0013-7227/92/1301-0093$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 1 Printed in U.S.A.
Society
Activation of Phospholipase Isolated Islet Cells Follows MARJORIE
DUNLOP
AND
STEWART
University of Melbourne (M.D.), Department 3050, Australia; and Section of Endocrinology Wisconsin, Madison, Wisconsin 53792
D by Glyceraldehyde in Protein Kinase C Activation*
A. METZ Medicine, Royal Melbourne Hospital, Parkville, Victoria (S.A.M.), Department of Medicine, The University of
of
ABSTRACT. Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, elvceraldehvde. induced formation of phosnhatidic acid in cells pielabeled with [“Clarachidonic acid or [3-H]myristic acid (164 f 7 and 210 + 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [“Clarachidonate-labeled diacylglycerol (139 + 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 f 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 f 5% of basal) which preceded the activation of PLD, as indicated by the time course of glycer-
aldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 PM 12-0tetradecanoylphorbol-13-acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 PM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an earlv rise in alvceraldehvde-stimulated diacylglycerol (which may be formed denovo or by the action of phospholipase C), suggests that PLD is recruited-by the activation of protein kinase C by this nutrient. (Endocrinology 130: 93-101,1992)
H
YDROLYSIS of membrane phospholipids makes a major contribution to intracellular molecules involved in metabolism and growth. In many cell systems there is now considerable evidence for direct receptoragonist-induced formation of diacylglycerol from phosphatidylinositols and for its formation indirectly from phosphatidylcholine after the activation of phospholipase D (PLD) and formation of phosphatidic acid (l-3, reviewed in Ref. 1). We have reported recently that intact adult islets and dispersed islet cells from the neonatal rat contain a PLD directed against phosphatidylcholine, which is activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (4,5), indicating a potential involvement of protein kinase C (PKC) in this activation. A potential functional correlate of this finding is seen in the fact that insulin release is stimulated in adult
rat islets by the addition of an exogenous PLD of bacterial origin, coincident with raised intracellular phosphatidic acid (6), and exogenous phosphatidic acid is a stimulus to insulin secretion in the neonatal islet cell (7). Little is known of the mechanism(s) and timing of phosphatidylcholine breakdown in islets and whether, by formation of phosphatidic acid, this may contribute to nutrient-stimulated insulin release. In the pancreatic islet there is considerable evidence for PLC-catalyzed hydrolysis of phosphatidylinositol(4,5)bisphosphate after receptor-agonist activation and exposure to nutrient secretagogues (3-10). Subsequent to PLC action, phosphatidic acid can be formed from diacylglycerol via diacylglycerol kinase but also can be formed de novo from nutrient sources (8, 11-13). Although a role for PKC in insulin release is contested (14-B), the activation of PKC by phorbol esters (14,1921) or cell-permeable diglycerides (22) is accompanied by insulin release in islets or insulin-secreting tumoral cell lines, suggesting that endogenous activation of PKC may be involved in the action of both receptor and nutrient secretagogues. Also, chelator-stable translocation of pro-
Received August 2, 1991. Address all correspondence and requests for reprints to: Dr. M. Dunlop, University of Melbourne, Department of Medicine, Clinical Science Building, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. *This work was supported by NIH Grant DK-37312 and by the National Health and Medical Research Council of Australia. 93
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94
PHOSPHOLIPASE
tein kinase C accompanies receptor-agonist stimulation (15) and was shown recently for the a-isoenzyme of PKC in glucose-perifused islet cells (23). Recently, a significant positive contribution of PKC activation to insulin release was shown after glyceraldehyde stimulation in the RINm5F islet P-cell subclone (24). Although the present study is not designed to address this aspect, as indicated above, activation of PLD may play a role in PKC-related insulin release. In the present study evidence is provided for glyceraldehyde-stimulated PLD activity in neonatal islet cells, an event which requires the activation of cellular PKC. PLD-mediated hydrolysis was investigated by the specific formation of phosphatidylethanol via a PLD-mediated transphosphatidylation in the presence of a primary alcohol (e.g. ethanol) (1, 4). Since phosphatidylethan01 formation (i.e. phosphatidyl transfer to alcohol) always occurs at the expense of PLD-derived phosphatidic acid (phosphatidyl transfer to water), this maneuver enabled us to discern the contribution made by PLD to cellular phosphatidic acid.
Materials
and Methods
[1-Y]Arachidonic acid, [9,10(n)-3H]myristic acid, [r”‘P] ATP, and reagents for the determination of PKC were obtained from Amersham (Amersham, UK). Insulin RIA reagents (Phadeseph) were from Pharmacia Diagnostics AB (Uppsala, Sweden). All other chemicals were from Sigma (St. Louis, MO). Cultured neonatal islet cells were prepared from l-day-old rat pancreases which were dissected and collagenase digested as described by Hellerstrom et al. (25). Digests were washed three times by suspension and sedimentation (5 min x 1 g) in medium RPM1 1640 containing 11.1 mM glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% (vol/vol) fetal bovine serum, buffered to pH 7.4 with HEPES to give discrete islet cell clusters when viewed by stereomicroscopy. These cells were cultured in the above medium containing 0.1 mM 3isobutyl-1-methylxanthine for 48 h (26). We have previously employed this technique to deter the rapid growth of fibroblasts and to promote formation of islet cell monolayers (27) in which over 80% (87 + 3%, four determinations) routinely give positive specific fluorescence when preincubated with an excess of insulin (1 pg/ml) before processing and incubation with guineapig antiporcine insulin immunoglobulin followed by fluorescein isothiocyanate-conjugated rabbit antiguinea pig globulin as described (28). Islet cell culture was continued for a further 48 h in RPM1 1640 containing 11.1 mM glucose and 10% (vol/vol) fetal bovine serum before cells were labeled in culture with [l“‘Clarachidonic acid, 54.7 mCi/mmol (final activity 50 nCi/ ml), or [9,10(n)3H]myristic acid, 53 Ci/mmol (final activity 1 rCi/ml) and preincubated as described (4, 5). We have determined previously that cells cultured in this manner maintain an insulin secretory response to nutrient stimuli. This is shown by a 4- and 6-fold increase, over insulin release at 1.7 mM glucose, when cells were incubated for 15 min with 16.7 mM glucose or 20 mM glyceraldehyde, respectively (29).
