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Pancreastatin: Characterization of biological activity
Vo1.173, No. 3,1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 0-
December 31, 1990
Pages 1157-1160
PANCREASTATIN: CHARACTERIZATION OF BIOLOGICAL ACTIVITY 1
2
3
Tao Zhang , Tohru Mochizuki , Masafumi Kogire , 2 Jin Ishizuka, Noboru Yanaihara , James C. Thompson and
George H. Greeley, Jr.
Department of Surgery The University of Texas Medical Branch Galveston, Texas 77550 Received October 26, 1990
SUMMARY: Pancreastatin (PST) (1-49) was first isolated from the porcine pancreas and can inhibit glucose-induced insulin release. PST (33-49), a PST C-terminal fragment, can also inhibit insulin release. The purpose of this study was to determine the shortest C-terminal biologically active fragment of PST, in terms of inhibition of insulin release from the isolated perfused rat pancreas. Porcine PST (1-49) and C-terminal fragments, PST (33-49), PST (35-49), PST (37-49) and PST (39-49) were synthesized by solid-phase methodology. PST (1-49), PST (33-49) and PST (35-49), at I0 nM, significantly (p <0.05) inhibited insulin release from isolated perfused rat pancreas: the first phase was inhibited by 15.6±2.4, 24.4±6.5 and 12.5±1.9% and the second phase, 18.9±2.7, 25.7±4.8 and 20.i±1.9% by PST (1-49), PST (33-49) and PST (35-49), respectively. PST (35-49) shows a dose-dependent inhibition of insulin release. PST (37-49) and PST (39-49) were, however, inactive. Our results indicate that the shortest C-terminal biologically active fragment is PST (35-49). These data further indicate that the C-terminal portion of PST is primarily responsible for the biological activity of PST. ©1990Ac~de~ioPress, Inc.
Pancreastatin (PST), a forty-nine residue peptide with a C-terminal am±de, was first isolated from the porcine pancreas by Tatemoto colleagues.
(i).
and
In the perfused rat pancreas PST (i-49) can inhibit
not only glucose-induced insulin release (1,2,3,4) but can also inhibit insulin release in response to arginine, tolbutamide, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), and cholecystokinin (CCK-8) (2,3,5).
In addition, two synthetic C-terminal
PST fragments, PST (14-49) and PST (33-49) inhibit insulin release with similar magnitudes from the isolated perfused rat pancreas (I).
These
I Visiting Scientist from the School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422, Japan. 2 School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422, Japan. s Visiting Scientist from the Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan.
1157
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
data suggest that biological activity of PST resides in the C-terminal portion of PST.
The purpose of this study was to determine the shortest
C-terminal biologically active fragment of PST in terms of inhibition of insulin release from isolated perfused rat pancreas.
METHODS Porcine PST C-terminal fragments, PST (33-49), PST (35-49), PST (37-49), and PST (39-49) were synthesized by solid-phase methodology using an automatic synthesizer (Beckman 990). Porcine PST (1-49) was purchased from Peninsula Laboratories, Belmont, CA. Male Sprague-Dawley rats weighing 250-350 g were permitted to eat and drink ad iibitum. Rats were anesthetized with pentobarbital (50 mg/kg) and the pancreas was isolated according to the method of Grodsky, and colleagues (6,7) with some modifications. The isolated pancreas was perfused through the celiac artery at a flow rate of 2 ml/min for 40 minutes with 95% 02-5% CO 2 saturated Krebs-Ringer bicarbonate buffer (KRBB, pH 7.4) containing 4% Dextran T-70 (Pharmacia Fine Chemical, NJ), 0.5% bovine serum albumin (BSA) (Sigma Chemical Company, MO), and 4.2 mM glucose. Peptide infusions were started 5 minutes before the perfusion of 16.7 mM glucose, and were given for 25 minutes. For each PST fragment tested, control pancreas were also perfused with glucose (16.7 mM) plus saline. Perfusates were collected from the portal vein and stored at -20 C for measurement of insulin levels. Perfusate insulin levels were measured by an insulin-specific radioimmunoassay as described previously (8). The intra and inter-assay variances at the IDSO were 9% and 10%, respectively. Integrated insulin outputs were divided into the first and second phases and analyzed by the Student's t test separately. A p value of <0.05 was considered statistically different.
