Vol.
167,
March
No.
16,
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
507-513
THE PUTATIVE HORMONE ISLET AMYLOID POLYPEPTIDE (IAPP) INDUCES IMPAIRED GLUCOSE TOLERANCE IN CATS K.H.
Johnson*,
*Department Medicine,
T.D.
of Veterinary Pathobiology, University of Minnesota, St.
'Department
of
Pathology,
#Department Received
O'Brien*, K. Jor an*, and P. Westermark c'
January
19,
University of
Pathology, Linkoping,
C. Betsholtz',
College of Veterinary Paul, Minnesota 55108
of Uppsala, University Sweden
Uppsala,
Sweden,
Hospital,
1990
Islet amyloid polypeptide (IAPP) has been implicated by in vitro studies as an inhibitor of insulin-stimulated glucose utilization by skeletal muscle cells and also as an inhibitor of insulin-stimulated insulin secretion by beta cells. Increased expression and production of IAPP by beta cells, as has been suggested to occur in cats with impaired glucose tolerance, could thus contribute substantially to the development of the insulin resistance and impaired insulin release which are the hallmarks of Type 2 diabetes mellitus. The effects of IAPP with respect to glucose metabolism in living animals, however, have not been previously reported. In the present in viva study we show that synthetic amidated IAPP induced impaired glucose tolerance in each with dramatic impairment (increases in of the 3 cats studied, glucose to T+ values of 124% and 234%) in 2 of the 3 cats. Impaired insulin responses were also evident in the 2 cats with the most dramatic states of glucose intolerance. These results provide the most direct evidence to-date that IAPP may have an important role 01990Academic Press, Inc. in the development of Type 2 diabetes mellitus. SUMMARY:
A previously undescribed protein identified as islet amyloid polypeptide (IAPP) was recently shown to be the major component of the amyloid deposits present in the pancreatic islet of Type 2 (non-insulin-dependent) diabetic humans and adult diabetic cats, and also in the amyloid deposits from a human insulinoma (l-4). IAPP is a 37 amino acid polypeptide with 43% to 46% amino acid homology with calcitonin gene-related peptide (CGRP)(5,6). IAPP and other animal species immunoreactivity in normal humans, cats, has been shown to be limited to pancreatic beta cells (2,7-lo), and ultrastructural studies in humans and cats have demonstrated that IAPE' is stored with insulin in beta cell granules (7,11,12). Nucleotide sequences of both human cDNA (10,13,14) and genomic DNA (15) clones encoding the IAPP precursor predict a prepro-IAPP 0006-291X/90 $1.50 507
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
2, 1990
BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
that is 89 amino acids in length. Prepro-IAPP has an N-terminal series of hydrophobic amino acids that are characteristic of a for transport through the membrane of the signal sequence The mature 37 amino acid IAPP polypeptide endoplasmic reticulum. (represented by positions 34-70 of prepro-IAPP) is generated by proteolytic cleavage from short N- and C-terminal propeptides, and The structure of IAPP predicted C-terminal amidation (10,13,15). from cDNA sequences is identical to the structure of IAPP derived from islet amyloid deposits. The capacity for conversion of IAPP to amyloid fibrils appears limited to certain species (e.g., humans, cats, and non-human primates) that also develop maturity-onset diabetes mellitus. The fibrillogenic nature of IAPP in these several species may be most importantly related to amino acid residues in the 20-29 region in that synthetic human and cat IAPP 20-29 peptides have an intrinsic capacity of forming amyloid fibrils in vitro (10,16). Also, IAPP molecules of several rodent species (which do not develop islet amyloid) diverge substantially from the human sequence in the 2029 region (17,18). The function of IAPP is not known at this time but its conservation in multiple animal species indicates biological importance. The hormonal nature of this polypeptide [also (19,20)] is supported identified as "amylin" b y some investigators by its co-localization with insulin in pancreatic beta cells, its homology with CGRP, and its reported role as an inhibitor of insulin-stimulated glucose uptake by skeletal muscle cells in vitro (19,20). Glucose-stimulated insulin secretion by rat pancreatic islets in vitro has also been reported to be inhibited by IAPP (21) -
The implications of the latter recent in vitro studies are significant in that increased expression and production of IAPP by beta cells [as has been suggested by recent studies with cats (22)] could contribute substantially to development of the insulin resistance (i.e., insulin insensitivity) and impaired insulin response which are the hallmarks of Type 2 diabetes mellitus. However, it remains to be shown whether the in vitro effects of IAPP can also be demonstrated in living animals. In the present in viva study, blood glucose and insulin responses to synthetic human IAPP (h-IAPP) were evaluated in domestic cats using the high dose intravenous glucose tolerance test. The results of this study indicate that IAPP can induce substantial impaired glucose tolerance and impaired insulin
Vol.
167,
No.