D IN ISLET CELLS
Endo. 1992 Voll30. No 1
In the following studies when present, 2 pM TPA in dimethylsulfoxide or dimethylsulfoxide (0.01%) alone were added for the final 18 h of culture. Islet cells were washed with Krebs Ringer bicarbonate buffer containing 5.6 mM glucose, 25 pM choline, and 3 pM arachidonic acid and preincubated in this buffer without further additions or with propranolol (100 pM) for 20 min. Timed incubations in the presence of glyceraldehyde (2.5-20 mM), ethanol (1.5%), or the phosphohydrolase inhibitor propranolol(lO0 pM) alone or in combination followed. Medium was removed rapidly and the cells extracted for lipid determination as described (5). For determination of diacylglycerol mass and PKC activity, islet cells were cultured and incubated in an identical manner, except that no radiolabeled compounds were present in the final overnight culture. For the determination of radiolabeled phosphatidic acid, phosphatidylethanol, and diacylglycerol the extracted lipids were applied to preactivated TLC plates and separated using multiple solvent systems as described (5). The radiolabeled compounds were localized by autoradiography, removed, and quantitated by liquid scintillation spectrometry. Values for individual metabolites were expressed as percentages of total labeled phospholipid to normalize values for experimental variations in cell number after it was established that none of the pretreatment conditions affected total phospholipid labeling. Diacylglycerol mass was determined by enzymatic conversion of extracted diacylglycerol to [32P]phosphatidic acid (30). Cellular lipid extracts were suspended with 1.5% (wt/vol) noctyl-@-glucopyranoside and 1 mM cardiolipin with 20 mM diethylaminepentaacetic acid in 10 mM imidazole buffer and treated with an excess of Escherichia coli diacylglycerol kinase in the presence of [-y3’P]ATP, specific activity 250,000 dpm/ nmol. [32P]Phosphatidic acid was extracted and separated by TLC. The mass of [32P]phosphatidic acid resulting from diacylglycerol phosphorylation of lipid extracts and sn-1,2 diacylglycerol standards was determined. PKC was assayed by the transfer of y-phosphate of [-$‘P] ATP to the threonine group of a specific PKC substrate octapeptide in a mixed micellar assay (31). PKC activity was determined on membrane and cytosolic fractions obtained from cells disrupted in 20 mM Tris HCl, 0.5 mM EGTA, and 0.25 M sucrose, containing 50 pM digitonin and 50 pg/ml leupeptin at pH 7.5. After rapid centrifugation at 13,000 x g cell membranes were solubilized with 1.0% Triton X-100 in 20 mM Tris HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, and 50 pg/ml leupeptin. PKC activity was determined by the addition of a reaction mixture containing 50 mM Tris HCl, 1 mM calcium acetate, 15 mM magnesium acetate, and 2.5 mM dithiothreitol in a 50-~1 micellar mixture of 375 mg L-cu-phosphatidylserine and 0.15 pg TPA containing 0.46 nmol substrate peptide and initiated by the addition of 50 WM[-y3’P]ATP (final activity, 53 &i/rmol). After a 15-min incubation period the phosphorylated peptide was precipitated by acid addition and recovered on paper filters which were washed extensively before determination of 32P content. PKC activity was calculated by subtracting the 32P incorporated into substrate in the absence of phospholipid micelle, and activity in the membrane fraction was expressed as nanomoles of 32Pphosphate incorporated per min/mg protein. Total protein in the membrane fraction was
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PHOSPHOLIPASE determined with Coomassie brilliant blue dye (32). Results are expressed as mean + SE with either the number of individual experimental determinations or degrees of freedom (df) indicated. The statistical difference between mean values was obtained using Student’s t test.
Results Labeling of phosphatidylcholine
in islet cells
Incorporation of the radiolabeled precursor fatty acids into phosphatidylcholine is shown in Table 1. Under the labeling conditions phosphatidylcholine was preferentially labeled with myristic acid. This incorporation reflected that of choline [determined previously (9), where the choline label distributes between phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin phospholipid species only]. Arachidonic acid was more widely distributed among phospholipid classes. Determination of phosphatidic phosphutidylethanol
acid and
Incubation of [14C]arachidonate-prelabeled islet cells with glyceraldehyde (2.5-20 InM) for 30 min resulted in a significant concentration-dependent increase in the production of labeled phosphatidic acid (Fig. 1). Ph_osTABLE
1.
Labeling of phosphatidylcholine
in neonatal islet cells
Radiolabeled precursor
Total phospholioids (%)
[“ClArachidonic acid [3H]Myristic acid IWlCholine
35.7 f 0.4 84.8 + 0.6 95.7 f 0.1
Values shown are mean + SEM the disintegrations per minute present in phosphatidylcholine expressed as a percentage of total disintegrations per minute in all phospholipids determined as in Materials and Methods. Radiolabeled precursors present for 18 h in culture. 200
D IN ISLET
CELLS
95
phatidic acid formation was increased at all concentrations reaching statistical significance at 7.5 mM glyceraldehyde, increasing from a basal level of 0.25 + 0.03% (mean & SE) of labeled phospholipids to 0.36 f 0.03% (P < 0.05, df = 12) and was maximal at 20 mM glyceraldehyde (0.44 + 0.05%, P < 0.01, df = 12). When ethanol (1.5%) was included during the incubation there was a concentration-dependent increase in phosphatidylethan01 formation which was maximal at 20 mM glyceraldehyde (0.20 + 0.03% of labeled cellular phospholipids, P < 0.05, df = 12) shown in Fig. 2. This ethanol concentration was shown to be maximally effective for the production of phosphatidylethanol by glyceraldehyde and by acute TPA stimulation (data not shown) and was employed in all subsequent experiments. The time course of arachidonate-labeled phosphatidic acid formation in the presence and absence of ethanol and the formation of phosphatidylethanol is shown in Fig. 3. Despite the concurrent presence of ethanol, at an early time point (5 min) after 20 mM glyceraldehyde phosphatidic acid production is increased significantly (Fig. 3, upper panel). This occurred without a concomitant increase in phosphatidylethanol (Fig. 3, lower panel). Preliminary experiments have shown that this failure of phosphatidylethanol to rise is not due to inadequate delivery of ethanol for transphosphatidylation. Prolonged preincubation with ethanol also failed to increase phosphatidylethanol at this time after glyceraldehyde, whereas no preincubation was required to demonstrate a rise in phosphatidylethanol within 3-5 min of TPA exposure (4). However, at later time points phosphatidic acid production remained raised, but in the presence of ethanol progressive formation of phosphatidylethanol was now seen which was significantly elevated at 20 and 30 min after glyceraldehyde. An increase of
1
190 -
l *
160 170 160 150 -
0
5
10 [Glycersldehyde].