RESULTS For each fragment of PST tested, a set of control pancreas (N=5) were infused with glucose and saline.
As shown at Figure i, insulin release
in response to 16..7 mM glucose occurred in two phases.
PST (1-49) as
well as the PST fragments, PST (33-49) and PST (35-49), inhibited the first and second phases of insulin release significantly (p <0.05) I).
(Table
PST (33-49) inhibited the first and second phases of insulin release
by 24.4±6.5 and 25.7±4.8%, respectively (Table i).
In addition,
PST (35-49) inhibited insulin release in a dose-dependent fashion. PST (35-49), at i00 nM, inhibited insulin release by 29.4±4.9% (Ist phase) and 26.2±1.6% (2nd phase), when compared to PST (35-49), at i0 ruM, for 12.5±1.9% (ist phase) and 20.1±1.9% (2nd phase) (Fig. 2).
PST (37-49)
and PST (39-49) were, however, inactive (Table i).
DISCUSSION In the present study, we have compared the inhibitory activities of PST (1-49) and C-terminal fragments, PST (33-49), (35-49),
(37-49) and
(39-49) on glucose-induced insulin release from the isolated perfused rat pancreas.
Our results show that both PST (1-49) and PST (33-49)
1158
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.0
E
2.0
1.5
1.5
Control
U) ¢: v
I-Z) Q,. I--
1.0
1.0
0.5
0.5
0.0
0.0
0 Z
z
~=~ I
-15 -10
I
I
I
I
I
I
l
I
-5
0
5
10
15
20
25
,50
~
,
-15 -10 -5
I
I
I
I
I
I
|
0
5
10
15
20
25
30
MINUTES
Figure i.
Effects of PST (33-49) (left panel) and PST (35-49) (right panel) on glucose-induced insulin release from the isolated perfused rat pancreas. PST (33-49) or PST (35-49), at i0 nM, were infused from -5 min to 20 min, and 16.7 mM glucose was infused from 0 min to 20 min. Saline was used as a control for the peptides. The flow rate of the perfusate was 2 ml/min. Insulin levels are expressed as mean i SEM (n = 5 pancreas/control and peptide).
significantly inhibit insulin release from the perfused rat pancreas response to 16.7 mM glucose, reports
(1,2,3,4).
PST (35-49),
in
These data confirm and extend the previous
We have also shown that a shorter C-terminal fragment,
inhibits the first and second phases of glucose-induced
insulin release in a dose-dependent fashion.
Since PST (37-49) and
PST (39-49) fail to suppress insulin release,
PST (35-49) is the shortest
C-terminal biologically active fragment,
at least in terms of inhibition
of insulin release from the isolated perfused rat pancreas. also suggests that the residues,
35-36
(Glu-Glu),
This finding
are essential
in exerting
the biological activity of PST (35-49). Our data also support the previous report that the biological portion of PST is located in the C-terminus.
Because pancreastatin may be an
Table i. Effects of PST (1-49) and PST C-terminal fragments on glucose-induced insulin release from the isolated perfused rat pancreas Peptide(10 nM) PST(I-49) PST(33-49) PST(35-49) PST(37-49) PST(39-49)
Inhibition of Insulin Release (9) Ist phase(0-5 min) 2nd phase(6-20 min) 15.6 24.4 12.5 1.8 3.8
+ 2.4* + 6.5* + 1.9" _+ 4.9 _+ 2.4
18.9 25.7 20.1 -6.8 5.7
± + + ± +
2.7* 4.8* 1.9" 6.8 5.1
Values are expressed as mean ± SEM (N = 5 pancreas); * P <0.05 vs. control. Values are shown as a magnitude of inhibition when compared to controls (saline with 16.7 mM glucose).
