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Glucose and carbachol synergistically stimulate phosphatidic acid accumulation in pancreatic islets
Vol. 180, No. 2, 1991
October 31, 1991
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 960-966
GLUCOSE AND CARBACHOL SYNERGISTICALLY STIMULATE PHOSPHATIDIC ACID ACCUMULATION IN PANCREATIC ISLETS"
Robert J. Konrad, Y. Camille Jolly, and Bryan A. Wolf "~
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6082 Received September 18, 1991
Summary: Phosphatidic acid has been previously implicated as an intracellular mediator of insulin secretion. Very little is known, however, about endogenous phosphatidic acid levels in islets. We now show, for the first time, that glucose and carbachol, at concentrations which stimulate insulin secretion, significantly increase endogenous phosphatidic acid levels in pancreatic islets by 2-fold at 1 min, nearly 3-fold at 2 min, and over 3-fold at 30 rain compared to control. Possible mechanisms include de novo synthesis from glucose and/or activation of phospholipase D. Our data, taken together with previous studies, suggest that phosphatidic acid may have a central role in insulin secretion as an intracellular mediator. ® 1991Ao~d~micPr.... Inc. Different classes of secretagogues are capable of stimulating insulin secretion from the islets of Langerhans (1). D-glucose is an example of a fuel secretagogue and is the major stimulus for insulin secretion (2,3). Carbachol which binds to the muscarinic receptor
in islets also stimulates
insulin secretion
(4). The exact
mechanism of action of each of these secretagogues is not completely elucidated (5). Oxidation of glucose is believed to increase the I~-cell intracellular ATP/ADP ratio which results in closure of the K ÷ channels, depolarization, influx of extracellular calcium through voltage-dependent Ca 2+ channels, increase in intracellular calcium and insulin exocytosis (6). Carbachol activates phospholipase C which generates the second messengers inositol 1,4,5-trisphosphate and 1,2-diacyl-sn-glycerol (7).
" This work was supported by National Institutes of Health Research Grant RO1 DK43354 (to B.A.W.), an American Diabetes Association Research and Development Award (to B.A.W.), and a University of Pennsylvania Diabetes Endocrinology Research Center Pilot Feasibility grant (to B.A.W., NIH DK19525-14). ""
To whom correspondence should be addressed.
0006-29lX/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
960
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Phosphatidic acid has recently been recognized as a potential mediator of insulin secretion (8). Exogenous phosphatidic acid increases insulin secretion from neonatal r$-cell (9). Phosphatidic acid in islets may be generated by three different mechanisms: 1 ) activation of phospholipase D (10), 2) d e n o v o synthesis from glucose ( 1 1 - 14), and 3) activation of phospholipase C and diacylglycerol kinase. Recent studies have shown that exogenous phospholipase D is capable of stimulating insulin release from islets at the same time as it increases intracellular phosphatidic acid levels (1 5). In order to implicate phosphatidic acid as an intracellular mediator of insulin secretion, it is necessary to demonstrate that insulin secretagogues increase endogenous levels of phosphatidic acid. Endogenous phosphatidic acid levels have, however, been difficult to measure in islets because of low levels (14), technical problems in separating phosphatidic acid from other phospholipids (16), and the small amounts of islets routinely obtainable (17).
We have therefore used a sensitive 2-dimensional TLC
system coupled to a very sensitive radioactivity detection instrument to measure endogenous phosphatidic acid levels in islets during stimulation with two important secretagogues, glucose and carbachol.