15
20
mM
1. Stimulatory effect of increasing concentrations of glyceraldehyde on [“Clarachidonate-labeled phosphatidic acid (PtdOH) formation. Values are mean f SE for 6-10 determinations at each point expressed as percentage of basal production (values stated in text). Statistically significant differences compared to basal values are indicated: *, P < 0.05; **, P < 0.01. FIG.
[Glycereldehyde],
mM
2. Stimulatory effect of increasing concentrations of glyceraldehyde on [“Clarachidonate-labeled phosphatidylethanol (PtdEtOH) formation in the presence of glyceraldehyde and 1.5% ethanol. Values are mean + SE for 6-10 determinations at each point expressed as percentage of basal production (values stated in text). Statistically significant differences compared to basal values are indicated: *, P < 0.05; **, P < 0.01. FIG.
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96
PHOSPHOLIPASE
D IN ISLET
Endo l 1992 Voll30 l No 1
CELLS El
I,
Glycoraldohydo
I
Glycorrldohydo
1
-, .a.
“0
0
5
10 Timo,
20
30
mlnutra
FIG. 3. Time course for the formation of [“Clarachidonate-labeled phosphatidic acid (PtdOH) and phosphatidylethanol (PtdEtOH). Cells were incubated with 20 mM glyceraldehyde alone or in the concurrent presence of ethanol, the labeled products determined and expressed as percentage of basal formation in the absence of glyceraldehyde. Values are mean f SE for 8-10 determinations at each point. Statistically significant differences compared to 0 time under appropriate control conditions are indicated: *, P < 0.05; **, P < 0.01; and ***, P < 0.005.
86% above basal phosphatidylethanol in the ethanolsubstituted transphosphatidylation product is seen after 20 mM glyceraldehyde stimulation for 30 min. The formation of phosphatidic acid after glyceraldehyde was shown to occur also in [3H]myristate-labeled cells in which phosphatidylcholine is preferentially labeled. In these cells phosphatidic acid increased throughout a 30-min incubation, and phosphatidylethanol was formed in the presence of ethanol with a significant increase above glyceraldehyde alone at 10 min (Fig. 4). When compared to the ethanol-free control at the 30min time point a significant difference is seen in phosphatidic acid production from glyceraldehyde-stimulated cells receiving ethanol. The significant inhibition of phosphatidic acid formation by ethanol coincides with maximum phosphatidylethanol production; the quantitative difference between the rise in phosphatidylethanol and decline in phosphatidic acid with provision of ethanol has been observed in adult rat islets stimulated with phorbol ester and considered due to slower meta-
10
s Tlmo,
0
+ EIhmol 1.5%
mlnutor
FIG. 4. Time course for the formation of [3H]myristate-labeled phosphatidic acid (PtdOH) and phosphatidylethanol (PtdEkOH). Cells were incubated with 20 mM glyceraldehyde alone or in the concurrent presence of ethanol, the labeled products determined and expressed as percentage of basal formation in the absence of glyceraldehyde. Values are mean f SE for 3-10 determinations at each point. Statistically significant differences compared to 0 time under appropriate control conditions are indicated: *, P < 0.05; **, P < 0.01; and ***, P < 0.005.
bolic utilization of phosphatidylethanol compared to phosphatidic acid (4). Figure 5 indicates that phosphatidic acid formation in both arachidonate- and myristate-prelabeled cells could be increased significantly in the presence of 100 PM propranolol. This was seen under both basal (after preincubation with propranolol) and stimulated conditions (propranolol continued throughout the incubation). Propranolol-enhanced phosphatidic acid formation after glyceraldehyde was cumulative. Furthermore, as shown in Table 2, although propranolol had no effect on basal phosphatidylethanol formation (indicating that it does not directly activate PLD), a potentiation in glyceraldehyde-stimulated phosphatidylethanol could be elicited by propranolol (from 0.20 k 0.03 to 0.50 f 0.05% of arachidonate-labeled phospholipids, P < 0.001). This latter figure represents an increase to 455% of basal PLD activity after glyceraldehyde in the presence of propran0101 and indicates that considerable catabolism of phosphatidylethanol as well as in phosphatidic acid is possible in these cells. This effect of propranolol was exploited to amplify PLD-derived metabolites after 18 h preexposure
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PHOSPHOLIPASE 0
n
Con1r0l
D IN ISLET
CELLS
97
dylethanol production was not significantly different from basal at any time point after TPA pretreatment (data not shown).