2, 1990
BIOCHEMICAL
responses. This in vivo evidence which supports the clinical development MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
study thus provides new and more direct a potentially important role for IAPP in of Type 2 diabetes mellitus.
AND METHODS
Animals: Three young adult domestic cats (ages estimated to be 2 to 4 years) were acquired from the University of Minnesota Animal Resources Center. The cats were acclimated to the holding facilities for a minimum of one week prior to the initiation of each study. Each cat was anesthetized 20 to 24 hours prior to the experiments and, using previously described procedures (23), an indwelling catheter was placed in one of the jugular veins. wide Svnthesis, Analvsis, and Preoaration for Injection. C-amidated h-IAPP was synthesized using solid phase techniques on a Biosearch 960 peptide synthesizer with MBHA (p-methylbenzhydrylamine) resin and standard t-butoxycarbonyl(BOC) chemistry. The peptide was purified by reverse-phase high performance liquid chromatography. Amino acid composition was determined after acid hydrolysis and sequence analysis was performed using an Applied Biosystems 477A protein sequencer. Synthetic amidated h-IAPP was prepared for intravenous injection (10, 20, or 50 nM/Kg body weight) either as a suspension in 10 ml isotonic saline with 0.1% BSA (Cat 251), or in isotonic saline with 0.1% BSA after dissolving the h-IAPP in 30% acetic acid (Cats 24'7 and 249). Acetic acid-dissolved h-IAPP was adjusted to pH 7.2 before injection. Control injections consisted of the same vehicles used for injection of h-IAPP in the respective cats. Control or h-IAPP injections were given 1 minute (Cats 247 and 249) or 15 minutes (Cat 251) prior to the start of each glucose tolerance test. Glucose Tolerance Tests and Insulin Assavs. Cats with indwelling jugular catheters were fasted for 12 hours prior to administration of the high dose intravenous glucose tolerance test as previously described (23). All glucose tolerance tests were and unsedated cats between 9 and 11 AM. performed on conscious Glucose (50% dextrose) was injected via the jugular catheter at the rate of 1 g anhydrous glucose/Kg body weight over a period of 1 minute. Blood samples taken immediately prior to infection of glucose, and at 5, 15, 30, 45 and 60 minutes after glucose injection, were obtained by jugular catheter. Blood collected in NaF-containing tubes was used for determination of blood glucose concentrations by the orthotoluidine method. Serum samples were stored at -20'~ until insulin assays were performed.
Linear concentrations for glucose
regression analyses of semilogarithmic versus time were used to calculate disappearance (T%) (23).
plots of glucose the one-half time
as previously Serum insulin concentrations were measured described (23) by radioimmunoassay using a commercially available kit (RSL Insulin Kit, Catalog No. 07-160102, ICN Biomedicals, Inc., Carson, CA). 509
Vol.
167,
No.
RESULTS
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION
These in vivo studies in cats [which are known to develop a form of age-related diabetes mellitus similar to Type 2 diabetes mellitus in humans (24,25)] reveal significant effects of IAPP. An impaired glucose tolerance test response following intravenous injection of synthetic amidated h-IAPP is apparent in each of the cats when that response is compared with its own initial pre-IAPP glucose tolerance test response (Table 1). When the T?i values obtained from each cat's first injection with synthetic h-IAPP are compared with each cat's respective pre-IAPP T$ values, decreases in blood glucose clearing ability of approximately 124% (Cat 247), 13% (Cat 249), and 234% (Cat 251) are apparent. Dramatically impaired glucose tolerance test responses are therefore obvious in 2 of the 3 cats. Mean glucose T+ values increased from 33.2 minutes in control tests to 64.6 minutes after injection of h-IAPP. The impaired glucose tolerance test responses associated with injection of h-IAPP are not likely to be significantly attributable
TABLE1 Effect of Synthetic Levels in Cats
Amidated Using the
Blood
h-IAPP on Blood High Dose Intravenous
Glucose
Concentration
Hieh DAY
Fastine’
Dose
Glucose and Serum Glucose Tolerance
(ma/d11
IV
Glucose
15
5
& Serum Tolerance
Test
30
Insulin Test
Insulin Levels’ (minutes)
fulIJ/mll
45
60
Tlf2
CAT 241: PRE-IAPP IAPP IAPP
Control3 (20 (10
POST-IAPP
nM/Kg)4 nM/Kg)4
CONTROL3
88(22) 88(19) 79(20) 96(19)
526(35) 556( 16) 587(20) 655(19)
403(45) 449(25) 492(40) 545(34)
3 12(40) 398(40) 427(42) 483(40)
241(60) 351(42) 371(60) 435(60)
167(50) 333(40) 347(60) 428(42)
34.7 77.9 73.9 92.2
73(IO) 89(18) 85(12) 76(12)
559(45) 511(70) 523(33) 479(50)
441(58) 407(88) 405(74) 391(43)
352(40) 339(54) 344(43) 330(35)
293(40) 279(70) 301(37) 281(30)
231(36) 234(58) 266(33) 243(43)
44.9 50.5 59.9 58.1
80
557(130)
86(37) 96(15) 82(28)
546(30) 603(50) 494( 110)
400(190) 463(49) 442(60) 405( 120)
256(130) 398(88) 372(42) 313(72)
157(180) 344( 130) 342(66) 254( 130)
78(100) 300(130) 301(120) 199(130)
19.9 65.5 60.4 42.0
CAT 249: PRE-IAPP IAPP (20 IAPP (50 POST-IAPP
I 8 IO 11
Control3 nM/K8)4 nM/K8)4 CONTROL3
CAT251;
PRE-IAPP IAPP (50 POST-IAPP POST-IAPP ’
Control3 nM/Kg)’ Control3 CONTROL3
Serum
insulin
levels
2 Fasting
blood
glucose
3 Control
injection
3 1
IO are
in
levels
parentheses.