1159
V o l . 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
5O I
Io
v
w
I-
40
,,< W ,.,.J W
Z -,,I
C O
30
Z --
20
O Z O __
lo
T T
m
z o
1ST PHASE ( 0 - 5 MIN)
Figure 2.
2ND PHASE ( 6 - 2 0 MIN)
Inhibitory effects of PST (35-49) at i0 and i00 nM on glucose-induced insulin release. The values (mean ± SEM) are expressed as an amount (magnitude) of inhibition of insulin release when compared to controls. * p <0.05.
important paracrine regulator of insulin secretion (4), the present study provides useful information in making biologically active agonists or antagonists of pancreastatin.
ACKNOWLEDGMENTS Supported by grants from the National Institutes of Health (DK 15241) and the John Sealy Memorial Endowment Fund - (2723).
REFERENCES i. Tatemoto K., Efendic S., Mutt V., Makk G., Feistner G.J., and Barchas J.D. (1986) Nature 324, 476-478. 2. Efendic S., Tatemoto K., Mutt V., Quan C., Chang D., and Ostenson C-G. (1987) Natl. Acad. Sci. U.S.A. 84, 7257-7260. 3. Silvestre R.A., Peiro E., Miralles P., Villanueva M.L., and Marco J. (1988) Life Sci. 42, 1361-1367. 4. Greeley Jr G.H., Thompson J.C., Ishizuka J., Cooper W.C., Levine M.A., Gorr S.U., and Cohn D.V. (1989) Endocrinology 124, 1235-1238. 5. Peiro E., Miralles P., Silvestre R.A., Villanueva M.L., and Marco J. (1989) Metabolism 38, 679-682. 6. Grodsky G.M., Batts A.A., Bennett L.L., Vcella C., McWilliams N.B., and Smith D.F. (1963) Am. J. Physiol. 205, 638-644. 7. Grodsky G.M., and Heldt H. (1984) In Methods in Diabetes Research (J. Larner, S.L. Pohl. Ed) Vol.l, pp. 137-146. 8. Greeley Jr G.H., and Thompson J.C. (1984) Regul. Peptides. 8, 97-103.
1160
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 0-
December 31, 1990
Pages 1157-1160
PANCREASTATIN: CHARACTERIZATION OF BIOLOGICAL ACTIVITY 1
2
3
Tao Zhang , Tohru Mochizuki , Masafumi Kogire , 2 Jin Ishizuka, Noboru Yanaihara , James C. Thompson and
George H. Greeley, Jr.
Department of Surgery The University of Texas Medical Branch Galveston, Texas 77550 Received October 26, 1990
SUMMARY: Pancreastatin (PST) (1-49) was first isolated from the porcine pancreas and can inhibit glucose-induced insulin release. PST (33-49), a PST C-terminal fragment, can also inhibit insulin release. The purpose of this study was to determine the shortest C-terminal biologically active fragment of PST, in terms of inhibition of insulin release from the isolated perfused rat pancreas. Porcine PST (1-49) and C-terminal fragments, PST (33-49), PST (35-49), PST (37-49) and PST (39-49) were synthesized by solid-phase methodology. PST (1-49), PST (33-49) and PST (35-49), at I0 nM, significantly (p <0.05) inhibited insulin release from isolated perfused rat pancreas: the first phase was inhibited by 15.6±2.4, 24.4±6.5 and 12.5±1.9% and the second phase, 18.9±2.7, 25.7±4.8 and 20.i±1.9% by PST (1-49), PST (33-49) and PST (35-49), respectively. PST (35-49) shows a dose-dependent inhibition of insulin release. PST (37-49) and PST (39-49) were, however, inactive. Our results indicate that the shortest C-terminal biologically active fragment is PST (35-49). These data further indicate that the C-terminal portion of PST is primarily responsible for the biological activity of PST. ©1990Ac~de~ioPress, Inc.
Pancreastatin (PST), a forty-nine residue peptide with a C-terminal am±de, was first isolated from the porcine pancreas by Tatemoto colleagues.