Proprsn0lol
Glyceraldehyde 0.8 -I
Determination
of diacylglycerol
Arachidonateand myristate-labeled diacylglycerol formation rose by 5 min after glyceraldehyde stimulation and remained elevated over the first 10 min (Table 3). These elevations were matched by a concomitant rise in cellular diacylglycerol mass which was sustained after 30 min exposure to 20 mM glyceraldehyde when no significant elevation in labeled product could be shown. PKC activity Glyceroldshyde 4.0
1
0
5 Time,
10
,,,I
30
minutes
FIG. 5. Time course for formation of phosphatidic acid (PtdOH) from [“Clarachidonateand [3H]myristate-labeled cells in response to glyceraldehyde and propranolol. Cells were incubated with 20 mM glyceraldehyde alone or after preincubation and incubation in the presence of 100 pM propranolol. Values are mean f SE for six determinations at each point and are expressed as percentage of the respective total labeled phospholipids. Statistically significant differences compared to timed values without propranolol are indicated: **, P < 0.01 and ***, P < 0.005.
to 2 pM TPA used to down-regulate PKC and thus to examine the possible intermediacy of PKC in PLD activation (see Introduction) (Table 2). As indicated previously, in the absence of TPA pretreatment a significant increase in labeled phosphatidylethanol followed glyceraldehyde stimulation in the presence of ethanol alone. This was enhanced in the presence of ethanol combined with propranolol. Production of phosphatidylethanol was prevented completely by TPA preexposure. Additionally, the data of Table 2 shows that TPA preexposure may reduce glyceraldehyde-stimulated phosphatidic acid. The rise in phosphatidic acid after 30 min stimulation with 20 mM glyceraldehyde in these cells (176 + 20% of appropriately controlled basal value, P < 0.05) was reduced (to 137 + 21%) in cells with TPA preexposure. This difference was not statistically significant probably due to PKC activation of phosphatidic acid reincorporation into phospholipids in the nondown-regulated cells (see Discussion). In myristate-labeled cells phosphati-
To determine directly whether glyceraldehyde-induced changes in diacylglycerol were associated with changes in membrane-associated PKC, chelator-stable PKC activity was measured over the time course of glyceraldehyde stimulation. As presented in Table 4, an increase in membrane-bound PKC activity was apparent after 5 min of stimulation and was returned to a value not significantly different from basal (0 time point) by 30 min. Total PKC activity (membrane + cytosol) was 8.07 f 0.74 nmol/min. mg protein and was unchanged throughout the period of glyceraldehyde stimulation (data not shown). Pretreatment of the cells with 2 pM TPA for 18 h reduced total PKC activity to 0.87 f 0.12 nmol/min. mg protein, which is a reduction of 89 + 4% in total activity. This decrease was reflected in membrane-bound activity which was reduced in the basal state and in which no change was seen after glyceraldehyde addition (Table 4). These data establish clearly that a rise in intracellular diacylglycerol and in PKC activity is present before the time at which phosphatidylethanol production is demonstrated in parallel incubations in labeled cells in the presence of ethanol. Inclusion of ethanol was without any significant effect on membrane-bound PKC activity at any time point (data not shown). Discussion These data are the first which show that stimulation of a phosphatidylcholine-directed phospholipase (specifically PLD) follows a nutrient stimulus. Formation of a specific PLD product (phosphatidylethanol) in both arachidonate- and myristate-labeled cells is stimulated by glyceraldehyde.’ Formation of myristate-labeled phosphatidylethanol is an indication that phosphatidylcholine, which preferentially incorporates exogenous myris1 We have observed that glucose itself activates PLD in these cells but only after pH reduction; this is in accordance with the known acidic pH optimum of PLD.
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98
PHOSPHOLIPASE
D IN ISLET CELLS
Endo l 1992
Voll30 No 1 l
TABLE 2. Effect of preexposure to TPA on phosphatidic incubation
acid and phosphatidylethanol
formation
in arachidonate-labeled
islet cells over 30 min
Pretreatment Nil Phosphatidic
Product Addition Nil Ethanol, 1.5% Ethanol, 1.5% + propranolol, 100 PM D,L-Glyceraldehyde, 20 mM D,L-Glyceraldehyde, 20 mM + ethanol, 1.5% D,L-Glyceraldehyde, 20 mM + ethanol, 1.5% + propranOlO1, 100 pM
0.25 0.27 0.41 0.44 0.37 0.64
+ f f f + +
0.03 0.03 0.04 0.05” 0.04 0.06
TPA TPA Nil (18 h) (18 h) acid Phosphatidylethanol Percent phospholipid 0.27 f 0.03 0.09 + 0.02 0.10 + 0.02 0.10 f 0.01 0.11 f 0.02 0.11 f 0.01 0.37 f 0.06 0.11 f 0.01 0.12 + O.Olb 0.35 f 0.06 0.20 f 0.03” 0.50 + 0.05’ 0.12 + 0.03d 0.58 f 0.07
Values are mean + SE for 6-10 experimental determinations. ‘Significantly different from appropriately controlled basal value, P < 0.01. * Significantly different from incubation without TPA pretreatment, P C 0.025. c P < 0.01. O-P < 0.001. TABLE 3. Time course of formation of diacylglycerol
after 20 mM glyceraldehyde
[“ClArachidonate-labeled diacylglycerol (% total labeling) Incubation time (min) 1.49 f 0.03 0 5 1.63 f 0.04” 10 1.81 f 0.07* 30 1.62 f 0.06 Values are mean + SE for six to eight determinations at each point. * Significantly different from 0 time point, P C 0.02. *P < 0.005. c P < 0.001. TABLE 4. Activity of membrane-bound hyde
Pretreatment
PKC after 20 mM glyceralde-
Phosphorus transferred (nmol/min . mg membrane protein) nil
TPA
(18 h)
Incubation time (min) 0 2.68 f 0.41 0.38 f 0.10 5 4.93 f 0.60* 0.34 f 0.06 4.33 f 0.546 0.30 f 0.07 10 30 3.19 * 0.41 0.31 f 0.11 Values are mean f SE for five experimental determinations. “Significantly different from activity without pre-treatment, 0.005. * Significantly different from activity at 0 time, P c 0.01.
P
Vol. 130, No. 1 Printed in U.S.A.
