Download Glycogenolytic effect of pancreastatin in the rat

Bioscience Reports, Vol. I0, No. 1, 1990
Glycogenolytic Effect of Pancreastatin
in the Rat
Victor S a n c h e z , t J u a n R . Calvo I and R a i m u n d o G o b e r n a l'z
Received October 13, 1989
Pancreastatin is a novel 49-amino acid peptide with a C-terminal glycine amide. The peptide was
isolated from porcine pancreatic extracts and shows a structural similarity to chromogranin A. The
effect of synthetic porcine pancreastatin on blood glucose levels and hepatic glycogen content was
investigated in rats in vivo. Pancreastatin (300pmol/kg) produced a time-dependent decrease in
glycogen content of liver and a slight hyperglycemia. Basal plasma insulin and glucagon levels were
not modified by pancreastatin. We suggest that pancreastatin could play a biological role in the
glucose metabolism through a glycogenolytic effect.
KEY WORDS: glycogen; pancreastatin.
INTRODUCTION
Pancreastatin, a new 49-amino acid peptide with a C-terminal gylcine amide, was
recently isolated from porcine pancreatic extracts by using the amide structure as
a marker during the isolation procedure (1). Pancreastatin is widely distributed
throughout the central nervous system (2) and in endocrine cells of the
gastrointestinal tract, islets and exocrine cells of pig pancreas (3), and possesses a
striking sequence homology with a part of bovine chromogranin A, a secretory
glycoprotein widely distributed throughout the neuroendocrine system (4). It has
been shown that pancreastatin inhibits glucose and arginine induced insulin
release, from the isolated perfused pancreas (1, 5, 6). In the mouse, pancreastatin
inhibits both glucose and carbachol induced insulin release, stimulates baseline
glucagon secretion, and induces a slight hyperglycemia (7). However, the effects
of pancreastatin in the rat under in vivo conditions have been very little studied.
Thus, Funakoshi et al. have found that an intravenous infusion of pancreastatin
inhibited the plasma insulin response to the intragastric infusion of glucose, and
increased the plasma glucagon response to the intravenous infusion of arginine,
but had not any effect on basal plasma insulin and glucagon concentrations (8).
The aim of this study is to investigate, in vivo, the effect of pancreastatin on
1 Department of Medical Biochemistry and Molecular Biology School of Medicine, University of
Sevilla, Auda Sanchez Pizjuan 4 41009, Sevilla, Spain.
2 To whom correspondence should be addressed.
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0144-8463/90/0200-008750600/0~ 1990PlenumPublishingCorporation
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Sanchez, Calvo and Goberna
glucose metabolism. We show that pancreastatin causes hepatic glycogenolysis
and hyperglycemia without altering basal plasma insulin and glucagon levels.
MATERIALS AND METHODS
Animals
Male Wistar rats weighing 250-350 g were used. The animals were fed a
standard diet ad libitum. The experiments were performed on anaesthetized
animals (pentobarbital sodium, 50 mg/kg intraperitoneally) after a short-term fast
(4-6 h) in the postabsortive state.
Experimental Design
Pancreastatin was obtained from Peninsula Laboratories Europe (Merseyside
UK); glucagon was from Novo Biolabs (Bagsvaerd, Denmark); bovine serum
albumin (BSA) from Sigma Chemical (St Louis, MO, USA). The peptides were
dissolved in 0.9% NaCI-I% BSA to prevent adsorption to the syringe.
Anaesthetized rats were injected intravenously in the superior mesenteric
vein with porcine pancreastatin or porcine glucagon. The doses were 300 pmol/kg
and the volume injected was l ml. Control rats were injected with 0.9%
NaCI-I% BSA.
Pieces of hepatic lobes from the same rat (caudate, left and right main
lobes), weighing approximately 0.3 g, were tied off and rapidly excised before, at
10 and 20min after the injection and immediately processed to obtain the
glycogen by alcoholic precipitation.
Blood samples (0.9 ml) were taken from the jugular vein before, at 5, 10 and
20 min after the injection. The blood obtained was heparinized and 20/,1 were
taken to measure blood glucose levels, the remaining blood was immediately
centrifuged and plasma separated and stored at -20~ for insulin and glucagon
determinations.
Determinations
ELISA (Enzyme linked immunosorbent assay, kit from Boerhinger Mannhein GmbH, FRG) was employed to measure plasma insulin. Radioimmunoassay
(kit from Medgenix, Brussels, Belgium) was employed to measure plasma
glucagon. Glycogen was determined by enzymatic conversion (amyloglucosidase
from Boehringer Mannhein GmbH, FRG) to glucose which was determined by
the glucose oxidase method. Blood glucose levels were also determined by this
method.
Student's t-test was used to test the degree of significance.
RESULTS
Effect of Pancreastatin and Glucagon on Hepatic Glycogenolysis
The injection in the superior mesenteric vein of 300 pmol/kg of pancreastatin
produced a decrease in glycogen content of liver. The basal content of hepatic
GlycogenolyticEffectof Pancreastatin
89
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Fig. 1. Glycogenolysisafter the injection of pancreastatin (300pmol/kg; vertically striped bars),
glucagon (300pmol/kg; horizontallystriped bars)
or saline-BSA-l% (open bars) in the superior
mesenteric vein. Means+ SEM are shown. *p <
0.05, **p <0.001 (n = 7), probabilitylevel of random differencebetween any group and the control
group.
o
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20
10
Time (rain)
glycogen was 51.3 + 5 mg/g liver. The results are expressed as the difference of
glycogen concentration between the basal conditions and after the injection of
saline-l%-BSA, glucagon or pancreastatin (Fig. 1). The glycogenolysis was
significantly enhanced by pancreastatin at 10 min (7.8 4- 1.4 mg/g compared with
2.85: 1.4mg/g in controls, p < 0 . 0 5 ) ; the enhancement was comparable in
magnitude to the increase caused by glucagon. At 20 rain the glycogenolysis was
enhanced from 3.4 + 1 mg/g in controls to 12.2 + 1.2 mg/g (p < 0.001) in animals
injected with pancreastatin. There were no significant differences between this
group and the animals injected with glucagon.
