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Carbachol stimulation of triacylglycerol lipase activity in pancreatic acinar cells
Vol.
184,
No.
April
30,
1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
CARBACHOL STIMULATION OF TRIACYLGLYCEROL LIPASE IN PANCREATIC ACINAR CELLS Thomas
R. Hundley
and Ronald
626-633
ACTIVITY
P. Rubin*
Department of Pharmacology & Therapeutics, School of Medicine & Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214
Received
March
9,
1992
SUMMARY: Carbachol (CCh) reduced the levels of [3H]arachidonic acid in triacylglycerol (TG) of pancreatic acinar cells. In cells prelabeled with [14C]glycerol, CCh reduced [14C]TG and increased [14C]diacylglycerol levels. Using [3H]triolein as exogenous substrate, CCh enhanced TG lipase activity 3-fold in a particulate fraction derived from intact acinar cells. These results portray a mechanism for generating diacylglycerol and arachidonic acid in exocrine pancreas involving agonist stimulation of TG hydrolysis. , 19'32AcadrmlcPress,IOC.
from Agonist stimulated secretion of digestive enzymes pancreatic acinar cells is accompanied by the activation of enzymes which hydrolyze phospholipids (1). In exocrine pancreas the stimulation of phospholipase C to increase phosphoinositide and inositol hydrolysis yields diacylglycerol (DAG) trisphosphate (l-4). In addition, phospholipase C catalyzes the hydrolysis of phosphatidylcholine (PtdCho) to produce DAG free arachidonic acid (AA) also increase The levels of (3,4). response to secretagogue-induced in pancreatic acinar cells in lipase (5,6). In addition to activation of PLA2 or diglyceride phospholipases, lipases catalyze the rate-limiting step in the and degradation of triacylglycerol (TG) to form acylglycerols long chain fatty acids such as AA (7,8).
XTo whom correspondence
should
be addressed.
triacylglycerol; Abbreviations: DAG, diacylglycerol; TG, phosphatidylcholine; arachidonic acid; CCh, carbachol; PtdCho, PtdIns, phosphatidylinositol; PMSF, phenylmethylsulfonylfluoride.
AA,
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
implicated as mediators of DAG and AA have been amylase secretion (1,9), it is important to consider sources from which secretagogues might promote the all possible The cellular messengers. production of these putative source of supposition that TG hydrolysis may be another support by the finding cellular DAG, and possibly AA, engenders fatty acid composition of DAG in mouse pancreas that the implying a stimulated with acetylcholine resembled that of TG, relationship This report presents product-precursor (10) * activity in TG lipase evidence implicating CCh stimulation of the biochemical processes associated with the production of DAG and AA in pancreatic acinar cells. Since exocytotic
METHODS Acinar cells were prepared from Sprague-Dawley rats by collagenase digestion of pancreatic tissue and dissociation of acini with EDTA as previously described (11). The cells were incubated in Krebs-Henseleit buffer (KHB) containing essential amino acids; 0.2% BSA; 0.25 mg/ml soyabean trypsin inhibitor: 1.28 mM Ca2+; 1.18 mM MgS04; and 1OmM HEPES (pH 7.4). All incubations were carried out at 37OC with 95% 02-5% CD2. li ids, cells For radioactive labeling of acinar cell were incubated with [3H]AA (0.75 pCi/ml) or [ P *C]glycerol (luci/ml) for various time intervals, and lipids were extracted from 500 ul aliquots into 9 ml of chloroform/methanol (2:l w/v) were separated from neutral lipids by a (12) * The phospholipids two-phase containing solvent system in a single dimension, chloroform:methanol.:acetic acid:water (50:40:6:0.5, v/v) and anhydrous ether:petroleum ether:glacial acetic acid (50:50:1, solvent system