Society
Activation of Phospholipase Isolated Islet Cells Follows MARJORIE
DUNLOP
AND
STEWART
University of Melbourne (M.D.), Department 3050, Australia; and Section of Endocrinology Wisconsin, Madison, Wisconsin 53792
D by Glyceraldehyde in Protein Kinase C Activation*
A. METZ Medicine, Royal Melbourne Hospital, Parkville, Victoria (S.A.M.), Department of Medicine, The University of
of
ABSTRACT. Our recent studies have demonstrated the presence in neonatal islet cells and intact adult islets of a phosphatidylcholine-directed phospholipase D (PLD) which is activated after phorbol ester stimulation. The present study describes PLD activation in the presence of a carbohydrate insulin secretagogue. At the highest concentration tested (20 mM) the triose, elvceraldehvde. induced formation of phosnhatidic acid in cells pielabeled with [“Clarachidonic acid or [3-H]myristic acid (164 f 7 and 210 + 9% of basal phosphatidic acid values, respectively). Experimental confirmation of a concentration-dependent specific activation of PLD was provided by the formation of a transphosphatidylation product, phosphatidylethanol, after stimulation with glyceraldehyde in the presence of added ethanol (1.5%). Additionally, there was an early (within 5 min) rise in [“Clarachidonate-labeled diacylglycerol (139 + 7% of basal) accompanied by an increase in intracellular diacylglycerol mass (51 f 2 pmol/mg protein) and an increase in membrane-associated protein kinase C activity (183 f 5% of basal) which preceded the activation of PLD, as indicated by the time course of glycer-
aldehyde-stimulated phosphatidylethanol formation in the presence of ethanol. Pretreatment of islet cells with 2 PM 12-0tetradecanoylphorbol-13-acetate for 18 h, to down-regulate protein kinase C, was without effect on diacylglycerol and phosphatidic acid production after 5 min but inhibited completely the production of phosphatidylethanol at 30 min. The phosphohydrolase inhibitor propranolol (100 PM) potentiated the accumulation of phosphatidic acid and phosphatidylethanol incubation following incubation with glyceraldehyde. These findings demonstrate for the first time that a physiological nutrient activates a phospholipase directed against endogenous phosphatidylcholine in intact islet cells; furthermore, they indicate a role for PLD in a delayed formation of phosphatidic acid in the islet cell. The finding of an earlv rise in alvceraldehvde-stimulated diacylglycerol (which may be formed denovo or by the action of phospholipase C), suggests that PLD is recruited-by the activation of protein kinase C by this nutrient. (Endocrinology 130: 93-101,1992)
H
YDROLYSIS of membrane phospholipids makes a major contribution to intracellular molecules involved in metabolism and growth. In many cell systems there is now considerable evidence for direct receptoragonist-induced formation of diacylglycerol from phosphatidylinositols and for its formation indirectly from phosphatidylcholine after the activation of phospholipase D (PLD) and formation of phosphatidic acid (l-3, reviewed in Ref. 1). We have reported recently that intact adult islets and dispersed islet cells from the neonatal rat contain a PLD directed against phosphatidylcholine, which is activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (4,5), indicating a potential involvement of protein kinase C (PKC) in this activation. A potential functional correlate of this finding is seen in the fact that insulin release is stimulated in adult
rat islets by the addition of an exogenous PLD of bacterial origin, coincident with raised intracellular phosphatidic acid (6), and exogenous phosphatidic acid is a stimulus to insulin secretion in the neonatal islet cell (7). Little is known of the mechanism(s) and timing of phosphatidylcholine breakdown in islets and whether, by formation of phosphatidic acid, this may contribute to nutrient-stimulated insulin release. In the pancreatic islet there is considerable evidence for PLC-catalyzed hydrolysis of phosphatidylinositol(4,5)bisphosphate after receptor-agonist activation and exposure to nutrient secretagogues (3-10). Subsequent to PLC action, phosphatidic acid can be formed from diacylglycerol via diacylglycerol kinase but also can be formed de novo from nutrient sources (8, 11-13). Although a role for PKC in insulin release is contested (14-B), the activation of PKC by phorbol esters (14,1921) or cell-permeable diglycerides (22) is accompanied by insulin release in islets or insulin-secreting tumoral cell lines, suggesting that endogenous activation of PKC may be involved in the action of both receptor and nutrient secretagogues. Also, chelator-stable translocation of pro-
Received August 2, 1991. Address all correspondence and requests for reprints to: Dr. M. Dunlop, University of Melbourne, Department of Medicine, Clinical Science Building, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. *This work was supported by NIH Grant DK-37312 and by the National Health and Medical Research Council of Australia. 93
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94
PHOSPHOLIPASE
tein kinase C accompanies receptor-agonist stimulation (15) and was shown recently for the a-isoenzyme of PKC in glucose-perifused islet cells (23). Recently, a significant positive contribution of PKC activation to insulin release was shown after glyceraldehyde stimulation in the RINm5F islet P-cell subclone (24). Although the present study is not designed to address this aspect, as indicated above, activation of PLD may play a role in PKC-related insulin release. In the present study evidence is provided for glyceraldehyde-stimulated PLD activity in neonatal islet cells, an event which requires the activation of cellular PKC. PLD-mediated hydrolysis was investigated by the specific formation of phosphatidylethanol via a PLD-mediated transphosphatidylation in the presence of a primary alcohol (e.g. ethanol) (1, 4). Since phosphatidylethan01 formation (i.e. phosphatidyl transfer to alcohol) always occurs at the expense of PLD-derived phosphatidic acid (phosphatidyl transfer to water), this maneuver enabled us to discern the contribution made by PLD to cellular phosphatidic acid.
Materials
and Methods
[1-Y]Arachidonic acid, [9,10(n)-3H]myristic acid, [r”‘P] ATP, and reagents for the determination of PKC were obtained from Amersham (Amersham, UK). Insulin RIA reagents (Phadeseph) were from Pharmacia Diagnostics AB (Uppsala, Sweden). All other chemicals were from Sigma (St. Louis, MO). Cultured neonatal islet cells were prepared from l-day-old rat pancreases which were dissected and collagenase digested as described by Hellerstrom et al. (25). Digests were washed three times by suspension and sedimentation (5 min x 1 g) in medium RPM1 1640 containing 11.1 mM glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% (vol/vol) fetal bovine serum, buffered to pH 7.4 with HEPES to give discrete islet cell clusters when viewed by stereomicroscopy. These cells were cultured in the above medium containing 0.1 mM 3isobutyl-1-methylxanthine for 48 h (26). We have previously employed this technique to deter the rapid growth of fibroblasts and to promote formation of islet cell monolayers (27) in which over 80% (87 + 3%, four determinations) routinely give positive specific fluorescence when preincubated with an excess of insulin (1 pg/ml) before processing and incubation with guineapig antiporcine insulin immunoglobulin followed by fluorescein isothiocyanate-conjugated rabbit antiguinea pig globulin as described (28). Islet cell culture was continued for a further 48 h in RPM1 1640 containing 11.1 mM glucose and 10% (vol/vol) fetal bovine serum before cells were labeled in culture with [l“‘Clarachidonic acid, 54.7 mCi/mmol (final activity 50 nCi/ ml), or [9,10(n)3H]myristic acid, 53 Ci/mmol (final activity 1 rCi/ml) and preincubated as described (4, 5). We have determined previously that cells cultured in this manner maintain an insulin secretory response to nutrient stimuli. This is shown by a 4- and 6-fold increase, over insulin release at 1.7 mM glucose, when cells were incubated for 15 min with 16.7 mM glucose or 20 mM glyceraldehyde, respectively (29).
