Vol. 276, No. 2, February
1, pp. 486-494,199O
Insulin Increases the Synthesis of Phospholipid Diacylglycerol and Protein Kinase C Activity in Rat Hepatocytes Denise
Jong Y. Kuo, and Robert V. Farese James A. Haley Veterans Hospital and the Departments of Medicine and Biochemistry, University of South Florida College of Medicine, Tampa, Florida 33612 Received July 26,1989, and in revised form October 10, 1989
The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30-60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de nova synthesis of PA. That de nova synthesis of PA was increased is supported by the fact that t3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulinand vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte 0 1990 Academic Press, Inc. metabolism.
Insulin has been found to increase the production of diacylglycerol (DAG)’ and/or activate protein kinase C ’ To whom correspondence should be addressed at Research Service 151, James A. Haley Veterans Hospital, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. ’ Abbreviations used: DAG, 1,2-diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PA, phosphatidic acid; PS, phosphatidylserine; PI, phosphatidylinositoh TAG, triacylglycerol; MAG, monoacylglycerol; AH, angiotensin II; VP, vasopressin; TPA,
in BC3H-1 myocytes (l-3), rat adipose tissue (4), and rat diaphragm (5). In the BC3H-1 myocyte, DAG is apparently increased through three mechanisms, viz., de nouo phosphatidic acid (PA) synthesis (2), hydrolysis of a phosphatidylinositol (PI)-glycan (6), and hydrolysis of phosphatidylcholine (PC) (7). Effects of insulin on hepatic DAG production have not been reported. Nevertheless, there are reported findings which suggest that insulin may increase de nouo PA synthesis and subsequent DAG production. For example, insulin has been found to increase hepatic de nouo fatty acid synthesis (8,9), as well as synthesis of triacylglycerol, PC, and phosphatidylethanolamine (PE) (8, 10). The halflife of PA hydrolase or phosphatase is decreased and the activity of glycerol-3-phosphate acyltransferase are increased by insulin (11-13). Glycolysis is increased through stimulation of phosphofructokinase (14), and levels of glycerol 3-phosphate (15) are increased by insulin. In short, insulin appears to increase both substrates and enzymatic activities that would favor de nouo PA synthesis in rat liver, and this would be expected to result in increases in DAG, which is an obligatory intermediate in the synthesis of triacylglycerol, PC, and PE. In view of these findings, and the potential importance of DAG as a signaling substance, we presently studied the effects of insulin on DAG and phospholipid metabolism, as well as on the activation of protein kinase C in rat liver, and compared these to the effects of angiotensin II and vasopressin, which have been more extensively studied (16-18).
12.0-tetradecanoyl phorbol13-acetate; PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, EGTA, ethylene glycol bis(fi-aminoethyl ether) N,N’-tetraacetic acid.
0003.9861/90 $3.00 1990 by Academic Press, Inc. of reproduction in any form reserved.
Copyright 0 All
General. Hepatocytes were prepared from fed, male, SpragueDawley rats (180-250 g) by methods described by Zaleski and Ontko (19). Experiments were performed between 0830 and 1100 hr. Washed hepatocytes were resuspended in gassed (95% 0,/5% CO,) medium 199 (Gibco formulation) containing 1 mM CaCl,, 26 mM HCO;, 25 mM Hepes (pH 7.4), 20 mM glucose, and 2 mg/ml radioimmunoassay grade bovine serum albumin (BSA). The cells (>95% viable by trypan blue exclusion) were then preincubated and incubated at 37°C under 95% 0,/5% CO2 with continual agitation (90 oscillations/min) in a metabolic shaker. In all cases, siliconized glass or polystyrene incubation tubes contained 0.5 ml of medium and approximately lo-15 mg (dry wt) of hepatocytes. Two basic protocols were followed. In [“H]Glycerol experiments. both cases, hepatocytes were preincubated batchwise for 60 min, washed, and resuspended in medium containing 5 mM glucose, and then distributed to incubation tubes. In protocol A, the cells were further preincubated for 30 min in the presence of 10 &i of [2-“H]glycerol. Over the next 10 min, insulin or other agonists were added in a retrograde manner to provide varying times (lo-O.5 min) of treatment. Control cells were treated by addition of comparable amounts of vehicle [medium 199 or, where indicated, dimethyl sulfoxide (DMSO)] at varying times of the treatment period (these additions did not alter results). In all cases, the durations of the two preincubations (i.e., 60 and 30 min) and the experimental period (10 min) were identical for all cells, and the only variable was the duration of exposure to insulin or other agonists during the 10.min “experimental period.” In protocol B, after