facilitated the separation of v/v) * The second neutral lipids. Lipid standards identified with iodine vapor and radiolabeled lipids were identified by autoradiography scraped from plates and counted by scintillation spectrometry. For assay of triglyceride lipase in subcellular fractions, acinar cells were pelleted by speed low centrifugation. The pellets were resuspended in homogenization buffer (HB) containing: 150 mM sucrose; 30 mM HEPES (pH 6.0): 1mM EGTA; 20mM MgC12; 1mM 2-mercaptoethanol; lOOug/ml trypsin inhibitor: lug/ml pepstatin A; and 0.3 mM 51-1g/ml leupeptin; PMSF. The homogenate was centrifuged twice at 600xg and the supernatants pooled. The pooled supernatants were centrifuged at 1300xg for 15 min; the resulting supernatant was layered over a discontinuous sucrose gradient of 10% (w/v) sucrose containing 600 mM NaCl in HB, over 20% (w/v) sucrose (without NaCl) and centrifuged at 100,OOOxg for 60 min. The TG lipase assay buffer contained: lo-20 ug protein: 1OOmM potassium phosphate buffer (pH 6.5): 1mM EGTA; fatty acid free glycerol (20% 23 uM [3H]triole!? (b', mg/ml) ; w/v) : and pCi/umol) in a final volume of 1 ml. Reactions were conducted for 20 min at 37OC and were terminated by the addition of chloroform/methanol (2:l). Lipids were extracted and neutral lipids separated as described above. Proteins were determined by the method of Lowry et al (13). 627
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS When pancreatic radioactivity min
in
reached
the
a plateau
of the incorporated was equivalent to PtdCho the
(data
[3H]AA
CCh
reduced
and
67%,
acinar
TG fraction (Fig. 1A). radiolabel
the
not
of
the
were
incubated
rose
dramatically
control,
of
label
CCh caused
TG (Fig.
amount
1A).
of
After
[3H]AA
5
into
20
into
reduction
min,
1
and
in 10
TG by an average Concurrent
(3fllTrig/ycerides
10
60
incorporated
of
with
30
40
TIME
(MINI
50
60
70
50
60
70
/3ffjDglycerides
0
10
20
30
40
TIME
CMIN)
Fig. 1. The effect of CCh on incorporation of C3H]arachidonic acid into acinar cells were incubated Pancreatic presence of circles), 10 1W CCh (filled
the (A)
with
time TG
course
[2iyAA(B:n
of "t:,
pM CCh (diamonds), or variance revealed zero CCh (open circles). Two-way analysis of that CCh caused a significant attenuation in (A) [3H]TG and a errors significant rise in (B) [3H]DAG (PC O.O5)(n=6). Standard clarity, but were within lo-15% of the mean are not shown for values. 628
uM 77 the
0
r
0
[3H]AA,
and after
a significant
respectively.
1OOr A
with
After 30 min an average of 35% was found in the TG pool, which
percentage
shown).
content of
cells
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
in cellular [3H]TG levels, there was a 32% CCh-induced decrease increase in [3H]DAG levels (Fig. 1B). The reduction in [3H]TG observed in the presence of [3H]AA plus CCh could be a consequence of stimulation of TG hydrolysis or a decrease in the activity of DAG acyltransferase, the enzyme that channels DAG into triacylglycerol (14). So, in the next series of experiments, acinar cells were prelabeled with [14C]glycerol for 60 min and then exposed to CCh. Within 15 min CCh caused a significant reduction in [14C]TG levels (Fig. 2A) and an increase in
A
50
/14ClTrig/ycerides
’ 0
1 10
20
TIME
30
40
30
40
(MIN)
>
50' 0
10
20
TIME
(MIN)
p;q. 2 * Time course of CCh-stimulated changes in and [ C]DAG. Acinar cells were prelabeled for 60 rn:~"~!.~~ [14C]glycerol prior to the addition of CCh (closed 1PM circles), 1OuM CCh (diamonds), and zero CCh (open circles). Data are expressed as percent of time zero, which for TG and DAG averaged 138 and 148 respectively. PcO.05, as dpm, assessed by two way analysis of variance (n=3). Standard errors were within lo-15% of the mean values.
