H]-2-DOG experiments) and

182

treated for 10 min with or without insulin. In each experiment, four controls or insulin-treated muscles were pooled, and PKC enzyme activity or immunoreactivity was determined in cytosol and membrane fractions as described below. Adipocyte experiments. Adipocytes were obtained by collagenase digestion of rat epididymal fat pads (20) in KrebsRinger phosphate buffer (pH 7.4) containing 127 mM NaCI, 12.3 mM NaH2PO4, 5.1 mM KCI, 1.3 mM MgSO4, 1.4 mM CaCI2, 3% BSA, and 2.5 mM glucose. Adipocytes were washed and preincubated at 37°C in glucose-free KrebsRinger phosphate buffer containing 1% BSA for 30 min and then incubated with or without 100 |xg/ml 1,2-dioleoyl-snglycerol (diolein) and other additions as indicated. [3H]-2DOG (0.08 jjtCi) and unlabeled 2-DOG (0.05 mM) were then added to a 6% (cell vol/total cell plus buffer vol) adipocyte suspension (final vol 300 |xl), and uptake of [3H]-2-DOG was measured over 1 min (21). BC3H1 myocyte experiments. As described previously ( 1 3,12), BC3H1 myocytes were grown in collagen-coated, 24well cluster plates (Costar) for 10-13 days and supplemented with 25 mM glucose 24-48 h before assay. Cells were treated with 100 (xg/ml 1,2-dioctanoyl-sn-glycerol (DiC8) or 0.5 U/ml phospholipase C and other substances as indicated for 20 min, after which the 6-min uptake of [3H]2-DOG was measured as described (12). PKC studies. Rat soleus muscle or epididymal adipose tissue was homogenized with a polytron homogenizer in 20 mM Tris-HCI buffer (pH 7.5) containing 0.25 M sucrose, 1.2 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 20 |xg/ml leupeptin, and 20 mM 2-mercaptoethanol. The homogenates were centrifuged for 60 min at 100,000 x g to obtain cytosol and membrane fractions. The latter was homogenized in buffer 1 containing 5 mM EGTA, 2 mM EDTA, and 1% Triton X-100. To measure PKC enzymatic activity of the rat soleus muscle, cytosol or solubilized membrane fractions were diluted fourfold with buffer 1, which contained 20 mM Tris-HCI buffer (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol. The sample was then applied to a Mono Q column (0.5 x 5 cm, Pharmacia HR 5/5, Piscataway, NJ) that was connected to a Pharmacia fast-protein liquid-chromatography system that had been equilibrated with buffer 1. PKC was eluted by application of a 20-ml linear concentration gradient of NaCI (0-0.7 M) in buffer 1 at a flow rate of 1 ml/min. Fractions of 1 ml were collected, and PKC activity of each fraction was assayed by measuring phosphorylation of histone Ill-S as described previously (3), except that 0.8 |xg/ml diolein was present in the assay mixtures. Activation of PKC in rat soleus muscles and adipocytes was also assessed by changes in the subcellular distribution (i.e., apparent translocation) of immunoreactive PKC with methods described previously (22). Equal amounts of cytosol or membrane fractions from control and stimulated soleus muscles or adipocytes were prepared as described above and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and incubated first with polyclonal antiserum raised to synthetic peptide specific to type II PKC (kindly supplied by J. Mehegan and B. Roth, Naval Medical Res. Inst., Bethesda, MD; 23) and second with goat anti-rabbit 7globulin complexed to horseradish peroxidase or alkaline

