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The effect of amylin and calcitonin gene-related peptide on insulin-stimulated glucose transport in the diaphragm
Vol. 169, No. 2, 1990 June 15, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 451-454
THE EFFECT OF AMYLIN AND CALCITONIN GENE-RELATED PEPTIDE ON INSULIN-STIMULATED GLUCOSETRANSPORT IN THE DIAPHRAGM John S. Hothersall, Roslyn P. Muirhead and Sunll Wimalawansa* Department of Biochemistry,University College and Middlesex Schoolof Medicine, Wmdeyer Building ClevelandSt, London W 1P 6DB, UK *BiochemicalEndocrinologyUnit, Department of Medicine, Royal PostgraduateMedical School, HammersmithHospital, London W12 ONN, UK Received April
20, 1990
The two peptidescalcitonin generelated peptide (CGRP) and amylin at 1uM levelsin an isolatedrat diaphragm preparationinhibited insulin stimulated 2-deoxy[3H]glucose transport by 30 and 60 percent, respectively;this wasthe caseat maximal(ImU) and sub-maximal(OSmU) insulinconcentrations.No effect wasmeasuredon the basal level of 2-deoxy[SH]glucosetransport. 01990 Academic Press,Inc.
Albumin preparedfrom the plasmaof diabetic patients hasbeen found by VallanceOwen (1) to have more antagonismto insulin than albuminpreparedfrom normal subjects.In each casethe antagonismis not due to albuminitself but to something associatedwith it, hencethe namesynalbumin.This antagonist,which did not affect insulin action on adiposetissue,wasfound when isolatedto be a polypeptide of molecularweight 4000. It has alsobeen known for sometime that the pancreasfrom non-insulin dependentdiabetics(XIDD) containsdepositsof amyloid, an extracellularprotein matrix (2). The major peptide componentof amyloid, known as amylin, hasbeen purified and sequenced.it has 37 amino acidsand a 46% homologvwith calcitonin generelated-peptide (CGRP) (3). Certain effects of amylin have been observedwhich may have a direct bearingon the pathology of diabetes,the most controvertial of these is a suppressionof basaland glucosestimulatedinsulin secretion(4), which in contrast hasnot beendemonstratedby Ghatei and co-workers (5) in both hi tivo and in vitro systems.Theseauthorshave confirmed the CGRP ?ffect on insulin secretion previously demonstratedby Petterssonet al (6). Inhibition of basaland
451
ooo6-291x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
Vol.
169,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
insulin stimulated glycogen synthesis in skeletal muscle has been reported and in common with earlier studies with synalbumin, no response was evident in adipose tissue (7). More recent studies have shown that induction of hyperglycaemia with dexamethasone induces a six fold increase in the mRNA of amylin (8). Little is known of the effects of amylin or CGRP on glucose transport, although Cooper et al (7) have shown that glucose transport, when evaluated as the sum of glycolysis and glycogen synthesis, is inhibited by amylin. As yet there is no experimental data on the direct effect of amylin on glucose transport in muscle although the above authors have stated. from their own unpublished experiments, that insulin-stimulated 3-0-methylglucose
transport is unaffected by CGRP at concentrations of lo-8M in
soleus muscle strips (9). In view of the remarkable similarities between synalbumin and amylin in their origin, tissue specificity and molecular weight the effects of synthetic amylin and CGRP on insulin stimulated glucose transport in isolated rat diaphragm was examined. Methods
The isolatedrat hemidiaphragmis a particularly well suitedmodel for transport studiesin muscledue to its uniform cross-sectionalthicknessand the fact that it can be excisedwith the minimumof damageto musclefibres, two factors which cannot be controlled in other musclepreparations.The advantagesof the 2-deoxyglucoseuptake procedurefor measuringglucosetransport are its sensitivity and specificity, and, once phosphorylated.it is trappedwithin tissues as the 2-deoxyglucose6-phosphate(10). Hemidiaphragms wereprepared from male Wistar rats (140- 160g)starved for 16 hours.Whole diaphragmswere removedand after trimming off connectivetissuewere cut into equalhalvesalong the central tendon, excluding the posterior tendon and xiphistemum.The hemidiaphragms were blotted dry and weighedand then placedin stopperedflaskscontaining Imi Krebs Ringer Bicarbonatecontaining O.OOloio fatty acid free bovine serumalbumin, in the absenceof glucoseand gassedwith OZ/COZ95%/5%for a 15 minute pre-incubation period. Glucose(1OmM) and 2-[sH]deoxyglucose( lOmM, 40 umoles/uCi)were addedat this stageaswere the insulin, amylin or CGRP (all in O.OOlNacetic acid) where required, and the incubation allowedto continue for a further 30 minutesin a shakingwater bath. After this time, the hemidiaphragms were removedand thoroughly rinsed in salineand homogenisedin 3ml of 1040trichloroaceric acid and, after centrifugation at 7~10~g mins, the total supematantwascounted. 2-deoxy[sH]glucose uptake wascalculatedand expressedasumolesof 2-deoxyglucose/g/hour. Resultsand Discussion In the absenceof amylin and CGRP, insulin (O.SmU)stimulates2deox@H]glucose transport in rat hemldiaphragmsby 50%(Table 1). In the presence 452
Vol.