629
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2, 1992
[l*C]DAG [l*C]DAG
BIOCHEMICAL
levels in levels
[14C]PtdIns
levels
(data
during
prolonged
of
DAG formed
reduction
is
in
and
lipase
with
Relatively
high
100,000
xg
the
with
1 ipase
activity
comparable acinar not
1OrM increase
cell shown),
was
similar
observed
in was
shown). in homogenate intact acinar
of
exogenous substrate. was the observed in
activity
fraction,
the
proportion A
monitored as
in in
radioactivity
which (data not
directly
rise rise
supports
CCh
stimulation
1 ipase
presumably
due to
the
presence
(Fig. 3). However, CCh-stimulated TG most predominant in the membrane fraction intact 100,000 xg pellet. Treatment of for
15 min produced
in
the
particulate
TG lipase
homogenate, or the
finding
CCh in
2B). The continuous
CCh a greater by
[3H]triolein
basal
associated
in
after
COMMUNICATIONS
phospholipids.
caused
was
supernatant lipase was
with
to
cells glycerol
using
of secretory TG lipase activity cells
This
levels
fractions CCh,
shown). inositol
RESEARCH
(Fig. a
exposure to
activity
subcellular
cells
converted
prelabeled excess cold
with TG
not
[l*C]TG
[l*C]glycerol
"chasedt'
BIOPHYSICAL
the same experiments was associated with
view
that
AND
supernatant
Homogenate
a
a 3-fold fraction
was activity 1300 xg particulate fraction
100,000 x Supernatant
increase
(Fig.
g
(Fig. not
in
3).
observed fraction
in (data
3).
100,000 Pellet
x
g
Fig. 3. Distribution and CCh-sensitivity of TG lipase in the subcellular fractions. Acinar cells were incubated in presence (hatched columns) or absence (open columns) of 1OuM CCh for 15 min and subcellular fractions isolated and assayed using exogenous [3H)triolein as substrate. Data 2;~ expressed 0.01 as as pmoles [3H]triolein hydrolyzed/min/mg protein. assessed by paired Student's t-test.
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No.
2,
1992
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AND
BIOPHYSICAL
RESEARCH
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DISCUSSION Although TG has been measured in exocrine pancreas mechanisms involved in its regulation, as well as (5,101 I the assessed. its functional significance, has not been The evidence for CCh-induced turnover of TG present study presents and demonstrates a CCh-stimulatable TG lipase. A TG lipase that is activated in fraction prepared from acinar a particulate cells previously indicative of a stable exposed to CCh is modification of the enzyme. This modification may involve a phosphorylation-dephosphorylation reaction, has as been established for hormone-sensitive adipocytes (8). lipase in Because CCh sensitivity was predominant in the 100, oooxg particulate fraction, the TG lipase acinar detected in cells appears to be membrane-bound. Further studies are required to determine whether the enzyme is an integral membrane protein or is reversibly associated with membranes. The demonstrated ability of CCh to reduce radiolabeled TG levels of cells exposed to r3H]AA or [14C]glycerol taken together with the increase in labeled DAG measured in the same experiments fortifies the evidence favoring lipase-mediated TG breakdown. Although the DAG formed in response to CCh in acinar cells is derived from TG, as well as PtdCho and phosphoinositides (1,3,4), the quantitative contribution of each source cannot be accurately assessed at this time. The findings that the continued fall in [14C]TG levels produced by CCh after 15 min was associated with a continuous rise in [14C]PtdIns levels, rather than in [14]DAG levels, support the view that CCh-stimulated TG breakdown contributes to the synthesis of PtdIns from DAG via the classical phosphoinositide pathway (9) , particularly during prolonged stimulation. The CCh-sensitive TG lipase detected in this study is of potential physiological significance with regard to the generation of AA and DAG. Although endogenous TG of rat pancreas is not particularly enriched in AA (15), our study reveals that TG rapidly incorporates this fatty acid. The prompt liberation of r3H]AA from TG when acinar cells are resuspended in medium devoid of added [3H]AA (unpublished findings) has also been observed in other cell types (16) and connotes a rapid turnover of the AA moiety in TG. Thus, TG may serve as an intracellular source of AA to provide free fatty acid for the turnover of membrane phospholipid acyl groups (16) 631
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1992
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and/or for utilization by cyclooxygenase and lipoxgenase pathways (17,18). Future studies are planned to determine whether the DAG arising from TG is involved in cellular signaling and whether distinct physiological functions are subserved by DAG generated by phospholipid and TG hydrolysis. The generation of DAG in different compartments of the acinar cell could lead to the differential activation of protein kinase C isozymes. Nevertheless, the present findings indicate that in exocrine pancreas a process involving lipase-mediated TG breakdown is operative for generating substances which may serve as second messengers. Acknowledgment.