the first 60 min preincubation, the cells were preincubated for another 30 min in the absence of isotope. [“HIGlycerol (10 or 20 &i) was then added simultaneously with or without insulin, and the experimental period was continued for the indicated times. In some experiments, insulin effects on DAG mass were examined without concurrent addition of [“HIglycerol. Reactions were stopped by addition of 3 ml of chilled CH,OH and 6 ml CHCl,. The organic phase of the lipid extract was washed three times with 3 ml of HzO, taken to dryness, and chromatographed for neutral lipids and phospholipids as described previously (20-22). Incorporation of [3H]glycerol into glycerol S-phosphate was determined by chromatography of delipidated aqueous extracts of hepatocytes by anion-exchange (Dowex-1) chromatography as described (21). After the columns were washed with water to remove the free glycerol, a single peak of radioactivity was found to elute at approximately 0.2 M ammonium formate in 0.1 M formic acid. Authentic [U‘“C]glycerol3-phosphate (New England Nuclear) was found to quantitatively coelute with samples containing [“HIglycerol X-phosphate which was generated in the hepatocyte incubations. It should be noted that [2-“HIglycerol loses its radioactivity if metabolized to dihydroxyacetone phosphate and other glycolytic intermediates. Therefore, glycerolipid synthesis from dihydroxyacetone phosphate directly would be precluded from the measurements reported here. [“H]lnositol experiments. Hepatocytes were preincubated batchwise for 90 min with [3H]inositol (200 &i/15 ml of medium 199). Cells were collected by centrifugation, washed and resuspended in isotopefree medium, and preincubated for another 20 min. A IO-min experimental treatment period was then used as described above in protocol A. Reactions were terminated by addition of 10% trichloroacetic acid (TCA). TCA extracts were extracted with ether, neutralized, and analyzed for [3H]inositol mono-, bis-, and trisphosphates, as described by Berridge (23). Diacylglycerol mass experiments. Diacyglycerol content was measured by incubating aliquots of lipid extracts with DAG kinase as described by Priess et al. (24). After incubation, [“‘Plphosphatidic acid was purified by thin-layer chromatography (22), and DAG content was calculated by comparing samples to DAG standards. Protein kinase C experiments. Cells were preincubated and incubated as described in protocol A (see above), except that [“HIglycerol
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L” I ~ ,uTAG DAG PC/PE
E 2 PI MAG
FIG. 1. Incorporation of [“HIglycerol into rat hepatocyte neutral and phospholipids. Rat hepatocytes were preincubated for 30 min in medium 199 with 0.1% BSA, and then incubated with [2-“HIglycerol for the indicated times. Results are the means of four determinations.
was omitted. After the 10.min experimental treatment period, the reaction mixtures were chilled, and cells were collected by centrifugation at 4°C. The cells were sonicated in 0.5 ml of buffer A [20 mM Tris-HCI, 1.2 mM EGTA, and pH 7.5,0.25 M sucrose, 50 mM p-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)], for 30 s at 50% output in a Heat Systems ultrasonicator. Sonicates were centrifuged at 105,OOOgfor 30 min at 4°C in a Beckman TL-100 ultracentrifuge. Supernatant fractions (cytosol) were decanted, and pellets (membrane fractions) were resuspended in buffer B (20 mM Tris-HCl, pH 7.5,0.25 M sucrose, 5 mM EGTA, 2 mM EDTA, 1% Triton X-100, and 0.1 mM PMSF). After standing for 20 min at 4”C, the solubilized membrane fractions were obtained by centrifugation as described above. Cytosol and membrane fractions were routinely assayed directly for protein kinase C activity as described previously (1). Protein kinase C reaction mixtures (0.25 ml) contained tissue extracts along with 5 wmol Tris HCI (pH, 7.5), 1.25 Kmol Mg acetate, 2.5 nmol of [+y-“P]ATP (lo-20 X lo* cpm/nmol), 10 Kg phosphatidylserine (PS), 125 nmol CaCl, (in excess of chelator concentrations), and 50 pg histone III-S. Reactions were initiated by addition of enzyme. After 3 min at 3O”C, reactions were terminated by adding 2 ml of 25% (w/v) TCA. Precipitates were collected on nitrocellulose filters, rinsed with 8 ml of 10% TCA, and counted for radioactivity. Materials. Highly purified porcine insulin (25.7 units/mg) was obtained from Elanco. [2-“HIGlycerol (sp act = 5 Ci/mmol) was obtained from ICN. [“H]Inositol (sp act = 15 Ci/mmol) was purchased from American Radiolabeled Chemicals. [y-a’P]ATP (sp act = 650 Ci/ mmol) was obtained from ICN. Medium 199 was purchased from Gibco. Vasopressin, angiotensin II, 12.0-tetradecanoyl phorbol13-acetate, phosphatidylserine, histone (type 111s). BSA (radioimmunoassay grade, fraction V), diolein, and other biochemicals were obtained from Sigma. Diglyceride kinase was purchased from Lipidex.