Effect of Pancreastatin and Glucagon on Baseline Levels of Blood Glucose,
Plasma Insulin and Plasma Glucagon
Blood glucose levels were significantly increased by pancreastatin
(300 pmol/kg), with the peak at 5 min (Fig. 2). Thus, at 5 min after the injection,
blood glucose levels were 4 . 3 + 0 . 2 m M in control rats and 5 . 1 + 0 . 2 m M in
animals injected with pancreastatin (p <0.01). At 10rain the differences were
still significant between controls (4.2 + 0.2 raM) and the animals injected with
pancreastatin (4.8 + 0.1, p < 0.05). At 20 min, blood glucose levels did not differ
from those in control rats.
Blood glucose levels constantly increased after the injection of glucagon. At
5 rain there were no significant differences between this group and that injected
with pancreastatin. At 10min the blood glucose levels were 6 . 2 + 0 . 2 m M
(p < 0.001) and at 20 min they were 6.7 + 0.3 mM (p < 0.00l).
Baseline levels of plasma insulin and glucagon levels were not significantly
affected by injection of pancreastatin (Fig. 2).
Glucagon slightly elevated basal plasma insulin levels. At 20 rain the increase
was significant (34 + 7/~U/ml compared with 16 + 3/~U/ml in the control group,
p < 0.05).
After glucagon injection, the plasma glucagon levels were 720 + 70 pg/ml at
5 min, 330 + 50 pg/ml at 10 min and 180 + 38 pg/ml at 20 min.
Sanchez, Calvo and Goberna
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Fig. 2. Blood glucose levels (top), plasma levels of
insulin (middle) and glucagon (bottom) before and at
various time points after the injectionof pancreastatin
(300pmol/kg; e, n = 8), glucagon (300pmol/kg; 9
n =8) or saline-BSA-1% (A, n = 6). Means :I:SEM
are shown. *p <0.05, **p <0.0t, ***p<0.001,
probability level of random difference between any
group and the control group.
T i m e (rain)
DISCUSSION
This paper describes the effect of pancreastatin, in vivo, on the hepatic
glycogen in rats. This 49-residue peptide with a C-terminal glycine amide was
isolated from porcine extracts by Tatemoto et al. (1). Recently, Eiden (4) and
Huttner and Benedum (9) independently drew attention to a striking similarity in
amino acid sequence between porcine pancreastatin and a segment of the peptide
chain of bovine chromogranin A. This similarity suggests a functional role of
chromogranin A as a precursor of pancreastatin or pancreastatin-like peptide.
Chromogranin A and its related proteins, chromogranin B and C and secretogranin I and II, are widely distributed throughout the neuroendocrine system and
other tissues (10, 11, 12). Thus, the role of pancreastatin may not be limited to
the pancreas. The foregoing results demonstrate that, in vivo, pancreastatin
(300 pmol/kg) injected via the portal vein stimulates the hepatic glycogenolysis,
and this effect could be direct since we have found no effect on basal insulin and
glucagon levels. Other authors have studied the effect of pancreastatin
(4 nmol/kg) in the mouse in v i v o ; Ahr6n et al. (7) have found that this peptide
weakly inhibits insulin secretion and stimulates glucagon secretion. These
contradictory results could be explained by the different animal species used, the
different doses employed and the different experimental models. On the other
hand, Lindskog et al. (13) reported that it slightly elevated basal plasma glucagon
levels without altering basal plasma insulin. Recently, Funakoshi et al. (8) have
Glycogenolytic Effect of Pancreastatin
91
found no effect of an intravenous infusion of pancreastatin (1 and 10 n m o l / k g / h )
on unstimulated insulin and glucagon release. H o w e v e r , unlike our study, they
found no effect on the basal plasma glucose levels. This difference m a y result
from the different metabolic situations, since they conducted the experiments
after an overnight fasting (18-24 h) when the glycogen stores are almost depleted,
and we suggest that the hyperglycemic effect is caused by glycogenolysis.
The glycogenolysis caused by pancreastatin was c o m p a r a b l e to that caused by
glucagon. The differences in the hyperglycemia caused by pancreastatin and
glucagon could be explained by a different effect of pancreastatin on gluconeogenesis and glycolysis (it has not b e e n studied yet), whereas glucagon increases
gluconeogensis as much as glycogenolysis (14) and inhibits glycolysis, in large part
mediated by changes in the level of fructose-2,6-biphosphate (15). H o w e v e r ,
further studies are needed to clarify this point.
ACKNOWLEDGEMENTS
Supported by a G r a n t f r o m Comision Asesora de Investigaci6n Cientifica y
T6cnica ( C A I C Y T no. 1106). V. Sanchez Margalet is a doctoral fellow of the
University of Sevilla, Spain, sponsored by M o n t e de Piedad Y Caja de A h o r r o s
de SeviUa.
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