MATERIALS AND METHODS Islet isolation, labeling with [ZH]palmitic acid, and incubation - Islets were isolated aseptically from the pancreas of 8 male Sprague-Dawley rats as previously described (18). Freshly isolated islets (approx. 3,000) were incubated 24 hours under sterile conditions at 37°C under an atmosphere of 95% air/5% CO2 in a sterile Petri dish containing 2.5 ml of complete CMRL-1066 and 50 pCi of [3H]palmitic acid (19). Following labeling, islets were washed five times in fresh modified Krebs'-Hepes buffer (25 mM Hepes pH 7.4, 115 mM NaCI, 24 mM NaHCO3, 5 mM KCI, 2.5 mM CaCI 2, 1 mM MgCI 2, 0 . 1 % bovine serum albumin) supplemented with 3 mM D-glucose and used immediately. [3H]palmitic acid-labeled islets (125-200 per condition) were preincubated in Krebs'-Hepes buffer (3 mM glucose) and then incubated for 1-30 min in fresh Krebs'-Hepes medium (3 mM or 28 mM glucose and/or 0.5 mM carbachol). The reaction was stopped by the addition of 2 ml of ice-cold chloroform/methanol (1:2, v/v) supplemented with 0.25% of the anti-oxidant butylated hydroxytoluene. The tubes were then chilled for 15 rain in a dry ice/ethanol bath, and stored at -20°C prior to analysis. Two-dimensional TLC analysis of phosphatidic acid - Prior to extraction, carrier amounts (5 pg) of phospholipids, neutral lipids and fatty acids were added to each tube to aid in recovery and endogenous phosphatidic acid was extracted essentially as described (20). Samples were applied to 10x10 cm high-performance HP-K silica gel TLC plates (Whatman Biosystems Inc, Clifton, N J) which had been activated 30 rain at 110°C. Plates were developed in the first dimension with chloroform/methanol/ammonium hydroxide (65:35:5.5; v/v) for 30 rain. Plates were carefully dried (60 rain) and then developed in the second dimension with chloroform/methanol/formic acid/water (55:28:5:1; v/v) for 30 rain. Radioactivity of 961
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
each plate was quantitated by 2-dimensional analysis with the Berthold Linear Analyzer 284 (Nashua, NH) equipped with a position-sensitive proportional highresolution counter tube (200 mm long, 1380 V) continuously flushed (0.5 L/min) with PIO gas (90% argon/10% methane) and fitted with a 2 mm entrance window. Each plate was scanned at least 12 hours. Resolution in the first-dimension was 0.5 mm and 2 mm in the second-dimension. Data analysis was performed using version 4.07 of the 2D-TLC software. Peaks were integrated after subtracting the background reading of a corresponding and adjacent region. In all cases, counts in the peaks of interest were at least 5-fold greater than background. Radioactivity in each peak was expressed as the % of total counts on the plate. The identity of the peaks was assigned by comparison with cold phospholipids standards, commercial radioactive standards, and labeling of the islet phospholipids with the appropriate radiolabeledhead group. The following peaks were identified and localized: phosphatidylcholine (Rf 1 in the first dimension: 0.19, Rf2 in the second dimension: 0.38), lysophosphatidylcholine (Rfl: 0.03, Rf2:0.11), phosphatidylethanolamine (Rfl: 0.31, Rf2: 0.63), sphingomyelin (Rfl: 0.09, Rf2: 0.23), phosphatidylserine (Rfl: 0.06, Rf2: 0.39), phosphatidylinositol (Rfl: 0.01, Rf2: 0.28), free fatty acid (Rf~: 0,28, R~2:0.85), phosphatidic acid (Rfl: 0.08, Rf2: 0.63). One-dimensional TLC analysis of fatty acids and diacylglycerol- Samples were spotted onto the pre-adsorbent zone of channeled silica gel G TLC 20x20 cm plates (Analtech, Newark, DE) which had been activated 30 min at 110°C. Plates were developed for 30-45 rain in petroleum ether (30-60°C)/diethyl ether/acetic acid (140:60:2, v/v/v) as previously described (21).
RESULTS Phosphatidic acid accumulation was measured by labeling islets 24 hours to isotopic equilibrium using [3H]palmitic acid. Labeled islets were then challenged with 0.5 mM carbachol and 28 mM glucose.
[3H-palmityl]phosphatidic
acid was extracted and then
separated from other phospholipids and neutral lipids by 2D-TLC followed by radioactivity detection with a high-resolution linear analyzer. As shown in figure 1,
[3H-palmityl]phosphatidic
acid is clearly separated from other radiolabeled lipids. The
combination of stimulatory concentrations of glucose (28 mM) and carbachol (0.5 raM) increased endogenous phosphatidic acid levels by 2-fold at 1 min, nearly 3-fold at 2 min, and over 3-fold at 30 min as compared to the non-stimulatory 3 mM glucose condition (Figure 2, p < 0.0001
by 2-way ANOVA). At 30 min, under these
experimental conditions, glucose and carbachol caused a robust insulin secretory response (4-6 fold increase, data not shown). The effects of glucose and carbachol alone on phosphatidic acid and diacylglycerol accumulation are shown in Table 1. Each secretagogue caused phosphatidic acid accumulation, but the combination of glucose and carbachol was synergistic.