To determine the pattern of labeling in the de nouo pathway, hepatocytes were incubated for short periods with [3H]glycerol (protocol B). As shown in Fig. 1, [“HIglycerol was rapidly incorporated into PA and DAG, and
FIG. 2. Time course of effects of insulin on [3H]glycerol incorporation into phospholipids and neutral lipids. Rat hepatocytes were preincubated for 30 min with [2-3H]glycerol (protocol A) and then insulin (20 nM) was added in a retrograde manner during a 10.min treatment period, i.e., 10,5,2,1, and 0.5 min prior to the end of the 10.min period. Control cells were treated by addition of small volumes of medium 199. Because of variation in control cpm in individual experiments, results are the means f SEM of the percentage increase above control for five separate experiments. Means f SEM of control cpm: DAG, 11,922 f 2030; TAG, 59,280 + 13,887; MAG, 446 f 72; PA, 5404 f 1232; PI, 1715 + 291; and PC/PE, 45,632 + 3402. *P < 0.05 by paired t test for the first significant data point in the time course (effects were significant at all subsequent time points).
labeling of PC and TAG followed after a short lag. Labeling of phosphatidylinositol (PI) or monoacylglycerol (MAG) was barely perceptible at early time points under these experimental conditions. Thus, entry of [3H]glycerol into the de novo pathway in isolated hepatocytes was identical to the labeling reported in the intact rat liver (25) and similar to what was found in BC3H-1 myocytes, where newly synthesized PA is metabolized primarily to DAG and then to PC or triacylglycerol (TAG) (2). After 30 min of prelabeling with [3H]glycerol (protocol A), treatment with 20 nM insulin provoked increases in the labeling of PA, PI, PC/PE, DAG, and TAG. Results from six separate experiments are summarized in Fig. 2. Using
ing of PA and DAG were significant within 30 s (the earliest point examined) of insulin addition and remained
elevated thereafter. Increases in PI, PC/PE, and TAG did not attain statistical significance until 2, 5, and 10 min, respectively. When [3H]glycerol and insulin were added simultaneously (protocol B), increases in DAG and PA were also evident after 1 min (the earliest point examined) of insulin addition (Tables I and II). Time-dependent effects of insulin on [3H]DAG production are shown in Table II and Fig. 3. With this experimental protocol, it seems likely that rapid, insulin-induced increases in [3H]DAG are derived largely from newly synthesized PA, rather than from preexisting (unlabeled) phospholipids such as PC, PE, and PI. This is further indicated by the results shown in Table I: PC + PE + PI contain only 10% of the [3H]glycerol incorporated into total glycerolipids during the first minute of labeling, and it is unlikely that observed effects of insulin, AII, or vasopressin on [3H]labeling of PA and DAG can be explained by accelerated hydrolysis of PC + PE + PI. Whereas insulin increased [3H]glycerol incorporation into neutral lipids and phospholipids of hepatocytes, uptake of [3H]glycerol and labeling of the precursor pool of glycerol 3-phosphate in aqueous extracts were not altered by insulin treatment: e.g., using protocol A (30-min prelabeling with 10 PCi [3H]glycerol/tube and lo-min treatment period with or without insulin), [3H]glycerol 3-phosphate was 2.58 f 0.16 X lo5 and 2.42 f 0.22 X lo5 cpm/tube, control vs insulin, respectively (mean -t SEM of four determinations); similarly, following the simultaneous addition of 20 &i [3H]glycerol/tube with (or without) insulin (protocol B), [3H]glycerol 3-phosphate was 4.4 + 0.1 X lo5 and 4.5 + 0.3 X lo5 cpm/tube, control vs insulin at 1 and 2 min. Thus, it appears that (a) [3H]glycerol3-phosphate is very rapidly labeled, (b) insulin does not affect glycerol 3-phosphate labeling, and (c) insulin effects on [3H]glycerol incorporation into lipids cannot be due merely to increases in glycerol uptake and/or glycerokinase activity. Furthermore, it should be noted that insulin increases glycolytic flux and glycerol 3phosphate levels (15); thus, specific activity of glycerol 3-phosphate