D IN ISLET CELLS
Endo. 1992 Voll30. No 1
In the following studies when present, 2 pM TPA in dimethylsulfoxide or dimethylsulfoxide (0.01%) alone were added for the final 18 h of culture. Islet cells were washed with Krebs Ringer bicarbonate buffer containing 5.6 mM glucose, 25 pM choline, and 3 pM arachidonic acid and preincubated in this buffer without further additions or with propranolol (100 pM) for 20 min. Timed incubations in the presence of glyceraldehyde (2.5-20 mM), ethanol (1.5%), or the phosphohydrolase inhibitor propranolol(lO0 pM) alone or in combination followed. Medium was removed rapidly and the cells extracted for lipid determination as described (5). For determination of diacylglycerol mass and PKC activity, islet cells were cultured and incubated in an identical manner, except that no radiolabeled compounds were present in the final overnight culture. For the determination of radiolabeled phosphatidic acid, phosphatidylethanol, and diacylglycerol the extracted lipids were applied to preactivated TLC plates and separated using multiple solvent systems as described (5). The radiolabeled compounds were localized by autoradiography, removed, and quantitated by liquid scintillation spectrometry. Values for individual metabolites were expressed as percentages of total labeled phospholipid to normalize values for experimental variations in cell number after it was established that none of the pretreatment conditions affected total phospholipid labeling. Diacylglycerol mass was determined by enzymatic conversion of extracted diacylglycerol to [32P]phosphatidic acid (30). Cellular lipid extracts were suspended with 1.5% (wt/vol) noctyl-@-glucopyranoside and 1 mM cardiolipin with 20 mM diethylaminepentaacetic acid in 10 mM imidazole buffer and treated with an excess of Escherichia coli diacylglycerol kinase in the presence of [-y3’P]ATP, specific activity 250,000 dpm/ nmol. [32P]Phosphatidic acid was extracted and separated by TLC. The mass of [32P]phosphatidic acid resulting from diacylglycerol phosphorylation of lipid extracts and sn-1,2 diacylglycerol standards was determined. PKC was assayed by the transfer of y-phosphate of [-$‘P] ATP to the threonine group of a specific PKC substrate octapeptide in a mixed micellar assay (31). PKC activity was determined on membrane and cytosolic fractions obtained from cells disrupted in 20 mM Tris HCl, 0.5 mM EGTA, and 0.25 M sucrose, containing 50 pM digitonin and 50 pg/ml leupeptin at pH 7.5. After rapid centrifugation at 13,000 x g cell membranes were solubilized with 1.0% Triton X-100 in 20 mM Tris HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, and 50 pg/ml leupeptin. PKC activity was determined by the addition of a reaction mixture containing 50 mM Tris HCl, 1 mM calcium acetate, 15 mM magnesium acetate, and 2.5 mM dithiothreitol in a 50-~1 micellar mixture of 375 mg L-cu-phosphatidylserine and 0.15 pg TPA containing 0.46 nmol substrate peptide and initiated by the addition of 50 WM[-y3’P]ATP (final activity, 53 &i/rmol). After a 15-min incubation period the phosphorylated peptide was precipitated by acid addition and recovered on paper filters which were washed extensively before determination of 32P content. PKC activity was calculated by subtracting the 32P incorporated into substrate in the absence of phospholipid micelle, and activity in the membrane fraction was expressed as nanomoles of 32Pphosphate incorporated per min/mg protein. Total protein in the membrane fraction was
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PHOSPHOLIPASE determined with Coomassie brilliant blue dye (32). Results are expressed as mean + SE with either the number of individual experimental determinations or degrees of freedom (df) indicated. The statistical difference between mean values was obtained using Student’s t test.
Results Labeling of phosphatidylcholine
in islet cells
Incorporation of the radiolabeled precursor fatty acids into phosphatidylcholine is shown in Table 1. Under the labeling conditions phosphatidylcholine was preferentially labeled with myristic acid. This incorporation reflected that of choline [determined previously (9), where the choline label distributes between phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin phospholipid species only]. Arachidonic acid was more widely distributed among phospholipid classes. Determination of phosphatidic phosphutidylethanol
acid and
Incubation of [14C]arachidonate-prelabeled islet cells with glyceraldehyde (2.5-20 InM) for 30 min resulted in a significant concentration-dependent increase in the production of labeled phosphatidic acid (Fig. 1). Ph_osTABLE
1.
