Diabetes Research and Clinical Practice, 18 (1992)
167- 17 1 0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-8227/92/$05.00
167
DIABET 00695
Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse Georgios
Panagiotidis
aDepartment of Pharmacology,
a, Albert A. Salehi”, Per Westermarkb
and Ingmar Lundquista’C
University of Lund. Sweden, b Department of Pathology, University Hospital, University of Link6ping. Sweden, ‘Department of Cell Biology, University of Linktiping, Sweden
(Received 31 March 1992) (Revision accepted 3 August 1992)
Summary We examined the effects of a single intravenous injection of homologous islet amyloid polypeptide (IAPP) on the plasma levels of glucagon, insulin and glucose in the freely fed mouse. It was observed that IAPP suppressed basal glucagon levels concomitant with a decrease of the blood glucose concentrations. Basal plasma insulin levels were not affected. IAPP did not appreciably modulate the plasma concentration of glucose, insulin or glucagon after an intravenous glucose load. Further, IAPP inhibited the insulin secretory response to &adrenoceptor stimulation. IAPP also lowered the plasma glucagon levels following &adrenoceptor stimulation, whereas no apparent effect on plasma levels of glucose was observed. The data suggest that IAPP suppresses glucagon secretion and lowers blood glucose levels in the freely fed mouse. It might also exhibit a negative feedback inhibition on &adrenoceptor-induced insulin secretion, but has little influence on glucose-induced insulin release. Since IAPP is co-secreted with insulin, it is not inconceivable, that in the freely fed mouse, IAPP may act to amplify the blood glucose lowering effect of insulin through a direct suppression of glucagon secretion via the islet microcirculation. Key words: Islet amyloid polypeptide; stimulation; Mouse
Plasma
Introduction Recent interest has focused on the putative role for islet amyloid polypeptide (IAPP) in the pathogenesis of non-insulin-dependent diabetes melliCorrespondence to: Dr. Georgios Panagiotidis,
Pharmacology,
Department of University of Lund, S-223 62 Lund, Sweden.
glucagon
and
insulin;
Glucose;
/I,-Adrenoceptor
tus [l-4]. IAPP, a 37-amino acid polypeptide, is the main component of amyloid deposits in the pancreatic islets [l-4]. It is co-localized with insulin in the secretory granules of the /Icells, and is apparently also co-secreted with insulin [ 5-91. There are conflicting reports with regard to the effects of IAPP on islet hormone secretion and
168 carbohydrate regulation (cf. [9]). In the present investigation we have studied the acute in vivo effects of injecting homologous IAPP on the plasma levels of glucagon, insulin, and glucose in the basal state as well as after administration of glucose or terbutaline (&adrenoceptor agonist).
Materials and Methods Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden) weighing 25-30 g were used. The animals were kept on a standard pellet diet (Astra-Ewos, Siidertalje, Sweden) and tap water ad libitum before and throughout the experiments. Experimental Mouse/rat islet amyloid polypeptide (IAPP) was custom synthesized by Multiple Peptide Systems (San Diego, CA). The peptide was C-terminally DL-Terbutaline amidated. sulfate (1-(3,5dihydroxyphenyl)-2-(tert-butylamino)ethanolsulfate) was a gift from Astra-Draco AB, Lund, Sweden. All other drugs or chemicals were obtained from Kebo Ltd, Stockholm, Sweden. IAPP was dissolved in 0.9% NaCl-0.2% gelatine (controls received 0.9% NaCl-0.2% gelatine alone) and administered i.v. in a tail vein either alone or 10 s before a rapid i.v. injection of D-glucose or terbutaline. Blood sampling was performed as previously described [ lo]. Plasma levels of insulin and glucagon were determined radioimmunochemitally [ 11,121 with the exception that the ethanol precipitation step in the glucagon assay was replaced by the charcoal separation procedure [ 131, which in our hands was found to be more convenient for routine use. The insulin RIA kit was obtained from Novo Research, Bagsvaerd, Denmark. The antiserum used for determination of plasma glucagon is highly selective against pancreatic glucagon (Milab Ltd, Malmii, Sweden). Plasma glucose levels were determined by a glucose oxidase method [ 141. Probability levels of random difference were calculated by Student’s two-tailed t-test.
Results The first set of experiments was designed to study the effect of an i.v. injection of IAPP on the basal plasma levels of glucagon, insulin, and glucose at 6, 15, and 30 min after administration of the peptide. Fig. 1 shows that basal levels of glucagon and glucose were reduced at 6 min by approximately 30% and 25 %, respectively. This effect was still apparent at 15 min. At 30 min the plasma levels of glucagon and glucose were restored to normal. No effect on basal insulin levels was recorded at any time-point after injection of IAPP. Fig. 2 shows the effect of IAPP on glucoseinduced changes in the plasma levels of glucagon, insulin and glucose at 2, 6, 15, and 30 min after an i.v. injection of IAPP + glucose. IAPP was
minutes
Fig. 1. Plasma
levels of glucagon.
ferent time points (5 nmol/kg) group.