would, if anything, be expected to decrease with insulin treatment, as well as with agents which increase glycogenolysis, such as angiotensin II and vasopressin (see below). As vasopressin and angiotensin II are known to increase inositol-phospholipid hydrolysis (16, 26), DAG generation (18), and kinase C activity (27) (at least for vasopressin) in rat hepatocytes, it was of interest to determine whether these agonists also activated de novo phospholipid synthesis. As shown in Table III, vasopressin increased [3H]DAG production in cells prelabeled for 30 min with [3H]glycerol. Levels of [3H]PA and [3H]PI were also significantly elevated. Increases in [3H]glycerol labeling of PC/PE and MAG and TAG were observed, but were only statistically significant for PC/PE after 10 min of vasopressin treatment,. Both vasopressin
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of Insulin, Angiotensin II (AII), and Vasopressin (VP) on Diacylglycerol (DAG) Mass and [3H]Glycerol Incorporation into DAG and Other Glycerolipids of Rat Hepatocytes
[3H]Glycerol DAG mass (nmol/tube)
Treatment Control Insulin, 20 AII, 100 nM VP, 100 nM
13.7 f 20.2 + 20.2 ? 20.2 *
1.4 1.9* 2.0* 1.4*
8,435+- 455 11,610 f 310*** 12,240-t 945** 13,120f 1,065***
8,525+ 530 10,360 + 575 10,665 f 1,125 10,450k 645
173Ok420 1635 t 60 1730*515 1630+160
638f103 56Ok 60 510f 85 480f 20
960f85 95Ok40 83Ok55 81Ok30
1230+180 1425k 25 1865k515 1475+ 85
Note. Hepatocytes were preincubated for 30 min in medium 199 + 0.1% BSA prior to the simultaneous addition of [3H]glycerol and insulin or control vehicle, AH, or vasopressin. Reactions were terminated after 1 min of labeling f hormone treatment. Results are the means f SE of four determinations. * P < 0.05, **P < 0.01, ***P < 0.005 (Student t test).
and AII, like insulin, were found to increase [3H]glycerol incorporation into DAG and other lipids in experiments in which agonist and isotope were added simultaneously (Table I, Figs. 4-6). Diacylglycerol
treatment with insulin and AII. the levels of DAG produced in angiotensin II treatment were ported by Bouscarel and Exton
It should be noted that response to insulin and comparable to those re(17) for angiotensin II.
We questioned whether increases in [3H]glycerol-labeled DAG were accompanied by increases in DAG mass. As shown in Table I and Figs. 4 and 5, relative increases in DAG mass were similar to those observed with [3H]glycerol labeling of DAG with all three agents. (The apparently greater increase in [3H]glycerol labeling of DAG at 10 min of treatment with vasopressin relative to insulin as shown in Fig. 4 was not observed in other experiments.) In most cases, the level of labeling with vasopressin plateaued, similar to the level noted with insulin. As also shown in Table IV, with the simultaneous addition of label and hormone, [3H]DAG and DAG mass were increased maximally within 5 min of
Effects of Insulin on [3H]Diacylglycerol and [3H]Phosphatidic Acid Synthesis from [3H]Glycerol following Simultaneous Addition of Isotope and Hormone % Increase over control Duration of insulin treatment (min)
91.5 k 32.0 (5)* 48.7 t 15.7 (3) 72.2 i 29.0(3)
45.8+- 15.2 (4) 23.7 k 1.0 (3)** 22.0+ 4.6(4)*
Note. Hepatocytes were preincubated for 30 min in medium 199 + 0.1% BSA prior to the simultaneous addition of [3H]glycerol and insulin. Reactions were terminated at the indicated times. Results are the means f SEM of percentage increases in three to five separate experiments (indicated in parentheses), each performed in triplicate. * P < 0.05, **P < 0.005 (paired t test).
lb TIME hIin)
FIG. 3. Effects of insulin on [3H]diacylglycerol synthesis from [3H]glycerol following simultaneous addition of isotope and hormone. Rat hepatocytes were preincubated for 30 min in medium 199 + 0.1% BSA prior to the simultaneous addition of insulin (20 nM) (0) or vehicle alone (controls, 0) and [2-3H]glycerol (protocol B). Reactions were terminated at the indicated times. Results are the means f SEM for four determinations from a single experiment. *P < 0.025, **P < 0.05 (by the Student t test).