This
study
was supported
by NIH grant
AM-28029.
REFERENCES 1. Rubin, R.P. (1986) In: PI Turnover and (J.W. Putney, Jr., Ed.); pp. 149-162; New York. 2. Streb, H., Heslop, J.P., Irvine, R-F., Berridge, M.J. (1985) J. Biol. Chem. 3. Matozaki, T. and Williams, J.A. (1989) 14729-14734. 4. Rubin, R.P., Hundley, T.R., and Adolf, 127-132. Biophys. Acta 1133, 5. Dixon, J.F. and Hokin, L.E. (1984) J.
Receptor Function Alan R. Liss, Schulz, I., and 260, 7309-7315. J. Biol. Chem. 264, M.A. Biol.
(1992)
Biochim.
Chem. 259,
14418-14425.
6.
Chaudhry, A., Laychock, S.G., and Rubin, R.P. (1987) J. Biol. Chem. 262, 17426-17431. 7. Stralfors, P., Olsson, H., and Belfrage, P. (1987) In: The Enzymes vol. XVIII, Part B; (P-D. Boyer & E.G. Krebs, Eds.); pp. 147-177, Academic Press, New York. 8. Yeaman, S.J. (1990) Biochim. Biophys. Acta 1052, 128-132. 9. Rana, R.S. and Hokin, L.E. (1990) Physiol. Rev. 70, 115-164. 10. Banschbach, M.W. Geison, R.L. and Hokin-Neaverson, M. (1981) Biochim. Biophys. Acta 663, 34-45. 11. Merritt, J.E., Bradford, P.G., and Rubin, R.P. (1987) In: In Vitro Methods for Studying Secretion (A.M. Poisner & J.M. Trifaro, Eds.) pp. 207-222; The Secretory Process, Vol.
12. 13.
3;
Elsevier,
Amsterdam.
Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 256-275. 632
Vol.
14. 15. 16. 17. 18.
184, No. 2, 1992
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Haagsman, H.P., DeHaas, C.G.M., Geelen M.J.H., and Van Golde, L.M.G. (1982) J. Biol. Chem. 257, 10593-10598. Calderon, P., Furnelle, J., and Christophe, J. (1979) Biochim. Biophys. Acta 574, 379-390. Denning, G.M., Figard, P.H., Kaduce, T.L., and Spector, A.A. (1983) J. Lipid Res. 24, 993-1001. Ragab-Thomas, J-M-F., Hullin, F., Chap, H., and DousteBlazy, L. (1987) Biochim. Biophys. Acta 917, 388-397. Fujimoto, Y., Shimada, S., Fujikawa, T., Sakuma, S., and Fujita, T. (1991) Prostaglandins, Leukotrienes, & Essential Fatty Acids 42, 251-256.
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No.