(i).
and
In the perfused rat pancreas PST (i-49) can inhibit
not only glucose-induced insulin release (1,2,3,4) but can also inhibit insulin release in response to arginine, tolbutamide, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), and cholecystokinin (CCK-8) (2,3,5).
In addition, two synthetic C-terminal
PST fragments, PST (14-49) and PST (33-49) inhibit insulin release with similar magnitudes from the isolated perfused rat pancreas (I).
These
I Visiting Scientist from the School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422, Japan. 2 School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422, Japan. s Visiting Scientist from the Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan.
1157
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
data suggest that biological activity of PST resides in the C-terminal portion of PST.
The purpose of this study was to determine the shortest
C-terminal biologically active fragment of PST in terms of inhibition of insulin release from isolated perfused rat pancreas.
METHODS Porcine PST C-terminal fragments, PST (33-49), PST (35-49), PST (37-49), and PST (39-49) were synthesized by solid-phase methodology using an automatic synthesizer (Beckman 990). Porcine PST (1-49) was purchased from Peninsula Laboratories, Belmont, CA. Male Sprague-Dawley rats weighing 250-350 g were permitted to eat and drink ad iibitum. Rats were anesthetized with pentobarbital (50 mg/kg) and the pancreas was isolated according to the method of Grodsky, and colleagues (6,7) with some modifications. The isolated pancreas was perfused through the celiac artery at a flow rate of 2 ml/min for 40 minutes with 95% 02-5% CO 2 saturated Krebs-Ringer bicarbonate buffer (KRBB, pH 7.4) containing 4% Dextran T-70 (Pharmacia Fine Chemical, NJ), 0.5% bovine serum albumin (BSA) (Sigma Chemical Company, MO), and 4.2 mM glucose. Peptide infusions were started 5 minutes before the perfusion of 16.7 mM glucose, and were given for 25 minutes. For each PST fragment tested, control pancreas were also perfused with glucose (16.7 mM) plus saline. Perfusates were collected from the portal vein and stored at -20 C for measurement of insulin levels. Perfusate insulin levels were measured by an insulin-specific radioimmunoassay as described previously (8). The intra and inter-assay variances at the IDSO were 9% and 10%, respectively. Integrated insulin outputs were divided into the first and second phases and analyzed by the Student's t test separately. A p value of <0.05 was considered statistically different.
RESULTS For each fragment of PST tested, a set of control pancreas (N=5) were infused with glucose and saline.
As shown at Figure i, insulin release
in response to 16..7 mM glucose occurred in two phases.
PST (1-49) as
well as the PST fragments, PST (33-49) and PST (35-49), inhibited the first and second phases of insulin release significantly (p <0.05) I).
(Table
PST (33-49) inhibited the first and second phases of insulin release
by 24.4±6.5 and 25.7±4.8%, respectively (Table i).
In addition,
PST (35-49) inhibited insulin release in a dose-dependent fashion. PST (35-49), at i00 nM, inhibited insulin release by 29.4±4.9% (Ist phase) and 26.2±1.6% (2nd phase), when compared to PST (35-49), at i0 ruM, for 12.5±1.9% (ist phase) and 20.1±1.9% (2nd phase) (Fig. 2).
PST (37-49)
and PST (39-49) were, however, inactive (Table i).
DISCUSSION In the present study, we have compared the inhibitory activities of PST (1-49) and C-terminal fragments, PST (33-49), (35-49),
(37-49) and
(39-49) on glucose-induced insulin release from the isolated perfused rat pancreas.
Our results show that both PST (1-49) and PST (33-49)
1158
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.0
E
2.0
1.5
1.5
Control
U) ¢: v
I-Z) Q,. I--
1.0
1.0
0.5
0.5
0.0
0.0
0 Z
z
~=~ I
-15 -10
I
I
I
I
I
I
l
I
-5
0
5
10
15
20
25
,50
~
,
-15 -10 -5
I
I
I
I
I
I
|
0
5
10
15
20
25
30
MINUTES
Figure i.