Effects of Vasopressin on [3H] Glycerol Incorporation into Lipids in Rat Hepatocytes % Increase (vasopressin
DAG Mass and [3H]DAG Synthesis from [3H]Glycerol following Insulin or Angiotensin II Treatment
DAG mass (nmol/tube)
5.24 k 0.41 8.41 k 0.15*** 7.44 t 0.68*
1507f 195 2955f368* 3137f277**
Treatment 1 min Phosphatidic acid Phosphatidylinositol Phosphatidylcholine and phosphatidylethanolamine Monoacylglycerol Diacylglycerol Triacylglycerol Note. Hepatocytes A (see Experimental sopressin for 1,5, or are shown. * P < 0.05 (paired
37+20 6+ 1 4 -t 10 22+ 7
21t 12 58 k lo*
ilk 2* 13 t 10 59 i 11* 15+ 7
18 t 13k 54-t 222
Control Insulin Angiotensin 3* 6 7* 8
were preincubated and incubated as per protocol Methods and treated with or without 100 nM va10 min as indicated. Results of four experiments
Furthermore, in addition to DAG, vasopressin and AII, like insulin, increased phospholipid and total glycerolipid labeling (Table I and Fig. 5) in experiments in which hormone and isotope were added simultaneously. Significant increases in labeling of TAG were not observed under these conditions. Increases in DAG mass and acute labeling with [3H]glycerol were also noted with doses of insulin less than 20 nM, which was routinely used (Table V). It may be noted that there were apparent dose-related increases in [3H]glycerol labeling of DAG, but not DAG mass. This disparity could reflect an inherent lag and variable labeling or dilution (from glycolysis) of the glycerol 3-phosphate precursor pool at various insulin levels and/or accelerated production of DAG from sources other than
Note. Hepatocytes were incubated for 5 min using protocol B, simultaneous addition of 10 PCi [3H]glycerol and insulin (20 nM) or angiotensin II (100 nM). Results are the means + SE for four determinations from a representative experiment which yielded similar data in three separate experiments. * P < 0.05, **P < 0.025, ***P < 0.01 (Student t test).
the de nouo pathway, e.g., hydrolysis of a PI-glycan (28) or PC (7). In any event, observed effects at 0.2 and 2 nM insulin indicated that these effects are apparent at more physiological insulin levels. The possibility that accelerated uptake and metabolism of [3H]glycerol were responsible for increases in DAG mass following insulin treatment was ruled out by the finding that in the absence of [3H]glycerol, DAG mass increased 80% within 1 min of insulin treatment (this compares to a mean increase of 60% in four other experiments). [3H]Inositol
In accordance with previous enhanced the production of nearly two- to threefold in our tocytes. Insulin, on the other inositol phosphate production
reports (16), vasopressin [3H]inositol phosphates preparations of rat hepahand, had little effect on (Table VI). In accordance
8 Control + insulin -m- VP
minutes FIG. 4. [“HIDAG synthesis from [3H]glycerol and DAG mass following insulin or vasopressin treatment. Rat hepatocytes were incubated using protocol B (simultaneous addition of 20 PCi [3H]glycerol and hormone). +, Insulin (20 nM); W, vasopressin; q , control vehicle. Results are means f SEM for three determinations from a representative experiment. *I’ < 0.05, **P < 0.001 (by the Student t test).
of Insulin and Vasopressin on [3H]Inositol Phosphate Production in Rat Hepatocytes
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Treatment None (control) Insulin, 20 nM Vasopressin, 100
2228 f 113 2548 f 180 4428 f 299*
114 + 14 150 f 13 424 f 41*
76-t 7 91+ 7 193 f 14*
10 6 6 4
Note. Hepatocytes were prelabeled with [“Hlinositol prior to treatment with hormones (see Experimental Methods). Results are means f SE or 15 or 16 separate experiments with three or four replicates in each experiment. * P -c 0.001 (t test).
FIG. 5. Insulin- and angiotensin II-induced increases in total [“HIglycerolipid labeling following simultaneous addition of [3H]glycerol and hormone. Rat hepatocytes were treated with 20 &i [3H]glycerol and 20 nM insulin, (0), or 100 nM AI1 (X), or control vehicle (0). Results are means + SE of four determinations from a representative experiment.
with published reports (16,29), we also found that vasopressin, but not insulin, increased cytosolic Ca’+, as measured by fluorescence of Quin-2-loaded hepatocytes (data not shown).
brane fractions of rat hepatocytes (Table VII). Histone (III-S) phosphorylation was stimulated upon addition of PS + Ca2+, whereas Ca” alone had either little or no effect. We did not routinely add diolein to the assay system as this could mask hormone-induced increases in protein kinase C activity caused by increases in endogenous diacylglycerol levels. In experiments reported here, protein kinase C is defined as histone (III-S) phosphorylation which is dependent upon PS + Ca*+ (i.e., the PS + Ca2+ value minus the value observed with the EGTA blank). Insulin provoked prompt increases in protein kinase C activity in both cytosol and membrane fractions within 30 s of treatment (Fig. 6). Activity remained elevated in membrane extracts, but, after 5 min, cytosolic activity diminished. Vasopressin also provoked prompt
Protein Kinase C Activity Protein kinase activity dependent upon Ca2’ and PS was readily demonstrable in crude cytosol and mem-
Effect of Insulin Concentration on Diacylglycerol Mass and [3H] Glycerol Incorporation into Diacylglycerol Insulin (nM)
DAG mass (nmol/tube)
0 0.2 2.0
16.60 -t 2.19 29.57 i 2.58’ 25.51 k 2.32
[“HIDAG (cpm/tube) 92,931+ 7254 107,499 * 3711 125,079 f 3927*
Hepatocytes were preincubated for 30 min in medium 199 + 0.1% BSA prior to the simultaneous addition of 20 &i [3H]glycerol per tube and insulin. Reactions were terminated 5 min later. DAG mass determinations were run on separate incubation tubes. Results are the means f SEM of four determinations. Note.