April
30,
1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
CARBACHOL STIMULATION OF TRIACYLGLYCEROL LIPASE IN PANCREATIC ACINAR CELLS Thomas
R. Hundley
and Ronald
626-633
ACTIVITY
P. Rubin*
Department of Pharmacology & Therapeutics, School of Medicine & Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214
Received
March
9,
1992
SUMMARY: Carbachol (CCh) reduced the levels of [3H]arachidonic acid in triacylglycerol (TG) of pancreatic acinar cells. In cells prelabeled with [14C]glycerol, CCh reduced [14C]TG and increased [14C]diacylglycerol levels. Using [3H]triolein as exogenous substrate, CCh enhanced TG lipase activity 3-fold in a particulate fraction derived from intact acinar cells. These results portray a mechanism for generating diacylglycerol and arachidonic acid in exocrine pancreas involving agonist stimulation of TG hydrolysis. , 19'32AcadrmlcPress,IOC.
from Agonist stimulated secretion of digestive enzymes pancreatic acinar cells is accompanied by the activation of enzymes which hydrolyze phospholipids (1). In exocrine pancreas the stimulation of phospholipase C to increase phosphoinositide and inositol hydrolysis yields diacylglycerol (DAG) trisphosphate (l-4). In addition, phospholipase C catalyzes the hydrolysis of phosphatidylcholine (PtdCho) to produce DAG free arachidonic acid (AA) also increase The levels of (3,4). response to secretagogue-induced in pancreatic acinar cells in lipase (5,6). In addition to activation of PLA2 or diglyceride phospholipases, lipases catalyze the rate-limiting step in the and degradation of triacylglycerol (TG) to form acylglycerols long chain fatty acids such as AA (7,8).
XTo whom correspondence
should
be addressed.
triacylglycerol; Abbreviations: DAG, diacylglycerol; TG, phosphatidylcholine; arachidonic acid; CCh, carbachol; PtdCho, PtdIns, phosphatidylinositol; PMSF, phenylmethylsulfonylfluoride.
AA,
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
implicated as mediators of DAG and AA have been amylase secretion (1,9), it is important to consider sources from which secretagogues might promote the all possible The cellular messengers. production of these putative source of supposition that TG hydrolysis may be another support by the finding cellular DAG, and possibly AA, engenders fatty acid composition of DAG in mouse pancreas that the implying a stimulated with acetylcholine resembled that of TG, relationship This report presents product-precursor (10) * activity in TG lipase evidence implicating CCh stimulation of the biochemical processes associated with the production of DAG and AA in pancreatic acinar cells. Since exocytotic
METHODS Acinar cells were prepared from Sprague-Dawley rats by collagenase digestion of pancreatic tissue and dissociation of acini with EDTA as previously described (11). The cells were incubated in Krebs-Henseleit buffer (KHB) containing essential amino acids; 0.2% BSA; 0.25 mg/ml soyabean trypsin inhibitor: 1.28 mM Ca2+; 1.18 mM MgS04; and 1OmM HEPES (pH 7.4). All incubations were carried out at 37OC with 95% 02-5% CD2. li ids, cells For radioactive labeling of acinar cell were incubated with [3H]AA (0.75 pCi/ml) or [ P *C]glycerol (luci/ml) for various time intervals, and lipids were extracted from 500 ul aliquots into 9 ml of chloroform/methanol (2:l w/v) were separated from neutral lipids by a (12) * The phospholipids two-phase containing solvent system in a single dimension, chloroform:methanol.:acetic acid:water (50:40:6:0.5, v/v) and anhydrous ether:petroleum ether:glacial acetic acid (50:50:1, solvent system facilitated the separation of v/v) * The second neutral lipids. Lipid standards identified with iodine vapor and radiolabeled lipids were identified by autoradiography scraped from plates and counted by scintillation spectrometry. For assay of triglyceride lipase in subcellular fractions, acinar cells were pelleted by speed low centrifugation. The pellets were resuspended in homogenization buffer (HB) containing: 150 mM sucrose; 30 mM HEPES (pH 6.0): 1mM EGTA; 20mM MgC12; 1mM 2-mercaptoethanol; lOOug/ml trypsin inhibitor: lug/ml pepstatin A; and 0.3 mM 51-1g/ml leupeptin; PMSF. The homogenate was centrifuged twice at 600xg and the supernatants pooled. The pooled supernatants were centrifuged at 1300xg for 15 min; the resulting supernatant was layered over a discontinuous sucrose gradient of 10% (w/v) sucrose containing 600 mM NaCl in HB, over 20% (w/v) sucrose (without NaCl) and centrifuged at 100,OOOxg for 60 min. The TG lipase assay buffer contained: lo-20 ug protein: 1OOmM potassium phosphate buffer (pH 6.5): 1mM EGTA; fatty acid free glycerol (20% 23 uM [3H]triole!? (b', mg/ml) ; w/v) : and pCi/umol) in a final volume of 1 ml. Reactions were conducted for 20 min at 37OC and were terminated by the addition of chloroform/methanol (2:l). Lipids were extracted and neutral lipids separated as described above. Proteins were determined by the method of Lowry et al (13). 627
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS When pancreatic radioactivity min
in
reached
the
a plateau
of the incorporated was equivalent to PtdCho the
(data
[3H]AA
CCh
reduced
and
67%,
acinar
TG fraction (Fig. 1A). radiolabel
the
not
of
the
were
incubated
rose
dramatically
control,
of
label
CCh caused
TG (Fig.