Labeling of phosphatidylcholine
in neonatal islet cells
Radiolabeled precursor
Total phospholioids (%)
[“ClArachidonic acid [3H]Myristic acid IWlCholine
35.7 f 0.4 84.8 + 0.6 95.7 f 0.1
Values shown are mean + SEM the disintegrations per minute present in phosphatidylcholine expressed as a percentage of total disintegrations per minute in all phospholipids determined as in Materials and Methods. Radiolabeled precursors present for 18 h in culture. 200
D IN ISLET
CELLS
95
phatidic acid formation was increased at all concentrations reaching statistical significance at 7.5 mM glyceraldehyde, increasing from a basal level of 0.25 + 0.03% (mean & SE) of labeled phospholipids to 0.36 f 0.03% (P < 0.05, df = 12) and was maximal at 20 mM glyceraldehyde (0.44 + 0.05%, P < 0.01, df = 12). When ethanol (1.5%) was included during the incubation there was a concentration-dependent increase in phosphatidylethan01 formation which was maximal at 20 mM glyceraldehyde (0.20 + 0.03% of labeled cellular phospholipids, P < 0.05, df = 12) shown in Fig. 2. This ethanol concentration was shown to be maximally effective for the production of phosphatidylethanol by glyceraldehyde and by acute TPA stimulation (data not shown) and was employed in all subsequent experiments. The time course of arachidonate-labeled phosphatidic acid formation in the presence and absence of ethanol and the formation of phosphatidylethanol is shown in Fig. 3. Despite the concurrent presence of ethanol, at an early time point (5 min) after 20 mM glyceraldehyde phosphatidic acid production is increased significantly (Fig. 3, upper panel). This occurred without a concomitant increase in phosphatidylethanol (Fig. 3, lower panel). Preliminary experiments have shown that this failure of phosphatidylethanol to rise is not due to inadequate delivery of ethanol for transphosphatidylation. Prolonged preincubation with ethanol also failed to increase phosphatidylethanol at this time after glyceraldehyde, whereas no preincubation was required to demonstrate a rise in phosphatidylethanol within 3-5 min of TPA exposure (4). However, at later time points phosphatidic acid production remained raised, but in the presence of ethanol progressive formation of phosphatidylethanol was now seen which was significantly elevated at 20 and 30 min after glyceraldehyde. An increase of
1
190 -
l *
160 170 160 150 -
0
5
10 [Glycersldehyde].
15
20
mM
1. Stimulatory effect of increasing concentrations of glyceraldehyde on [“Clarachidonate-labeled phosphatidic acid (PtdOH) formation. Values are mean f SE for 6-10 determinations at each point expressed as percentage of basal production (values stated in text). Statistically significant differences compared to basal values are indicated: *, P < 0.05; **, P < 0.01. FIG.
[Glycereldehyde],
mM
2. Stimulatory effect of increasing concentrations of glyceraldehyde on [“Clarachidonate-labeled phosphatidylethanol (PtdEtOH) formation in the presence of glyceraldehyde and 1.5% ethanol. Values are mean + SE for 6-10 determinations at each point expressed as percentage of basal production (values stated in text). Statistically significant differences compared to basal values are indicated: *, P < 0.05; **, P < 0.01. FIG.
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96
PHOSPHOLIPASE
D IN ISLET
Endo l 1992 Voll30 l No 1
CELLS El
I,
Glycoraldohydo
I
Glycorrldohydo
1
-, .a.
“0
0
5
10 Timo,
20
30
mlnutra
FIG. 3. Time course for the formation of [“Clarachidonate-labeled phosphatidic acid (PtdOH) and phosphatidylethanol (PtdEtOH). Cells were incubated with 20 mM glyceraldehyde alone or in the concurrent presence of ethanol, the labeled products determined and expressed as percentage of basal formation in the absence of glyceraldehyde. Values are mean f SE for 8-10 determinations at each point. Statistically significant differences compared to 0 time under appropriate control conditions are indicated: *, P < 0.05; **, P < 0.01; and ***, P < 0.005.
86% above basal phosphatidylethanol in the ethanolsubstituted transphosphatidylation product is seen after 20 mM glyceraldehyde stimulation for 30 min. The formation of phosphatidic acid after glyceraldehyde was shown to occur also in [3H]myristate-labeled cells in which phosphatidylcholine is preferentially labeled. In these cells phosphatidic acid increased throughout a 30-min incubation, and phosphatidylethanol was formed in the presence of ethanol with a significant increase above glyceraldehyde alone at 10 min (Fig. 4). When compared to the ethanol-free control at the 30min time point a significant difference is seen in phosphatidic acid production from glyceraldehyde-stimulated cells receiving ethanol. The significant inhibition of phosphatidic acid formation by ethanol coincides with maximum phosphatidylethanol production; the quantitative difference between the rise in phosphatidylethanol and decline in phosphatidic acid with provision of ethanol has been observed in adult rat islets stimulated with phorbol ester and considered due to slower meta-
10
s Tlmo,
0
+ EIhmol 1.5%
mlnutor
FIG. 4. Time course for the formation of [3H]myristate-labeled phosphatidic acid (PtdOH) and phosphatidylethanol (PtdEkOH). Cells were incubated with 20 mM glyceraldehyde alone or in the concurrent presence of ethanol, the labeled products determined and expressed as percentage of basal formation in the absence of glyceraldehyde. Values are mean f SE for 3-10 determinations at each point. Statistically significant differences compared to 0 time under appropriate control conditions are indicated: *, P < 0.05; **, P < 0.01; and ***, P < 0.005.
bolic utilization of phosphatidylethanol compared to phosphatidic acid (4). Figure 5 indicates that phosphatidic acid formation in both arachidonate- and myristate-prelabeled cells could be increased significantly in the presence of 100 PM propranolol. This was seen under both basal (after preincubation with propranolol) and stimulated conditions (propranolol continued throughout the incubation). Propranolol-enhanced phosphatidic acid formation after glyceraldehyde was cumulative. Furthermore, as shown in Table 2, although propranolol had no effect on basal phosphatidylethanol formation (indicating that it does not directly activate PLD), a potentiation in glyceraldehyde-stimulated phosphatidylethanol could be elicited by propranolol (from 0.20 k 0.03 to 0.50 f 0.05% of arachidonate-labeled phospholipids, P < 0.001). This latter figure represents an increase to 455% of basal PLD activity after glyceraldehyde in the presence of propran0101 and indicates that considerable catabolism of phosphatidylethanol as well as in phosphatidic acid is possible in these cells. This effect of propranolol was exploited to amplify PLD-derived metabolites after 18 h preexposure
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PHOSPHOLIPASE 0
n
Con1r0l
D IN ISLET
CELLS
97
dylethanol production was not significantly different from basal at any time point after TPA pretreatment (data not shown).