* P < 0.05 (t
Effect of Ca2+ and Phosphatidylserine (PS) on Kinase Activity from Insulin and Vasopressin-Treated Hepatocytes kinase activity
Addition Ca2+ + PS Ca2+ EGTA
0 min Control 44 35 36
5 min Insulin
10 min VP
77 54 56
0 min Control 85 53 56
1 min Insulin
5 min VP
Note. After preincubation for 30 min, hepatocytes were incubated with either insulin (20 nM) or vasopressin (VP, 100 nM) for 1,5, or 10 min. Cytosolic and membrane fractions (25 pg protein) were assayed as described under Experimental Methods. Where indicated, PS (40 pg/ml), CaCl, (0.5 mM in excess of chelator concentrations), or 0.5 mM EGTA was added. Results are means from selected incubation points (see Fig. 6) assayed in duplicate.
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FIG. 6. Effects of insulin, vasopressin, and TPA on hepatocyte protein kinase C activity. Rat hepatocytes were treated with insulin (20 nM), vasopressin (200 nM), or TPA (1 FM) for the indicated times. Aliquots of cytosol and membrane extracts containing 10 pg of protein were assayed for protein kinase C activity. Results are the means + SEM of four separate experiments for insulin, and are from a representative experiment with vasopressin and TPA, which evoked similar changes in two separate experiments. *P < 0.05 by the student t test.
increases in protein kinase activity of cytosol and membrane fractions, and both increases were sustained for 10 min. Thus, insulinand vasopressin-induced increases in DAG were reflected by increases in protein kinase C activity. TPA, on the other hand, provoked a very rapid and virtually complete disappearance of cytosolic protein kinase C activity, and this was associated with an initial, small increase, followed by a decrease in membrane-associated protein kinase C activity. More significant increases in membrane protein kinase C activity following TPA treatment may not have been apparent in these studies, as there may have been an increased rate of degradation of membrane-bound enzyme (and conversion to kinase M) under the present experimental conditions. DISCUSSION
Although insulin has little or no effect on phospholipase C-mediated hydrolysis of inositol phospholipids in rat hepatocytes [as reported earlier (10-12) and confirmed presently], insulin rapidly increased [3H]glycerol incorporation into PA and DAG in this tissue. This effect on [3H]DAG production appeared to be due at least partly to an increase in de nouo [3H]PA synthesis, as it occurred promptly following simultaneous addition of insulin and [3H]glycerol, and was accompanied by increases, rather than decreases, in the labeling of other
lipids and phospholipids. Increases in [3H]glycerol labeling of total glycerolipids, provided clear evidence that insulin increased de novo PA synthesis, and much of the newly synthesized PA was obligatorily converted to DAG, and then to other lipids such as TAG and PC/PE. The rapidity and extent of labeling of lipids and phospholipids in rat hepatocytes by [3H]glycerol were remarkable. Clearly, glycerokinase and the de nouo PA synthesis pathway are exceedingly active in this tissue, even in the basal state, and this pathway can be rapidly perturbed by insulin. The insulin effect on [3H]glycerol labeling of PA and DAG appeared to be maximally established within 1 min, and DAG labeling and levels remain elevated for 20 min. These findings are similar to those observed in the BC3H-1 myocyte (2). As alluded to above, the flux of radioactive precursors through the de novo pathway in hepatocytes appeared to be directed primarily (>99%) from PA to DAG and then to PC/PE and TAG. As in the perfused rat liver (25) and BC3H-1 myocyte (2), PI labeling accounts for less than 1% of total labeling by [3H]glycerol during the presently employed time courses, and it seemed unlikely that significant amounts of [3H]glycerol labeling of [3H]DAG were derived from hydrolysis of inositol phospholipids under the present experimental conditions. Interestingly, insulin-induced increases in DAG mass were also noted after 1 min of treatment, and were comparable to those observed with angiotensin II and vasopressin (18). This is surprising since both vasopressin and angiotensin II increase inositol phospholipid hydrolysis in hepatocytes (16), as well as de nova synthesis of PA and DAG, as demonstrated presently. Since insulin does not increase inositol phospholipid hydrolysis significantly, it is possible that AII- and vasopressin-induced increases in DAG are largely derived from sources other than inositol lipid hydrolysis. In addition to stimulation of the de novo pathway observed presently, it has been suggested that AII, vasopressin, and insulin increase PC hydrolysis (30-33). It is also possible that insulin may also increase DAG through PC hydrolysis (34) and/or PI-glycan hydrolysis in the liver (28). Further studies are needed to quantitate the contributions of each pathway to observed increases in DAG content and subsequent activation of protein kinase C. The present finding that vasopressin stimulates de novo DAG synthesis is at odds with the conclusions of Polverino and Barritt (33). These authors reported that they were unable to observe stimulation of the initial rate of incorporation of radioactivity in the presence of a high concentration of [3H]glycerol, and concluded that later increases in labeled DAG were due to increased hydrolysis of labeled PC. However, it is difficult to compare our results to theirs as they provided no information on the labeling of the glycerol 3-phosphate pool (high concentrations of glycerol as used in their experiments may not allow for the pool to be labeled sufficiently rapidly)
C BY INSULIN
kinase C and may have masked decreases in enzyme content caused by its translocation to the membrane fraction.