169, No. 2, 1990
Table 1. The effect
BIOCHEMICAL
of amylin
Condition
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and OXP on the insulin stimulated into rat hemidiaphr~
rate
2-deoxyglucose uptake
of 2-deoxy[3H]gluccee
trausport
insulin stimulation
umolas/g/hour
(n)
7.05 f 10.42 f 10.50 f
0.22 0.32 0.60
(25) (37) (22)
amylin/CGRP effect on insulin stimulation
umoles/g/hour
(Xl
Control Insulin Insulin
(l.CmLJ) (0.5d)
ax@ Insulin In!mlin
MlnU + CGRP (1uM) (0. !inlJ) + m (1uM)
6.58 f 9.35 f 9.58 f
0.27 0.30 0.27
(81 (7)
+2X i 0.12 +2.53 f 0.10
-32Ff -2z**
AOl&4iIl Insulin Insulin
(l.MJ) (OhlJ)
7.39 f 8.23 f 8.73 f
0.20 0.14 0.12
(7) (8) (19)
+1.18 f 0.10 +1.68 f 0.15
-64x”” -51x**
+ f!mylio + blylin
WI1 (lull)
+3.37 f 0.11 +3.45 f 0.18
(8)
Values are expressed as means f standard error of the means for the number of values in parenthesis. The insulin and insulin with amylin or CGRP were analysed by comparison of corresponding pairs, *** p< 0.001 and ** p< 0.01
of CGRP (1uM) this level of stimulationis decreasedby 30%.Amylin (1uM) producesa greater response,with 60%inhibition and neither peptide alone, hasany effect on the basallevel of 2-deoxy13H]glucosetransport (Table 1). In the presenceof 1.0 mU of insulin a similarstimulationis evident, and the antagonisticeffects of the two peptidesremain essentiallythe same even at this saturatinglevel of insulin. These result are very similarto the those obtainedby Valiance-Owen(1) using plasmasynalbuminisolatedfrom NIDD patients in the rat diaphragmsystem.The possibility that synalbuminand amylin are, in fact, the samepeptidehasbeen further strengthenedby the reported lack of effect of thesepeptideson insulin stimulatedmetabolismin adiposetissue(1,7). Our data indicatesamylin and CGRP action is on insulin stimulatedglucosetransport rather than basaluptake (Table 1). The action of insulin on glucosetransport is through translocationof a pool of specific transporter protein molecules,located within insulin sensitivetissues,to the cell surface(11) cardiac
and
skeletal
and thesetransporter molecules,found predominantlyin muscle,
are identical
to those
expressed
in
adipose
tissue
(12)
The implications,therefore, of our resultsare that amylin and CGRP are able to regulate
the
translocation
of insulin
sensitive
glucose
transporters
in muscle
and
indicate the presenceof specificbinding sitesfor thesepeptideson the muscle 453
Vol.
169,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cell surface. This is in contrast to the later results of Leighton and Cooper which indicate that the insulin antagonism of amylin is directed at glycogen synthesis. both
basal
and
insulin stimulated, and that glucose transpon is unaffected (9).
There is some contention over the effect of amylin on insulin secretion. the increase in the ratio of amylin/insulin mRNA with experimental models of diabetes indicates that the role of amylin in the aetiology of NIDD
is a complex one.