amount
1A).
of
After
[3H]AA
5
into
20
into
reduction
min,
1
and
in 10
TG by an average Concurrent
(3fllTrig/ycerides
10
60
incorporated
of
with
30
40
TIME
(MINI
50
60
70
50
60
70
/3ffjDglycerides
0
10
20
30
40
TIME
CMIN)
Fig. 1. The effect of CCh on incorporation of C3H]arachidonic acid into acinar cells were incubated Pancreatic presence of circles), 10 1W CCh (filled
the (A)
with
time TG
course
[2iyAA(B:n
of "t:,
pM CCh (diamonds), or variance revealed zero CCh (open circles). Two-way analysis of that CCh caused a significant attenuation in (A) [3H]TG and a errors significant rise in (B) [3H]DAG (PC O.O5)(n=6). Standard clarity, but were within lo-15% of the mean are not shown for values. 628
uM 77 the
0
r
0
[3H]AA,
and after
a significant
respectively.
1OOr A
with
After 30 min an average of 35% was found in the TG pool, which
percentage
shown).
content of
cells
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
in cellular [3H]TG levels, there was a 32% CCh-induced decrease increase in [3H]DAG levels (Fig. 1B). The reduction in [3H]TG observed in the presence of [3H]AA plus CCh could be a consequence of stimulation of TG hydrolysis or a decrease in the activity of DAG acyltransferase, the enzyme that channels DAG into triacylglycerol (14). So, in the next series of experiments, acinar cells were prelabeled with [14C]glycerol for 60 min and then exposed to CCh. Within 15 min CCh caused a significant reduction in [14C]TG levels (Fig. 2A) and an increase in
A
50
/14ClTrig/ycerides
’ 0
1 10
20
TIME
30
40
30
40
(MIN)
>
50' 0
10
20
TIME
(MIN)
p;q. 2 * Time course of CCh-stimulated changes in and [ C]DAG. Acinar cells were prelabeled for 60 rn:~"~!.~~ [14C]glycerol prior to the addition of CCh (closed 1PM circles), 1OuM CCh (diamonds), and zero CCh (open circles). Data are expressed as percent of time zero, which for TG and DAG averaged 138 and 148 respectively. PcO.05, as dpm, assessed by two way analysis of variance (n=3). Standard errors were within lo-15% of the mean values.
629
Vol.
184,
No.
2, 1992
[l*C]DAG [l*C]DAG
BIOCHEMICAL
levels in levels
[14C]PtdIns
levels
(data
during
prolonged
of
DAG formed
reduction
is
in
and
lipase
with
Relatively
high
100,000
xg
the
with
1 ipase
activity
comparable acinar not
1OrM increase
cell shown),
was
similar
observed
in was
shown). in homogenate intact acinar
of
exogenous substrate. was the observed in
activity
fraction,
the
proportion A
monitored as
in in
radioactivity
which (data not
directly
rise rise
supports
CCh
stimulation
1 ipase
presumably
due to
the
presence
(Fig. 3). However, CCh-stimulated TG most predominant in the membrane fraction intact 100,000 xg pellet. Treatment of for
15 min produced
in
the
particulate
TG lipase
homogenate, or the
finding
CCh in
2B). The continuous
CCh a greater by
[3H]triolein
basal
associated
in
after
COMMUNICATIONS
phospholipids.