or on the incorporation of [3H]glycerol into phospholipids or total glycerolipids. Furthermore, inspection of their data reveals statistically significant effects of vasopressin on DAG synthesis at all time points examined. The greater effects of vasopressin at later time points could reflect better labeling of the glycerol 3-phosphate pool and better utilization for de novo PA/DAG synthesis, as well as labeling of PC/PE to serve as substrate for hydrolytic reactions. Needless to say, as with insulin (2), vasopressin may simultaneously stimulate de novo synthesis of PA, DAG, and PC and hydrolysis of PC. The presently observed increases in both cytosolic and membrane-bound protein kinase C activity after insulin treatment of hepatocytes are, in many respects similar to those observed in BCSH-1 myocytes (l), and with AI1 treatment of cultured rat aortic smooth muscle cells (35). Insulin-induced increases in membranebound protein kinase C activity may logically be postulated to be due to insulin-induced increases in membrane-associated DAG. However, the mechanism whereby insulin increases cytosolic protein kinase C activity is uncertain. We have found (unpublished observations) that sizable amounts of DAG are recovered in cytosol (probably artefactually), as well as membrane fractions after cell disruption by several methods, and insulin-induced increases in DAG are apparent in both fractions. Furthermore, cytosolic DAG is associated with protein kinase C after partial purification of the latter by DEAE-Sephacel chromatography and sucrose density gradient centrifugation (unpublished observations). It is therefore plausible to suggest that increases in DAG in both membrane and cytosol fractions contribute to observed increases in protein kinase C enzyme activity after insulin or vasopressin treatment.” The presently reported increase in cytosolic protein kinase C enzyme activity by insulin may explain why other investigators (36) were able to demonstrate a decrease in cytosolic activity with TPA, but not with insulin treatment. Along these lines, it is of interest that a number of hormones and growth factors provoke increases in total enzyme activity, but do not seem to “translocate” protein kinase C from the cytosol to plasma membranes (37-39). In addition, a number of agonists have also been reported to provoke increases in cytosolic protein kinase C enzyme activity, with (40) or without (41) associated increases in membrane-bound protein kinase C enzyme activity. As with insulin, large increases in DAG may have activated cytosolic protein
11. Pittner, R. A., Fears, R., and Brindley, D. N. (1985) Biochem. J. 230,525-534. 12. Bates, E. J., Topping, D. L., Sooranna, S. P., Saggerson, D., and Mayes, P. A. (1977) FEBS Lett. 84,225-228. 13. Pittner, R. A., Fears, R., and Brindley, D. N. (1986) Biochem. J. 240,253-262. 14. Castano, J. G., Nieto, A., and Feliu, J. E. (1979) J. Biol. Chem. 264,5626-5629. 15. Beynan, A. C., Vaartjes, W. J., and Geelen, M. J. H. (1980) Horm. Metab.Res. 12,425-440. 16. Charest, R., Prpic, V., Exton, J. H., and Blackmore, P. F. (1985) Biochem. J. 227,79-90. 17. Bouscarel, B., and Exton, J. H. (1986) Biochim. Biophys. Acta 888,126~134. 18. Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260,14201-14207. 19. Zaleski, J., and Ontko, J. A. (1985) Biochim. Biophys. Acta 876, 134-142.
” Studies in the BC3H-1 myocyte further indicate that the actual level of cytosolic protein kinase C as measured by immunoblotting with polyclonal antibody decreases by approximately 70% (42), while the enzyme activity increases 50-100% following insulin treatment (1). These results suggest that insulin stimulates the translocation of protein kinase C from cytosol to membrane, but decreases in cytosolic enzyme content are paradoxically masked by increases in enzyme activity.