No
effect
of
either
secretagogue 962
was
observed
on
[3H-
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
¢3 ¢3 G" ¢3 Z
w Z
7=== -5"O
ol
L, <
-= 2nd dimension CHCI3 / CH3OH / HCOOH / H20 (55:28:5:1, v / v)
Figure 1. Two-dimensional thin-layer chromatography of phosphatidic acid from [3H]palmityl-labeled islets. PanelA: G3 (3 m M glucose, 30 min); Panel B: G28 + CCH (28 m M glucose + 0.5 m M carbachol, 30 rain). PA: phosphatidic acid, PS: phosphatidylserine, Ph phosphatidylinositol, LPC: lysophosphatidylcholine, SM: sphingomyelin, PC: phosphatidylcholine, PE: phosphatidylethanolamine, C16:0: palmitic acid.
palmityl]diacylglycerol levels after 30 min incubation (Table 1 ) nor at early time points (1 min, 2 rain, data not shown).
DISCUSSION We demonstrate, for the first time, that the combination of glucose and carbachol causes an almost immediate increase in levels of phosphatidic acid. Three
o
G28+CCH
400
//-!
o
~,~
300
o
.~
200
/
'Z.
~
G3
100
012
30 TIME [mini
Figure 2. Effect of glucose and carbachol on phosphatidic acid accumulation in islets labeled 24 h with [~H]palmitic acid. Results are shown as the Mean ± S.E.M. of phosphatidic acid accumulation expressed as a percentage of the 3 mM glucose control from 2 to 7 observations per condition. G 3 : 3 mM glucose, G28 + CCH: 28 mM glucose + 0.5 mM carbachol. 963
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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table 1. Effect of glucose and carbachol on diacylglycerol and phosphatidic acid accumulation in islets labeled with [3H]palmitic acid. islets prelabeled 24 h with
[3H]palmitic acid were incubated 30 rain with glucose and/or carbachol. Results are shown as the Mean ± S.E.M. expressed as a % of 3 mM glucose control from 4 to 7 observations per condition. *: p < 0.05 compared to 3 mM glucose control. Diacylglycerol (% of control) 3 mM glucose 28 mM glucose
Phosphatidic acid (% of control)
100.0 + 2.5
100.0 + 6.2
99.6 ± 1.8
144.6 + 8.3
3 mM glucose + 0.5 mM carbachol
113.7 4- 5.7
28 mM glucose + 0.5 mM carbachol
118.6 4- 4.3
200.0 + 17.7" 314.7 ± 43.7"
different mechanisms may result in phosphatidic acid accumulation. One possibility is that carbachol
and glucose stimulate
diacylglycerol
accumulation
following
phospholipase C activation (7). Diacylglycerol is then phosphorylated by diacylglycerol kinase to phosphatidic acid. Diacylglycerol levels, however, did not significantly increase. Previous studies have also failed to detect any glucose-induced increases in diacylglycerol
(19,21).
Our observations can not rule out the possibility that
diacylglycerol is so rapidly phosphorylated to phosphatidic acid that any increases are masked, although this appears unlikely. Alternatively, phosphatidic acid may be synthesized de n o v o from glucose according to the following scheme: metabolism of glucose to the triose phosphate dihydroxyacetone phosphate, acylation to acyldihydroxyacetone phosphate, reduction to lysophosphatidic acid, and acylation to phosphatidic acid (22). Previous studies have s h o w n that this pathway of de n o v o synthesis from glucose is present in islets but it is not clear h o w much this pathway contributes to phosphatidic acid accumulation (21).
Phospholipase D activation results in phosphatidic acid accumulation (23). The addition
of
exogenous phospholipase
D to
islets
results
in accumulation
of
endogenous phosphatidic acid while stimulating insulin secretion (1 5). Phospholipase D activity, measured as the production of phosphatidylethanol in the presence of ethanol, is present in islets (24,25). Furthermore, a number of pharmacological probes (but not glucose nor carbachol) have been shown to stimulate phospholipase D in islets (10). 964
Vol.
180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Regardless of the mechanisms of phosphatidic acid accumulation, phosphatidic acid appears to be a potential mediator of insulin secretion since exogenous phosphatidic acid stimulates insulin secretion and increases intracellular Ca 2+ levels (9). Furthermore, inhibition of phosphatidic acid metabolism with propranolol also stimulates
insulin secretion
(25).