DIABETES, VOL. 39, FEBRUARY 1990

T. 1SHIZUKA AND ASSOCIATES

phosphatase. As reported (22), this immunoblotting method detected a single major immunoreactive band that comigrated on SDS-PAGE and blotted identically with purified rat brain 80,000-Mr PKC. In addition, we have validated the use of type II PKC antiserum for studies of rat soleus muscle and epididymal adipose tissue by finding that type II PKC is the major PKC isozyme present in these tissues, as shown by chromatography on hydroxyapatite columns (24; unpublished observations). [2-3H)glycerol (sp act 5 Ci/mmol) and [1,2-3H]-2-DOG (sp act 50 Ci/mmol) were obtained from ICN (Lisle, IL). L[1-14C]glucose (sp act 47 mCi/mmol) was obtained from Du Pont-NEN (Boston, MA). Highly purified insulin was purchased from Elanco (Indianapolis, IN). M-199 was obtained from Gibco (Grand Island, NY). Collagenase (type I) was obtained from Worthington (Freehold, NJ). DiC8 was purchased from Life Science (Milwaukee, Wl). Staurosporine was obtained from Boehringer Mannheim (Indianapolis, IN). 1-(5-lsoquinolinesulfonyl)-2-methypiperazine (H 7) was obtained from Seikagaki (St. Petersburg, FL). DAG kinase was purchased from Lipidex (Westfield, NJ). TPA, histone Ill-S, radioimmunoassay-grade BSA, diolein, phospholipids, phospholipase C (type IX, Clostridium perfringens), and other biochemicals were obtained from Sigma (St. Louis, MO). RESULTS RAT DIAPHRAGM DAG production. In diaphragms that were prelabeled during a preliminary incubation for 60 min with [3H]glycerol in glucose-containing M-199, subsequent addition of insulin provoked a rapid increase in labeling of DAG, which reached a level - 3 5 % above that of the controls within 10 min of stimulation (Fig. 1). (Note that there was considerable labeling of lipids during the 60-min prelabeling period, medium glucose may decrease glycerol-3-phosphate specific radioactivity, especially in insulin-treated cells, and the observed 35% increase in overall DAG labeling reflects a much greater increase in the rate of labeling of DAG.) Stimulatory effects of insulin on labeling of total phospholipids (PL) and triacylglycerol (TAG) were also observed, and it may be surmised that total glycerolipid labeling was increased by insulin. (Monoacylglycerol labeling was extremely low, and results are not shown.) Experiments were also conducted in which hormone and [3H]glycerol were added simultaneously to avoid extensive labeling of DAG precursors, e.g., PI and PC. In this case, PA and DAG were labeled more rapidly than other lipids, and insulin stimulated this labeling at each time point (Fig. 2). Except for a transient increase at 2 min in the labeling of combined PC and phosphatidylethanolamine (PE; note that - 9 0 % of this label was in PC and 10% in PE), labeling of other lipids was not altered significantly by insulin during the course of a 10-min incubation, but total glycerolipid labeling was nevertheless increased. In addition to increases in [3H]glycerol labeling of DAG, insulin also provoked increases in DAG content during incubations of the rat diaphragm (Table 1). Increases of —40% were evident at 2 and 10 min of insulin treatment. Glucose transport. [3H]-2-DOG uptake in rat diaphragms

DIABETES, VOL. 39, FEBRUARY 1990

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MINUTES OF TREATMENT FIG. 1. Effects of insulin on [3H]glycerol labeling of diacylglycerol (DAG), total phospholipids (PL), and triacylglycerol in prelabeled rat diaphragms. Hemidiaphragms were first incubated with 20 jiCi pHJglycerol for 60 min in medium 199 and then treated with 100 nM insulin in buffer (INS) or buffer alone (CON) for 2, 5, or 10 min. Values are means ± SE of 16-18 determinations, cpm, Counts per minute. •P < 0.05 by t test.

was measured with two buffer systems, i.e., M-199, which contained 5.8 mM glucose (Table 2), and glucose-free KRBB (Table 3). In M-199, [3H]-2-DOG uptake proceeded linearly or near linearly for 30 min of incubation (Fig. 3). (Similar linearity was reported by Sowell et al. [18] with glucose-free Gey's medium.) Maximally effective doses of 100 nM insulin and 500YiM TPA provoked increases in the 10-min uptake

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FIG. 2. Effects of insulin on incorporation of simultaneously added [3H]glycerol into phosphatidic acid (PA), diacylglycerol (DAG), phosphatidylinositol (PI), monoacylglycerol (MAG), combined phosphatidylcholine/phosphatidylethanolamine (PC/PE), and triacylglycerol (TAG) in rat diaphragms. After preincubation for 60 min in medium 199, 20 jiCi [3H]glycerol and either 100 nM insulin in buffer ( • ) or buffer alone (O) were added simultaneously, and incubation was continued for 2, 5, or 10 min. Values are means ± SE of 4 determinations, cpm, Counts per minute.

of [3H]-2-DOG of - 5 2 and 29%, respectively, above control values (Table 2). At concentrations of 5 and 50 nM, TPA provoked similar increases in [3H]-2-DOG uptake, i.e., -25%, but lower concentrations were not effective (data not shown). Addition of 500 nM TPA with 100 nM insulin did not increase [3H]-2-DOG uptake above that observed with 100 nM insulin alone (data not shown).