Effects of PST (33-49) (left panel) and PST (35-49) (right panel) on glucose-induced insulin release from the isolated perfused rat pancreas. PST (33-49) or PST (35-49), at i0 nM, were infused from -5 min to 20 min, and 16.7 mM glucose was infused from 0 min to 20 min. Saline was used as a control for the peptides. The flow rate of the perfusate was 2 ml/min. Insulin levels are expressed as mean i SEM (n = 5 pancreas/control and peptide).
significantly inhibit insulin release from the perfused rat pancreas response to 16.7 mM glucose, reports
(1,2,3,4).
PST (35-49),
in
These data confirm and extend the previous
We have also shown that a shorter C-terminal fragment,
inhibits the first and second phases of glucose-induced
insulin release in a dose-dependent fashion.
Since PST (37-49) and
PST (39-49) fail to suppress insulin release,
PST (35-49) is the shortest
C-terminal biologically active fragment,
at least in terms of inhibition
of insulin release from the isolated perfused rat pancreas. also suggests that the residues,
35-36
(Glu-Glu),
This finding
are essential
in exerting
the biological activity of PST (35-49). Our data also support the previous report that the biological portion of PST is located in the C-terminus.
Because pancreastatin may be an
Table i. Effects of PST (1-49) and PST C-terminal fragments on glucose-induced insulin release from the isolated perfused rat pancreas Peptide(10 nM) PST(I-49) PST(33-49) PST(35-49) PST(37-49) PST(39-49)
Inhibition of Insulin Release (9) Ist phase(0-5 min) 2nd phase(6-20 min) 15.6 24.4 12.5 1.8 3.8
+ 2.4* + 6.5* + 1.9" _+ 4.9 _+ 2.4
18.9 25.7 20.1 -6.8 5.7
± + + ± +
2.7* 4.8* 1.9" 6.8 5.1
Values are expressed as mean ± SEM (N = 5 pancreas); * P <0.05 vs. control. Values are shown as a magnitude of inhibition when compared to controls (saline with 16.7 mM glucose).
1159
V o l . 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o
5O I
Io
v
w
I-
40
,,< W ,.,.J W
Z -,,I
C O
30
Z --
20
O Z O __
lo
T T
m
z o
1ST PHASE ( 0 - 5 MIN)
Figure 2.
2ND PHASE ( 6 - 2 0 MIN)
Inhibitory effects of PST (35-49) at i0 and i00 nM on glucose-induced insulin release. The values (mean ± SEM) are expressed as an amount (magnitude) of inhibition of insulin release when compared to controls. * p <0.05.
important paracrine regulator of insulin secretion (4), the present study provides useful information in making biologically active agonists or antagonists of pancreastatin.
ACKNOWLEDGMENTS Supported by grants from the National Institutes of Health (DK 15241) and the John Sealy Memorial Endowment Fund - (2723).
REFERENCES i. Tatemoto K., Efendic S., Mutt V., Makk G., Feistner G.J., and Barchas J.D. (1986) Nature 324, 476-478. 2. Efendic S., Tatemoto K., Mutt V., Quan C., Chang D., and Ostenson C-G. (1987) Natl. Acad. Sci. U.S.A. 84, 7257-7260. 3. Silvestre R.A., Peiro E., Miralles P., Villanueva M.L., and Marco J. (1988) Life Sci. 42, 1361-1367. 4. Greeley Jr G.H., Thompson J.C., Ishizuka J., Cooper W.C., Levine M.A., Gorr S.U., and Cohn D.V. (1989) Endocrinology 124, 1235-1238. 5. Peiro E., Miralles P., Silvestre R.A., Villanueva M.L., and Marco J. (1989) Metabolism 38, 679-682. 6. Grodsky G.M., Batts A.A., Bennett L.L., Vcella C., McWilliams N.B., and Smith D.F. (1963) Am. J. Physiol. 205, 638-644. 7. Grodsky G.M., and Heldt H. (1984) In Methods in Diabetes Research (J. Larner, S.L. Pohl. Ed) Vol.l, pp. 137-146. 8. Greeley Jr G.H., and Thompson J.C. (1984) Regul. Peptides. 8, 97-103.
1160