Proprsn0lol
Glyceraldehyde 0.8 -I
Determination
of diacylglycerol
Arachidonateand myristate-labeled diacylglycerol formation rose by 5 min after glyceraldehyde stimulation and remained elevated over the first 10 min (Table 3). These elevations were matched by a concomitant rise in cellular diacylglycerol mass which was sustained after 30 min exposure to 20 mM glyceraldehyde when no significant elevation in labeled product could be shown. PKC activity Glyceroldshyde 4.0
1
0
5 Time,
10
,,,I
30
minutes
FIG. 5. Time course for formation of phosphatidic acid (PtdOH) from [“Clarachidonateand [3H]myristate-labeled cells in response to glyceraldehyde and propranolol. Cells were incubated with 20 mM glyceraldehyde alone or after preincubation and incubation in the presence of 100 pM propranolol. Values are mean f SE for six determinations at each point and are expressed as percentage of the respective total labeled phospholipids. Statistically significant differences compared to timed values without propranolol are indicated: **, P < 0.01 and ***, P < 0.005.
to 2 pM TPA used to down-regulate PKC and thus to examine the possible intermediacy of PKC in PLD activation (see Introduction) (Table 2). As indicated previously, in the absence of TPA pretreatment a significant increase in labeled phosphatidylethanol followed glyceraldehyde stimulation in the presence of ethanol alone. This was enhanced in the presence of ethanol combined with propranolol. Production of phosphatidylethanol was prevented completely by TPA preexposure. Additionally, the data of Table 2 shows that TPA preexposure may reduce glyceraldehyde-stimulated phosphatidic acid. The rise in phosphatidic acid after 30 min stimulation with 20 mM glyceraldehyde in these cells (176 + 20% of appropriately controlled basal value, P < 0.05) was reduced (to 137 + 21%) in cells with TPA preexposure. This difference was not statistically significant probably due to PKC activation of phosphatidic acid reincorporation into phospholipids in the nondown-regulated cells (see Discussion). In myristate-labeled cells phosphati-
To determine directly whether glyceraldehyde-induced changes in diacylglycerol were associated with changes in membrane-associated PKC, chelator-stable PKC activity was measured over the time course of glyceraldehyde stimulation. As presented in Table 4, an increase in membrane-bound PKC activity was apparent after 5 min of stimulation and was returned to a value not significantly different from basal (0 time point) by 30 min. Total PKC activity (membrane + cytosol) was 8.07 f 0.74 nmol/min. mg protein and was unchanged throughout the period of glyceraldehyde stimulation (data not shown). Pretreatment of the cells with 2 pM TPA for 18 h reduced total PKC activity to 0.87 f 0.12 nmol/min. mg protein, which is a reduction of 89 + 4% in total activity. This decrease was reflected in membrane-bound activity which was reduced in the basal state and in which no change was seen after glyceraldehyde addition (Table 4). These data establish clearly that a rise in intracellular diacylglycerol and in PKC activity is present before the time at which phosphatidylethanol production is demonstrated in parallel incubations in labeled cells in the presence of ethanol. Inclusion of ethanol was without any significant effect on membrane-bound PKC activity at any time point (data not shown). Discussion These data are the first which show that stimulation of a phosphatidylcholine-directed phospholipase (specifically PLD) follows a nutrient stimulus. Formation of a specific PLD product (phosphatidylethanol) in both arachidonate- and myristate-labeled cells is stimulated by glyceraldehyde.’ Formation of myristate-labeled phosphatidylethanol is an indication that phosphatidylcholine, which preferentially incorporates exogenous myris1 We have observed that glucose itself activates PLD in these cells but only after pH reduction; this is in accordance with the known acidic pH optimum of PLD.
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98
PHOSPHOLIPASE
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Voll30 No 1 l
TABLE 2. Effect of preexposure to TPA on phosphatidic incubation
acid and phosphatidylethanol
formation
in arachidonate-labeled
islet cells over 30 min
Pretreatment Nil Phosphatidic
Product Addition Nil Ethanol, 1.5% Ethanol, 1.5% + propranolol, 100 PM D,L-Glyceraldehyde, 20 mM D,L-Glyceraldehyde, 20 mM + ethanol, 1.5% D,L-Glyceraldehyde, 20 mM + ethanol, 1.5% + propranOlO1, 100 pM
0.25 0.27 0.41 0.44 0.37 0.64
+ f f f + +
0.03 0.03 0.04 0.05” 0.04 0.06
TPA TPA Nil (18 h) (18 h) acid Phosphatidylethanol Percent phospholipid 0.27 f 0.03 0.09 + 0.02 0.10 + 0.02 0.10 f 0.01 0.11 f 0.02 0.11 f 0.01 0.37 f 0.06 0.11 f 0.01 0.12 + O.Olb 0.35 f 0.06 0.20 f 0.03” 0.50 + 0.05’ 0.12 + 0.03d 0.58 f 0.07
Values are mean + SE for 6-10 experimental determinations. ‘Significantly different from appropriately controlled basal value, P < 0.01. * Significantly different from incubation without TPA pretreatment, P C 0.025. c P < 0.01. O-P < 0.001. TABLE 3. Time course of formation of diacylglycerol
after 20 mM glyceraldehyde
[“ClArachidonate-labeled diacylglycerol (% total labeling) Incubation time (min) 1.49 f 0.03 0 5 1.63 f 0.04” 10 1.81 f 0.07* 30 1.62 f 0.06 Values are mean + SE for six to eight determinations at each point. * Significantly different from 0 time point, P C 0.02. *P < 0.005. c P < 0.001. TABLE 4. Activity of membrane-bound hyde
Pretreatment
PKC after 20 mM glyceralde-
Phosphorus transferred (nmol/min . mg membrane protein) nil
TPA
(18 h)
Incubation time (min) 0 2.68 f 0.41 0.38 f 0.10 5 4.93 f 0.60* 0.34 f 0.06 4.33 f 0.546 0.30 f 0.07 10 30 3.19 * 0.41 0.31 f 0.11 Values are mean f SE for five experimental determinations. “Significantly different from activity without pre-treatment, 0.005. * Significantly different from activity at 0 time, P c 0.01.
P