However, the demonstration that the insulin sensitive glucose transporter present in muscle is in some way impaired by amylin and CGRP could explain why elevated glucose levels are perpetuated during elevated pancreatic amylin production and output, thus maintaining an increase in the amylin to insulin ratio and eventually leading to amyloid deposits. Acknowledgments We thank Professors J. Valiance-Owen, P. N. Campbell, P. Mclean and I. McIntyre for initiating this project and The British diabetic Association and Basil Samuel Charitable Trust who have supplied grants to support this work. References 1. Valiance-Owen, J. Pfeiffer Diabetes Mellitus Vol II Edit0rsJ.F. Lehmamrs Munich: 105-122 (1971). 2. Opie, E.L.: J. Exp. Med. 5: 527-540 (1901). 3. Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M.: Proc. Natl. Acad. Sci. 84: 8628-8632 (1987). 4. Ohsawa, H., Kanasuka, A., Yamaguchi, T., Makino, H. and Yoshida, S.: Biochem. Biophys. Res. Commun. 160: 961-967 (1989). 5. Ghatei, MA., Datta, H.K., Zaidi, M., Bretherton-Watt, D., Wimalawansa, S.J., McIntyre, I. and Bloom, S.R.: J. Endocrin. 124: R9-11 (1990). 6. Pettersson, M., Ahren, B., Bottcher, G. and Sundler, F.: Endochrinology 119: 865-869 (1986). 7. Cooper, G.J.S., Leighton, B., Dimitriadis, G.D., ParryBillings, M., Kowalchuk, J.M., Howland, K., Rothbard, J.B., Willis, A.C. and Reid, K.B.M.: Proc. Natl. Acad. Sci. 85: 7763-7766 (1988). 8. Bretherton-Watt, D., Ghatei, M.A., Bloom, S.R., Jamal,H., Ferrier, G.J.M., Girgis, S.I. and Legon, S.: Diabetologia 32:881-883 (1989). 9. Leighton, B. and Cooper, G.J.S.: Nature 335: 632-635 (1988). 10. Sokoloff, L.J.: J. Neurochem. 29: 13-19 (1977). 11. Suzuki, K. and Kono, T.: Proc. Natl. Acad. Sci. 79: 3777-3779 (1980) 12. Charron, M.J., Brosius, F.C., Alper, S.L. and Lodish, H.F.: Proc. Natl. Acad. Sci. 86: 2535-2539 (1989).
454
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 451-454
THE EFFECT OF AMYLIN AND CALCITONIN GENE-RELATED PEPTIDE ON INSULIN-STIMULATED GLUCOSETRANSPORT IN THE DIAPHRAGM John S. Hothersall, Roslyn P. Muirhead and Sunll Wimalawansa* Department of Biochemistry,University College and Middlesex Schoolof Medicine, Wmdeyer Building ClevelandSt, London W 1P 6DB, UK *BiochemicalEndocrinologyUnit, Department of Medicine, Royal PostgraduateMedical School, HammersmithHospital, London W12 ONN, UK Received April
20, 1990
The two peptidescalcitonin generelated peptide (CGRP) and amylin at 1uM levelsin an isolatedrat diaphragm preparationinhibited insulin stimulated 2-deoxy[3H]glucose transport by 30 and 60 percent, respectively;this wasthe caseat maximal(ImU) and sub-maximal(OSmU) insulinconcentrations.No effect wasmeasuredon the basal level of 2-deoxy[SH]glucosetransport. 01990 Academic Press,Inc.
Albumin preparedfrom the plasmaof diabetic patients hasbeen found by VallanceOwen (1) to have more antagonismto insulin than albuminpreparedfrom normal subjects.In each casethe antagonismis not due to albuminitself but to something associatedwith it, hencethe namesynalbumin.This antagonist,which did not affect insulin action on adiposetissue,wasfound when isolatedto be a polypeptide of molecularweight 4000. It has alsobeen known for sometime that the pancreasfrom non-insulin dependentdiabetics(XIDD) containsdepositsof amyloid, an extracellularprotein matrix (2). The major peptide componentof amyloid, known as amylin, hasbeen purified and sequenced.it has 37 amino acidsand a 46% homologvwith calcitonin generelated-peptide (CGRP) (3). Certain effects of amylin have been observedwhich may have a direct bearingon the pathology of diabetes,the most controvertial of these is a suppressionof basaland glucosestimulatedinsulin secretion(4), which in contrast hasnot beendemonstratedby Ghatei and co-workers (5) in both hi tivo and in vitro systems.Theseauthorshave confirmed the CGRP ?ffect on insulin secretion previously demonstratedby Petterssonet al (6). Inhibition of basaland
451
ooo6-291x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
Vol.