20. Nomura, T., Tachibana, M., Nomura, H., and Hagino, Y. (1986) Lipids 21,366-367. 21. Vaartjes, W. J., de Haas, C. G. M., Geelen, M. J. H., and Bijleveld, C. (1987) Biochem. Biophys. Res. Commun. 142,135m140. 22. Farese, R. V., Sabir, M. A., and Larson, R. F. (1981) Biochemistry 20,6047-6051. 23. Berridge, M. ,J. (1983) Biochem. J. 212,864-863. 24. Priess, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261,8597-8600.
ACKNOWLEDGMENTS We thank Dr. Jan Zaleski, Laboratory for Cellular and Biochemical Toxicology, Rutgers University, for his advice on preparing hepatocytes. This work was supported by funds from the Research Service of the Veterans Administration and National Institutes of Health Grant DK-38079.
REFERENCES 1 Cooper, D. R., Konda, T. S., Standaert, M. L., Davis, J. S., Pollet, R. J., and Farese, R. V. (1987) J. Biol. Chem. 262,363333639. 2. Farese, R. V., Konda, T. S., Davis, J. S., Standaert, M. L., Pollet, R. J., and Cooper, D. R. (1987) Science 236,636-638. 3. Farese, R. V., Standaert, M. L., Barnes, D. E., Davis, J. S., and 116, 2650-2655. Pollet, R. J. (1985) Endocrinology 4. Draznin, B., Leitner, J. W., Sussman, K. E., and Sherman, N. A. (1988) Biochem. Biophys. Res. Commun. 156,570-575. 5. Walaas, S. I., Horn, R. S., Adler, A., Albert, K. A., and Walaas, 0. (1987) FEBS Lett. 220,311-318. 6. Saltiel, A. R., Fox, J. E., Sherline, P., and Cuatrecasas, P. (1986) Science 233,967-972. 7. Nair, G. P., Standaert, M. L., Pollet, R. J., Cooper, D. R., and Farese, R. V. (1988) Biochem. Biophys. Res. Commun. 154,13451349. 8. Beynen, A. C., Math, J. H., van den Berg, G., and van den Berg, S. G. (1980) Trends Biochem. Sci. 5,288-290. 9. Vaartjes, W. J., de Haas, C. G. M., and van den Berg, S. G. (1985) Biochem. Biophys. Res. Commun. 131,449-455. 10. Geelen, M. J. H., Groener, J. E. M., de Haas, C. G. M., Wisserhof, T. A., and van Golde, L. M. G. (1978) FEBS Lett. 90,57-60.
25. Akesson, B., Elovson, J., Arvidson, G. (1970) Biochim. Biophys. Acta 210,15-27. 26. Uhing, R. J., Prpic, V., Jiang, H., and Exton, J. H. (1986) J. Biol.
Chem.261,2140-2147. 27. Cooper, R. H., Kobayashi,
K., and Williamson,
J. R. (1984) FEBS
Lett.166,125-130. 28. Saltiel, A. R., and Cuatrecasas, P. (1986) Proc. N&l. Acad. Sci. USA 83,5793-5797. 29. Thomas, A. P., Martin-Requero, A., and Williamson, J. R. (1985) J. Biol. Chem. 260,5963-5973. 30. Sakai, M., and Wells, W. W. (1986) J. Biol. Chem. 261, 1005810062. 31. Allan, D., and Cockcroft, S. (1983) Biochem. J. 213,555~557. 32. Bocckino, S. T., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chen. 262,15309-15315. 33. Polverino, A. J., and Barritt, G. J. (1988) Biochim. Biophys. Acta
ET AL. 34. Nair, G. P., Standaert, M. L., Pollet, R. J., Cooper, D. R., and Farese, R. V. (1988) Biochem. Biophys. Res. Commun. 154,13451349. 35. Lang, U., and Vallotton, M. B. (1989) Biochem. J. 259,477-484. 36. Vaartjes, W. J., de Hass, C. G. M., and van den Bergh, S. G. (1986) Biochem. Biophys. Res. Commun. 138,1328-1333. 37. Pelech, S., Meier, K. E., and Krebs, E. G. (1986) Biochemistry
8348-8364. 38. Costa-Casnellie, 39. 40. 41. 42.
M. R., Segel, G. B., and Lichtman, M. A. (1986) J. Cell. Physiol. 129,336-342. Shinohara, O., Knecht, M., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133,468-474. Averdunk, R., and Gunther, T. (1986) FEBS Lett. 195,357-361. Widmaier, E. P., and Hall, P. F. (1985) Mol. Cell Endocrinol. 43, 181-188. Acevedo-Duncan, M., Cooper, D. R., Standaert, M. L., and Farese, R. V. (1989) FEBS Lett. 244,174-176.