In diaphragm experiments in which glucose-free KRBB was used, insulin increased [3H]-2-DOG uptake by 77% (Table 3). On the other hand, TPA effects on [3Hp2-DOG uptake were considerably less (mean ± SE increase above control, 8 ± 1%) in glucose-free KRBB than in glucose-containing M-199 (mean ± SE increase above control, 30 ± 7%; P < 0.005 vs. glucose-free KRBB by standard t test). Similar differences in effects of insulin and TPA on 2-DOG uptake were observed by Sowell et al. (18), who used glucose-free Gey's medium in their [3H]-2-DOG-uptake studies. To further evaluate a potential role for DAG during activation of [3H]-2-DOG uptake, phospholipase C was used to generate increases in cellular DAG from endogenous phospholipids (in agreement with results reported by Sowell et al. [18], we were unable to stimulate [3H]-2-DOG uptake by exogenous addition of diolein, DiC8, or 1-oleoyl-2-acetyl-snglycerol to intact muscle cells). As shown in Table 2, phospholipase C stimulated [3H]-2-DOG uptake in the rat diaphragm by 154%. This effect of phospholipase C was apparent even in the presence of high concentrations of protease inibitors (e.g., 100 (xg/ml leupeptin) and did not appear to be explained by insulinlike effects of proteases that theoretically might contaminate the phospholipase C preparation (data not shown). To evaluate the role of PKC during insulin-stimulated glucose transport in the rat diaphragm, two inhibitors of PKC, staurosporine (25) and polymixin B (26), were used in concentrations that were found to be effective in rat adipocytes and BC3H1 myocytes (unpublished observations) and mouse soleus muscle (26), respectively. (Note that extensive dose-response studies were not conducted in rat skeletal muscles because of difficulties in preparing large numbers of tissue samples for direct comparison in each experiment.) Both inhibitors largely inhibited insulin-stimulated [3H]-2DOG uptake. These inhibitors also decreased basal [3H]-2DOG uptake in some but not all cases, suggesting that this activity may also be variably and mildly stimulated by a PKCdependent mechanism. RAT SOLEUS MUSCLE DAG production. As in the rat diaphragm, insulin rapidly stimulated [3H]glycerol labeling of DAG, PL, and TAG in rat soleus muscles that had been prelabeled during a 30-min incubation with [3H]glycerol (Fig. 4). (Monoacylglycerol la-

TABLE 1 Effects of insulin on diacylglycerol content (nm/100 mg tissue wet wt) of rat diaphragm and soleus muscles Diaphragm* Incubation 2 Min 10 Min

Soleus musclef

Control

Insulin

Increase (%)

Control

Insulin

Increase (%)

7.12 ± 0.76 6.53 ± 0.53

9.20 ± 0.93 8.52 ± 0.60||

39 ± 10* 40 ± 13H

15.10 ± 0.89 21.03 ± 0.91

18.31 ± 0.71§ 26.73 ± 1.26#

22 ± 7 27 ± 3*

Hemidiaphragms or soleus muscles were equilibrated for 30 min in medium 199 or glucose-free Krebs-Ringer bicarbonate buffer (see RESEARCH DESIGN AND METHODS), respectively, and then treated for 2 or 10 min with vehicle alone (controls) or 100 nM insulin in vehicle. Diacylglycerol mass was determined as described in RESEARCH DESIGN AND METHODS. In each case, 1 control and 1 insulin-treated muscle

were directly compared, and data are expressed as means ± SE of both absolute values and insulin-induced percentage changes. *n = 22 for 2 min, 14 for 10 min. •fn = 4 for 2 min, 7 for 10 min. tP < 0.001, HP < 0.01, by paired t test. §P < 0.05, ||P < 0.025, #P < 0.005, by standard t test.

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T. ISHIZUKA AND ASSOCIATES TABLE 2 Effects of insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) on [3H]-2-deoxyglucose (DOG) uptake by rat diaphragms incubated in medium 199 [3H]-2-DOG uptake (cpm/100 mg tissue)

Increase due to treatment (%)

7 7

1543 ± 96 2345 ± 156

52 ± 6t

16 16

1385 ± 59 1790 ± 74*

30 ± 7t

Treatment Insulin 0 100 nM TPA 0 500 nM

Hemidiaphragms were preincubated in medium 199 containing 5.8 mM glucose for 30 min, and after changing to fresh medium, treatments were added as indicated. After 30 min of treatment, [3H]-2DOG uptake was determined over 10 min as described in RESEARCH DESIGN AND METHODS, n, Number of separate experiments. In each experiment, there were 3-4 control hemidiaphragms and a corresponding number of treated hemidiaphragms. Mean values of 3-4 determinations (SE usually