169,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
insulin stimulated glycogen synthesis in skeletal muscle has been reported and in common with earlier studies with synalbumin, no response was evident in adipose tissue (7). More recent studies have shown that induction of hyperglycaemia with dexamethasone induces a six fold increase in the mRNA of amylin (8). Little is known of the effects of amylin or CGRP on glucose transport, although Cooper et al (7) have shown that glucose transport, when evaluated as the sum of glycolysis and glycogen synthesis, is inhibited by amylin. As yet there is no experimental data on the direct effect of amylin on glucose transport in muscle although the above authors have stated. from their own unpublished experiments, that insulin-stimulated 3-0-methylglucose
transport is unaffected by CGRP at concentrations of lo-8M in
soleus muscle strips (9). In view of the remarkable similarities between synalbumin and amylin in their origin, tissue specificity and molecular weight the effects of synthetic amylin and CGRP on insulin stimulated glucose transport in isolated rat diaphragm was examined. Methods
The isolatedrat hemidiaphragmis a particularly well suitedmodel for transport studiesin muscledue to its uniform cross-sectionalthicknessand the fact that it can be excisedwith the minimumof damageto musclefibres, two factors which cannot be controlled in other musclepreparations.The advantagesof the 2-deoxyglucoseuptake procedurefor measuringglucosetransport are its sensitivity and specificity, and, once phosphorylated.it is trappedwithin tissues as the 2-deoxyglucose6-phosphate(10). Hemidiaphragms wereprepared from male Wistar rats (140- 160g)starved for 16 hours.Whole diaphragmswere removedand after trimming off connectivetissuewere cut into equalhalvesalong the central tendon, excluding the posterior tendon and xiphistemum.The hemidiaphragms were blotted dry and weighedand then placedin stopperedflaskscontaining Imi Krebs Ringer Bicarbonatecontaining O.OOloio fatty acid free bovine serumalbumin, in the absenceof glucoseand gassedwith OZ/COZ95%/5%for a 15 minute pre-incubation period. Glucose(1OmM) and 2-[sH]deoxyglucose( lOmM, 40 umoles/uCi)were addedat this stageaswere the insulin, amylin or CGRP (all in O.OOlNacetic acid) where required, and the incubation allowedto continue for a further 30 minutesin a shakingwater bath. After this time, the hemidiaphragms were removedand thoroughly rinsed in salineand homogenisedin 3ml of 1040trichloroaceric acid and, after centrifugation at 7~10~g mins, the total supematantwascounted. 2-deoxy[sH]glucose uptake wascalculatedand expressedasumolesof 2-deoxyglucose/g/hour. Resultsand Discussion In the absenceof amylin and CGRP, insulin (O.SmU)stimulates2deox@H]glucose transport in rat hemldiaphragmsby 50%(Table 1). In the presence 452
Vol.
169, No. 2, 1990
Table 1. The effect
BIOCHEMICAL
of amylin
Condition
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and OXP on the insulin stimulated into rat hemidiaphr~
rate
2-deoxyglucose uptake
of 2-deoxy[3H]gluccee
trausport
insulin stimulation
umolas/g/hour
(n)
7.05 f 10.42 f 10.50 f
0.22 0.32 0.60
(25) (37) (22)
amylin/CGRP effect on insulin stimulation
umoles/g/hour
(Xl
Control Insulin Insulin
(l.CmLJ) (0.5d)
ax@ Insulin In!mlin
MlnU + CGRP (1uM) (0. !inlJ) + m (1uM)
6.58 f 9.35 f 9.58 f
0.27 0.30 0.27
(81 (7)
+2X i 0.12 +2.53 f 0.10
-32Ff -2z**
AOl&4iIl Insulin Insulin
(l.MJ) (OhlJ)
7.39 f 8.23 f 8.73 f
0.20 0.14 0.12
(7) (8) (19)
+1.18 f 0.10 +1.68 f 0.15
-64x”” -51x**
+ f!mylio + blylin
WI1 (lull)
+3.37 f 0.11 +3.45 f 0.18
(8)
Values are expressed as means f standard error of the means for the number of values in parenthesis. The insulin and insulin with amylin or CGRP were analysed by comparison of corresponding pairs, *** p< 0.001 and ** p< 0.01