Whether
phosphatidic acid phosphorylates
endogenous substrates in islets as reported in heart, liver, brain, lung, and testis remains to be determined (26). In summary, we have demonstrated, for the first time, that the muscarinic agonist and insulin secretagogue carbachol stimulates phosphatidic acid accumulation in islets. We have also demonstrated a similar effect with glucose and a synergistic effect when islets are challenged with the combination of glucose and carbachol. Our data, taken together with that of previous studies (8), suggests that phosphatidic acid may play a central role in the process of insulin secretion.
REFERENCES
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
Ashcroft, S.J. (1980) Diabetologia 18, 5-15. Hedeskov, C.J. (1980) Physiol. Rev. 60, 442-509. Meglasson, M.D. and Matschinsky, F.M. (1986) Diabetes Metab. Rev. 2, 163-214. Prentki, M. and Matschinsky, F.M. (1987) Physiol. Rev. 67, 1185-1248. Matschinsky, F.M. (1990) Diabetes 39, 647-652. Dunne, M.J. and Petersen, O.H. (1991) Biochim. Biophys. Acta 1071, 67-82. Turk, J., Wolf, B.A. and McDaniel, M.L. (1987) Prog. Lipid Res. 26, 125-181. Metz, S. and Dunlop, M. (1991) Adv. Prostaglandin. Thromboxane.Leukotriene. Res. 21A, 287-290. Dunlop, M.E. and Larkins, R.G. (1989) Diabetes 38, 1187-1192. Dunlop, M. and Metz, S.A. (1989) Biochem. Biophys. Res. Commun. 163, 922-928. Best, L. and Malaisse, W.J. (1984) Arch. Biochem. Biophys. 234, 253-257. Dunlop, M. and Larkins, R.G. (1985) FEBS Lett. 193, 231-235. Dunlop, M.E. and Larkins, R.G. (1985) Biochem. Biophys. Res. Commun. 132, 467-473. Farese, R.V., DiMarco, P.E., Barnes, D.E., Sabir, M.A., Larson, R.E., Davis, J.S. and Morrison, A.D. (1986) Endocrinology 118, 1498-1503. Metz, S.A. and Dunlop, M. (1990) Biochem. J. 270, 427-435. Wolf, B.A., Turk, J., Sherman, W.R. and McDaniel, M.L. (1986) J. Biol. Chem. 261, 3501-3511. Wolf, B.A., Easom, R.A., Hughes, J.H., McDaniel, M.L. and Turk, J. (1989) Biochemistry 28, 4291-4301. Wolf, B.A., Pasquale, S.M. and Turk, J. (1991) Biochemistry 30, 6372-6379. Wolf, B.A., Easom, R.A., McDaniel, M.L. and Turk, J. (1990) J. Clin. Invest. 85, 482-490. 965
Vol. 180, No. 2, 1 9 9 1 [20]
[21] [22] [23] [24]
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Vance, D.E. Phospholipid Metabolism in Eucaryotes. In: Biochemistry of Lipids and Membranes, edited by Vance, D.E. and Vance, J,E. Menlo Park: The Benjamin/Cummings Publishing Company, Inc., 1985, p. 242-270. Dennis, E.A., Rhee, S.G., Billah, M.M. and Hannun, Y.A. (1991) FASEB J. 5, 2068-2077. Metz, S.A. and Dunlop, M. (1990) Arch. Biochem. Biophys. 283, 417-428. Metz, S.A. and Dunlop, M. (1991) Biochem. Pharmacol. 41, R1-R4. Bocckino, S.Bo, Wilson, P.B. and Exton, J.H. (1991) Proc. Natl. Acad. Sci. U, S. A. 88, 6210-6213.