Means
after a rapid
or saline + SEM
ity level of random
insuhn
and glucose
intravenous
(controls). are shown. difference,
There
injection
at difof IAPP
were 7 mice in each
Asterisks *p
167- 17 1 0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-8227/92/$05.00
167
DIABET 00695
Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse Georgios
Panagiotidis
aDepartment of Pharmacology,
a, Albert A. Salehi”, Per Westermarkb
and Ingmar Lundquista’C
University of Lund. Sweden, b Department of Pathology, University Hospital, University of Link6ping. Sweden, ‘Department of Cell Biology, University of Linktiping, Sweden
(Received 31 March 1992) (Revision accepted 3 August 1992)
Summary We examined the effects of a single intravenous injection of homologous islet amyloid polypeptide (IAPP) on the plasma levels of glucagon, insulin and glucose in the freely fed mouse. It was observed that IAPP suppressed basal glucagon levels concomitant with a decrease of the blood glucose concentrations. Basal plasma insulin levels were not affected. IAPP did not appreciably modulate the plasma concentration of glucose, insulin or glucagon after an intravenous glucose load. Further, IAPP inhibited the insulin secretory response to &adrenoceptor stimulation. IAPP also lowered the plasma glucagon levels following &adrenoceptor stimulation, whereas no apparent effect on plasma levels of glucose was observed. The data suggest that IAPP suppresses glucagon secretion and lowers blood glucose levels in the freely fed mouse. It might also exhibit a negative feedback inhibition on &adrenoceptor-induced insulin secretion, but has little influence on glucose-induced insulin release. Since IAPP is co-secreted with insulin, it is not inconceivable, that in the freely fed mouse, IAPP may act to amplify the blood glucose lowering effect of insulin through a direct suppression of glucagon secretion via the islet microcirculation. Key words: Islet amyloid polypeptide; stimulation; Mouse
Plasma
Introduction Recent interest has focused on the putative role for islet amyloid polypeptide (IAPP) in the pathogenesis of non-insulin-dependent diabetes melliCorrespondence to: Dr. Georgios Panagiotidis,
Pharmacology,
Department of University of Lund, S-223 62 Lund, Sweden.
glucagon
and
insulin;
Glucose;
/I,-Adrenoceptor
tus [l-4]. IAPP, a 37-amino acid polypeptide, is the main component of amyloid deposits in the pancreatic islets [l-4]. It is co-localized with insulin in the secretory granules of the /Icells, and is apparently also co-secreted with insulin [ 5-91. There are conflicting reports with regard to the effects of IAPP on islet hormone secretion and
168 carbohydrate regulation (cf. [9]). In the present investigation we have studied the acute in vivo effects of injecting homologous IAPP on the plasma levels of glucagon, insulin, and glucose in the basal state as well as after administration of glucose or terbutaline (&adrenoceptor agonist).
Materials and Methods Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden) weighing 25-30 g were used. The animals were kept on a standard pellet diet (Astra-Ewos, Siidertalje, Sweden) and tap water ad libitum before and throughout the experiments. Experimental Mouse/rat islet amyloid polypeptide (IAPP) was custom synthesized by Multiple Peptide Systems (San Diego, CA). The peptide was C-terminally DL-Terbutaline amidated. sulfate (1-(3,5dihydroxyphenyl)-2-(tert-butylamino)ethanolsulfate) was a gift from Astra-Draco AB, Lund, Sweden. All other drugs or chemicals were obtained from Kebo Ltd, Stockholm, Sweden. IAPP was dissolved in 0.9% NaCl-0.2% gelatine (controls received 0.9% NaCl-0.2% gelatine alone) and administered i.v. in a tail vein either alone or 10 s before a rapid i.v. injection of D-glucose or terbutaline. Blood sampling was performed as previously described [ lo]. Plasma levels of insulin and glucagon were determined radioimmunochemitally [ 11,121 with the exception that the ethanol precipitation step in the glucagon assay was replaced by the charcoal separation procedure [ 131, which in our hands was found to be more convenient for routine use. The insulin RIA kit was obtained from Novo Research, Bagsvaerd, Denmark. The antiserum used for determination of plasma glucagon is highly selective against pancreatic glucagon (Milab Ltd, Malmii, Sweden). Plasma glucose levels were determined by a glucose oxidase method [ 141. Probability levels of random difference were calculated by Student’s two-tailed t-test.
Results The first set of experiments was designed to study the effect of an i.v. injection of IAPP on the basal plasma levels of glucagon, insulin, and glucose at 6, 15, and 30 min after administration of the peptide. Fig. 1 shows that basal levels of glucagon and glucose were reduced at 6 min by approximately 30% and 25 %, respectively. This effect was still apparent at 15 min. At 30 min the plasma levels of glucagon and glucose were restored to normal. No effect on basal insulin levels was recorded at any time-point after injection of IAPP. Fig. 2 shows the effect of IAPP on glucoseinduced changes in the plasma levels of glucagon, insulin and glucose at 2, 6, 15, and 30 min after an i.v. injection of IAPP + glucose. IAPP was
minutes
Fig. 1. Plasma
levels of glucagon.
ferent time points (5 nmol/kg) group.
Means
after a rapid
or saline + SEM
ity level of random
insuhn
and glucose
intravenous
(controls). are shown. difference,
There
injection
at difof IAPP
were 7 mice in each
Asterisks *p