of CGRP (1uM) this level of stimulationis decreasedby 30%.Amylin (1uM) producesa greater response,with 60%inhibition and neither peptide alone, hasany effect on the basallevel of 2-deoxy13H]glucosetransport (Table 1). In the presenceof 1.0 mU of insulin a similarstimulationis evident, and the antagonisticeffects of the two peptidesremain essentiallythe same even at this saturatinglevel of insulin. These result are very similarto the those obtainedby Valiance-Owen(1) using plasmasynalbuminisolatedfrom NIDD patients in the rat diaphragmsystem.The possibility that synalbuminand amylin are, in fact, the samepeptidehasbeen further strengthenedby the reported lack of effect of thesepeptideson insulin stimulatedmetabolismin adiposetissue(1,7). Our data indicatesamylin and CGRP action is on insulin stimulatedglucosetransport rather than basaluptake (Table 1). The action of insulin on glucosetransport is through translocationof a pool of specific transporter protein molecules,located within insulin sensitivetissues,to the cell surface(11) cardiac
and
skeletal
and thesetransporter molecules,found predominantlyin muscle,
are identical
to those
expressed
in
adipose
tissue
(12)
The implications,therefore, of our resultsare that amylin and CGRP are able to regulate
the
translocation
of insulin
sensitive
glucose
transporters
in muscle
and
indicate the presenceof specificbinding sitesfor thesepeptideson the muscle 453
Vol.
169,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cell surface. This is in contrast to the later results of Leighton and Cooper which indicate that the insulin antagonism of amylin is directed at glycogen synthesis. both
basal
and
insulin stimulated, and that glucose transpon is unaffected (9).
There is some contention over the effect of amylin on insulin secretion. the increase in the ratio of amylin/insulin mRNA with experimental models of diabetes indicates that the role of amylin in the aetiology of NIDD
is a complex one.
However, the demonstration that the insulin sensitive glucose transporter present in muscle is in some way impaired by amylin and CGRP could explain why elevated glucose levels are perpetuated during elevated pancreatic amylin production and output, thus maintaining an increase in the amylin to insulin ratio and eventually leading to amyloid deposits. Acknowledgments We thank Professors J. Valiance-Owen, P. N. Campbell, P. Mclean and I. McIntyre for initiating this project and The British diabetic Association and Basil Samuel Charitable Trust who have supplied grants to support this work. References 1. Valiance-Owen, J. Pfeiffer Diabetes Mellitus Vol II Edit0rsJ.F. Lehmamrs Munich: 105-122 (1971). 2. Opie, E.L.: J. Exp. Med. 5: 527-540 (1901). 3. Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M.: Proc. Natl. Acad. Sci. 84: 8628-8632 (1987). 4. Ohsawa, H., Kanasuka, A., Yamaguchi, T., Makino, H. and Yoshida, S.: Biochem. Biophys. Res. Commun. 160: 961-967 (1989). 5. Ghatei, MA., Datta, H.K., Zaidi, M., Bretherton-Watt, D., Wimalawansa, S.J., McIntyre, I. and Bloom, S.R.: J. Endocrin. 124: R9-11 (1990). 6. Pettersson, M., Ahren, B., Bottcher, G. and Sundler, F.: Endochrinology 119: 865-869 (1986). 7. Cooper, G.J.S., Leighton, B., Dimitriadis, G.D., ParryBillings, M., Kowalchuk, J.M., Howland, K., Rothbard, J.B., Willis, A.C. and Reid, K.B.M.: Proc. Natl. Acad. Sci. 85: 7763-7766 (1988). 8. Bretherton-Watt, D., Ghatei, M.A., Bloom, S.R., Jamal,H., Ferrier, G.J.M., Girgis, S.I. and Legon, S.: Diabetologia 32:881-883 (1989). 9. Leighton, B. and Cooper, G.J.S.: Nature 335: 632-635 (1988). 10. Sokoloff, L.J.: J. Neurochem. 29: 13-19 (1977). 11. Suzuki, K. and Kono, T.: Proc. Natl. Acad. Sci. 79: 3777-3779 (1980) 12. Charron, M.J., Brosius, F.C., Alper, S.L. and Lodish, H.F.: Proc. Natl. Acad. Sci. 86: 2535-2539 (1989).
454