966
October 31, 1991
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 960-966
GLUCOSE AND CARBACHOL SYNERGISTICALLY STIMULATE PHOSPHATIDIC ACID ACCUMULATION IN PANCREATIC ISLETS"
Robert J. Konrad, Y. Camille Jolly, and Bryan A. Wolf "~
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6082 Received September 18, 1991
Summary: Phosphatidic acid has been previously implicated as an intracellular mediator of insulin secretion. Very little is known, however, about endogenous phosphatidic acid levels in islets. We now show, for the first time, that glucose and carbachol, at concentrations which stimulate insulin secretion, significantly increase endogenous phosphatidic acid levels in pancreatic islets by 2-fold at 1 min, nearly 3-fold at 2 min, and over 3-fold at 30 rain compared to control. Possible mechanisms include de novo synthesis from glucose and/or activation of phospholipase D. Our data, taken together with previous studies, suggest that phosphatidic acid may have a central role in insulin secretion as an intracellular mediator. ® 1991Ao~d~micPr.... Inc. Different classes of secretagogues are capable of stimulating insulin secretion from the islets of Langerhans (1). D-glucose is an example of a fuel secretagogue and is the major stimulus for insulin secretion (2,3). Carbachol which binds to the muscarinic receptor
in islets also stimulates
insulin secretion
(4). The exact
mechanism of action of each of these secretagogues is not completely elucidated (5). Oxidation of glucose is believed to increase the I~-cell intracellular ATP/ADP ratio which results in closure of the K ÷ channels, depolarization, influx of extracellular calcium through voltage-dependent Ca 2+ channels, increase in intracellular calcium and insulin exocytosis (6). Carbachol activates phospholipase C which generates the second messengers inositol 1,4,5-trisphosphate and 1,2-diacyl-sn-glycerol (7).
" This work was supported by National Institutes of Health Research Grant RO1 DK43354 (to B.A.W.), an American Diabetes Association Research and Development Award (to B.A.W.), and a University of Pennsylvania Diabetes Endocrinology Research Center Pilot Feasibility grant (to B.A.W., NIH DK19525-14). ""
To whom correspondence should be addressed.
0006-29lX/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
960
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Phosphatidic acid has recently been recognized as a potential mediator of insulin secretion (8). Exogenous phosphatidic acid increases insulin secretion from neonatal r$-cell (9). Phosphatidic acid in islets may be generated by three different mechanisms: 1 ) activation of phospholipase D (10), 2) d e n o v o synthesis from glucose ( 1 1 - 14), and 3) activation of phospholipase C and diacylglycerol kinase. Recent studies have shown that exogenous phospholipase D is capable of stimulating insulin release from islets at the same time as it increases intracellular phosphatidic acid levels (1 5). In order to implicate phosphatidic acid as an intracellular mediator of insulin secretion, it is necessary to demonstrate that insulin secretagogues increase endogenous levels of phosphatidic acid. Endogenous phosphatidic acid levels have, however, been difficult to measure in islets because of low levels (14), technical problems in separating phosphatidic acid from other phospholipids (16), and the small amounts of islets routinely obtainable (17).
We have therefore used a sensitive 2-dimensional TLC
system coupled to a very sensitive radioactivity detection instrument to measure endogenous phosphatidic acid levels in islets during stimulation with two important secretagogues, glucose and carbachol.
MATERIALS AND METHODS Islet isolation, labeling with [ZH]palmitic acid, and incubation - Islets were isolated aseptically from the pancreas of 8 male Sprague-Dawley rats as previously described (18). Freshly isolated islets (approx. 3,000) were incubated 24 hours under sterile conditions at 37°C under an atmosphere of 95% air/5% CO2 in a sterile Petri dish containing 2.5 ml of complete CMRL-1066 and 50 pCi of [3H]palmitic acid (19). Following labeling, islets were washed five times in fresh modified Krebs'-Hepes buffer (25 mM Hepes pH 7.4, 115 mM NaCI, 24 mM NaHCO3, 5 mM KCI, 2.5 mM CaCI 2, 1 mM MgCI 2, 0 . 