caused
was
supernatant lipase was
with
to
cells glycerol
using
of secretory TG lipase activity cells
This
levels
fractions CCh,
shown). inositol
RESEARCH
(Fig. a
exposure to
activity
subcellular
cells
converted
prelabeled excess cold
with TG
not
[l*C]TG
[l*C]glycerol
"chasedt'
BIOPHYSICAL
the same experiments was associated with
view
that
AND
supernatant
Homogenate
a
a 3-fold fraction
was activity 1300 xg particulate fraction
100,000 x Supernatant
increase
(Fig.
g
(Fig. not
in
3).
observed fraction
in (data
3).
100,000 Pellet
x
g
Fig. 3. Distribution and CCh-sensitivity of TG lipase in the subcellular fractions. Acinar cells were incubated in presence (hatched columns) or absence (open columns) of 1OuM CCh for 15 min and subcellular fractions isolated and assayed using exogenous [3H)triolein as substrate. Data 2;~ expressed 0.01 as as pmoles [3H]triolein hydrolyzed/min/mg protein. assessed by paired Student's t-test.
630
A
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184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
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DISCUSSION Although TG has been measured in exocrine pancreas mechanisms involved in its regulation, as well as (5,101 I the assessed. its functional significance, has not been The evidence for CCh-induced turnover of TG present study presents and demonstrates a CCh-stimulatable TG lipase. A TG lipase that is activated in fraction prepared from acinar a particulate cells previously indicative of a stable exposed to CCh is modification of the enzyme. This modification may involve a phosphorylation-dephosphorylation reaction, has as been established for hormone-sensitive adipocytes (8). lipase in Because CCh sensitivity was predominant in the 100, oooxg particulate fraction, the TG lipase acinar detected in cells appears to be membrane-bound. Further studies are required to determine whether the enzyme is an integral membrane protein or is reversibly associated with membranes. The demonstrated ability of CCh to reduce radiolabeled TG levels of cells exposed to r3H]AA or [14C]glycerol taken together with the increase in labeled DAG measured in the same experiments fortifies the evidence favoring lipase-mediated TG breakdown. Although the DAG formed in response to CCh in acinar cells is derived from TG, as well as PtdCho and phosphoinositides (1,3,4), the quantitative contribution of each source cannot be accurately assessed at this time. The findings that the continued fall in [14C]TG levels produced by CCh after 15 min was associated with a continuous rise in [14C]PtdIns levels, rather than in [14]DAG levels, support the view that CCh-stimulated TG breakdown contributes to the synthesis of PtdIns from DAG via the classical phosphoinositide pathway (9) , particularly during prolonged stimulation. The CCh-sensitive TG lipase detected in this study is of potential physiological significance with regard to the generation of AA and DAG. Although endogenous TG of rat pancreas is not particularly enriched in AA (15), our study reveals that TG rapidly incorporates this fatty acid. The prompt liberation of r3H]AA from TG when acinar cells are resuspended in medium devoid of added [3H]AA (unpublished findings) has also been observed in other cell types (16) and connotes a rapid turnover of the AA moiety in TG. Thus, TG may serve as an intracellular source of AA to provide free fatty acid for the turnover of membrane phospholipid acyl groups (16) 631
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and/or for utilization by cyclooxygenase and lipoxgenase pathways (17,18). Future studies are planned to determine whether the DAG arising from TG is involved in cellular signaling and whether distinct physiological functions are subserved by DAG generated by phospholipid and TG hydrolysis. The generation of DAG in different compartments of the acinar cell could lead to the differential activation of protein kinase C isozymes. Nevertheless, the present findings indicate that in exocrine pancreas a process involving lipase-mediated TG breakdown is operative for generating substances which may serve as second messengers. Acknowledgment.
This
study
was supported
by NIH grant
AM-28029.
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