1 % bovine serum albumin) supplemented with 3 mM D-glucose and used immediately. [3H]palmitic acid-labeled islets (125-200 per condition) were preincubated in Krebs'-Hepes buffer (3 mM glucose) and then incubated for 1-30 min in fresh Krebs'-Hepes medium (3 mM or 28 mM glucose and/or 0.5 mM carbachol). The reaction was stopped by the addition of 2 ml of ice-cold chloroform/methanol (1:2, v/v) supplemented with 0.25% of the anti-oxidant butylated hydroxytoluene. The tubes were then chilled for 15 rain in a dry ice/ethanol bath, and stored at -20°C prior to analysis. Two-dimensional TLC analysis of phosphatidic acid - Prior to extraction, carrier amounts (5 pg) of phospholipids, neutral lipids and fatty acids were added to each tube to aid in recovery and endogenous phosphatidic acid was extracted essentially as described (20). Samples were applied to 10x10 cm high-performance HP-K silica gel TLC plates (Whatman Biosystems Inc, Clifton, N J) which had been activated 30 rain at 110°C. Plates were developed in the first dimension with chloroform/methanol/ammonium hydroxide (65:35:5.5; v/v) for 30 rain. Plates were carefully dried (60 rain) and then developed in the second dimension with chloroform/methanol/formic acid/water (55:28:5:1; v/v) for 30 rain. Radioactivity of 961
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
each plate was quantitated by 2-dimensional analysis with the Berthold Linear Analyzer 284 (Nashua, NH) equipped with a position-sensitive proportional highresolution counter tube (200 mm long, 1380 V) continuously flushed (0.5 L/min) with PIO gas (90% argon/10% methane) and fitted with a 2 mm entrance window. Each plate was scanned at least 12 hours. Resolution in the first-dimension was 0.5 mm and 2 mm in the second-dimension. Data analysis was performed using version 4.07 of the 2D-TLC software. Peaks were integrated after subtracting the background reading of a corresponding and adjacent region. In all cases, counts in the peaks of interest were at least 5-fold greater than background. Radioactivity in each peak was expressed as the % of total counts on the plate. The identity of the peaks was assigned by comparison with cold phospholipids standards, commercial radioactive standards, and labeling of the islet phospholipids with the appropriate radiolabeledhead group. The following peaks were identified and localized: phosphatidylcholine (Rf 1 in the first dimension: 0.19, Rf2 in the second dimension: 0.38), lysophosphatidylcholine (Rfl: 0.03, Rf2:0.11), phosphatidylethanolamine (Rfl: 0.31, Rf2: 0.63), sphingomyelin (Rfl: 0.09, Rf2: 0.23), phosphatidylserine (Rfl: 0.06, Rf2: 0.39), phosphatidylinositol (Rfl: 0.01, Rf2: 0.28), free fatty acid (Rf~: 0,28, R~2:0.85), phosphatidic acid (Rfl: 0.08, Rf2: 0.63). One-dimensional TLC analysis of fatty acids and diacylglycerol- Samples were spotted onto the pre-adsorbent zone of channeled silica gel G TLC 20x20 cm plates (Analtech, Newark, DE) which had been activated 30 min at 110°C. Plates were developed for 30-45 rain in petroleum ether (30-60°C)/diethyl ether/acetic acid (140:60:2, v/v/v) as previously described (21).
RESULTS Phosphatidic acid accumulation was measured by labeling islets 24 hours to isotopic equilibrium using [3H]palmitic acid. Labeled islets were then challenged with 0.5 mM carbachol and 28 mM glucose.
[3H-palmityl]phosphatidic
acid was extracted and then
separated from other phospholipids and neutral lipids by 2D-TLC followed by radioactivity detection with a high-resolution linear analyzer. As shown in figure 1,
[3H-palmityl]phosphatidic
acid is clearly separated from other radiolabeled lipids. The
combination of stimulatory concentrations of glucose (28 mM) and carbachol (0.5 raM) increased endogenous phosphatidic acid levels by 2-fold at 1 min, nearly 3-fold at 2 min, and over 3-fold at 30 min as compared to the non-stimulatory 3 mM glucose condition (Figure 2, p < 0.0001
by 2-way ANOVA). At 30 min, under these
experimental conditions, glucose and carbachol caused a robust insulin secretory response (4-6 fold increase, data not shown). The effects of glucose and carbachol alone on phosphatidic acid and diacylglycerol accumulation are shown in Table 1. Each secretagogue caused phosphatidic acid accumulation, but the combination of glucose and carbachol was synergistic.
No
effect
of
either
secretagogue 962
was
observed
on
[3H-
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
¢3 ¢3 G" ¢3 Z
w Z
7=== -5"O
ol
L, <
-= 2nd dimension CHCI3 / CH3OH / HCOOH / H20 (55:28:5:1, v / v)
Figure 1. Two-dimensional thin-layer chromatography of phosphatidic acid from [3H]palmityl-labeled islets. PanelA: G3 (3 m M glucose, 30 min); Panel B: G28 + CCH (28 m M glucose + 0.5 m M carbachol, 30 rain). PA: phosphatidic acid, PS: phosphatidylserine, Ph phosphatidylinositol, LPC: lysophosphatidylcholine, SM: sphingomyelin, PC: phosphatidylcholine, PE: phosphatidylethanolamine, C16:0: palmitic acid.
palmityl]diacylglycerol levels after 30 min incubation (Table 1 ) nor at early time points (1 min, 2 rain, data not shown).
DISCUSSION We demonstrate, for the first time, that the combination of glucose and carbachol causes an almost immediate increase in levels of phosphatidic acid. Three
o
G28+CCH
400
//-!
o
~,~
300
o
.~
200
/
'Z.
~
G3
100
012
30 TIME [mini
Figure 2. Effect of glucose and carbachol on phosphatidic acid accumulation in islets labeled 24 h with [~H]palmitic acid. Results are shown as the Mean ± S.E.M. of phosphatidic acid accumulation expressed as a percentage of the 3 mM glucose control from 2 to 7 observations per condition. G 3 : 3 mM glucose, G28 + CCH: 28 mM glucose + 0.5 mM carbachol. 963
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table 1. Effect of glucose and carbachol on diacylglycerol and phosphatidic acid accumulation in islets labeled with [3H]palmitic acid. islets prelabeled 24 h with
[3H]palmitic acid were incubated 30 rain with glucose and/or carbachol. Results are shown as the Mean ± S.E.M. expressed as a % of 3 mM glucose control from 4 to 7 observations per condition. *: p < 0.05 compared to 3 mM glucose control. Diacylglycerol (% of control) 3 mM glucose 28 mM glucose
Phosphatidic acid (% of control)
100.0 + 2.5
100.0 + 6.2
99.6 ± 1.8
144.6 + 8.3
3 mM glucose + 0.5 mM carbachol
113.7 4- 5.7
28 mM glucose + 0.5 mM carbachol
118.6 4- 4.3
200.0 + 17.7" 314.7 ± 43.7"
different mechanisms may result in phosphatidic acid accumulation. One possibility is that carbachol
and glucose stimulate
diacylglycerol
accumulation
following
phospholipase C activation (7). Diacylglycerol is then phosphorylated by diacylglycerol kinase to phosphatidic acid. Diacylglycerol levels, however, did not significantly increase. Previous studies have also failed to detect any glucose-induced increases in diacylglycerol
(19,21).
Our observations can not rule out the possibility that
diacylglycerol is so rapidly phosphorylated to phosphatidic acid that any increases are masked, although this appears unlikely. Alternatively, phosphatidic acid may be synthesized de n o v o from glucose according to the following scheme: metabolism of glucose to the triose phosphate dihydroxyacetone phosphate, acylation to acyldihydroxyacetone phosphate, reduction to lysophosphatidic acid, and acylation to phosphatidic acid (22). Previous studies have s h o w n that this pathway of de n o v o synthesis from glucose is present in islets but it is not clear h o w much this pathway contributes to phosphatidic acid accumulation (21).
Phospholipase D activation results in phosphatidic acid accumulation (23). The addition
of
exogenous phospholipase
D to
islets
results
in accumulation
of
endogenous phosphatidic acid while stimulating insulin secretion (1 5). Phospholipase D activity, measured as the production of phosphatidylethanol in the presence of ethanol, is present in islets (24,25). Furthermore, a number of pharmacological probes (but not glucose nor carbachol) have been shown to stimulate phospholipase D in islets (10). 964
Vol.
180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Regardless of the mechanisms of phosphatidic acid accumulation, phosphatidic acid appears to be a potential mediator of insulin secretion since exogenous phosphatidic acid stimulates insulin secretion and increases intracellular Ca 2+ levels (9). Furthermore, inhibition of phosphatidic acid metabolism with propranolol also stimulates
insulin secretion
(25).
Whether
phosphatidic acid phosphorylates
endogenous substrates in islets as reported in heart, liver, brain, lung, and testis remains to be determined (26). In summary, we have demonstrated, for the first time, that the muscarinic agonist and insulin secretagogue carbachol stimulates phosphatidic acid accumulation in islets. We have also demonstrated a similar effect with glucose and a synergistic effect when islets are challenged with the combination of glucose and carbachol. Our data, taken together with that of previous studies (8), suggests that phosphatidic acid may play a central role in the process of insulin secretion.
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