prior
(IV)
of vehicle
to injection
of h-IAPP
or vehicle.
only.
4 Amidated
h-IAPP
was
injected
(IV)
after
solubilization
5 Amidawd
h-IAPP
was
injected
(IV)
after
sonication
in in
acetic
isotonic
acid saline
510
and
dilution
with
0.1%
with BSA.
isotonic
saline
containing
0.1%
BSA.
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2, 1990
BIOCHEMICAL
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to excitement-induced catecholamine release (causing subsequent inhibition of glucose-stimulated insulin release, hepatic glycogenolysis and gluconeogenesis). Use of jugular catheters, inserted in laboratory acclimated cats the day prior to the initial glucose tolerance test, virtually eliminated any discomfort or excitement during the test procedures. Additionally, a glucose tolerance test performed on each cat prior to the injection of hIAPP served as a pre-IAPP control for each respective cat. Of considerable interest is the apparent sustained effect of injected synthetic h-IAPP in maintaining a state of impaired glucose tolerance. A sustained or progressive state of impaired glucose tolerance was still observed at 24 and 48 hours after injection of h-IAPP in Cats 249 and 247, respectively. In Cat 251, a single injection of h-IAPP (50 nM/Kg body weight) produced a markedly impaired glucose tolerance test response that was still apparent at 4 days after injection of IAPP (T+ = 60.4). In the latter cat at 7 days after IAPP injection, the T$ value (42.0) was still slightly higher than the maximum normal T+ value (T% = 40) for cats. When Cat 251 was re-evaluated 11 weeks later, the glucose T+ value was within normal range prior to injection of h-IAPP. After injection of h-IAPP (50 nM/Kg body weight), a dramatically impaired glucose tolerance test (Tt = 65.5) and a markedly impaired first-phase insulin response were again observed. Comparison of glucose-stimulated insulin responses before and after injections of synthetic h-IAPP revealed markedly impaired first-phase insulin responses in the 2 cats that also had the most dramatic states of glucose intolerance (i.e., Cats 247 and 251) (Table 1). However, an impaired insulin response was not observed following injection of synthetic h-IAPP in CAT 249. The latter cat also had the most modest increase (13%) in glucose T4 value associated with injection of h-IAPP. The reason(s) for the sustained effects of synthetic h-IAPP the physiological in this study are not known. In that concentration of IAPP in serum is not known, the amounts of h-IAPP injected were selected on the basis of amounts of CGRP used for bolus to obtain in w physiological injection in a single responses in rats and mice. It is feasible that the sustained of h-IAPP noted in our study are related to serum affects concentrations that are well above physiological levels. Greater than physiological concentrations of hormone could conceivably down-regulate the glucose sensory apparatus present in IAPPskeletal muscle cells and/or pancreatic beta sensitive cells (e.g., 511
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causing sustained effects even after circulating levels of cells), IAPP have diminished. The actual physiological concentration of the amidated hormone in this study also is not known in that only a part of the synthetic h-IAPP may have achieved the conformation of the natural hormone. Also, cat IAPP is known to differ from h-IAPP at 4 positions (residues 17,18,23, and 29) (18,26). The specific mechanism(s) accounting for the impaired glucose tolerance induced by synthetic h-IAPP in cats cannot be determined on the basis of this preliminary study. However, the fact that an impaired insulin response was not evident in one of the cats suggests that the IAPP-induced glucose intolerance in the cat is not solely attributable to impairment of glucose-stimulated insulin secretion. Further studies are needed to clarify these mechanisms, but the results of this study provide the most direct evidence todate that IAPP exerts physiological changes that may play an important role in the development of Type 2 diabetes mellitus.
Acknowledgments: The authors are grateful to Ms. Kathleen Burg for valuable technical assistance. This study was supported by Grant ROl DK36734 of the National Institute of Diabetes and Digestive and Kidney Disease, the Swedish Medical Research Council (Project No. 5941) the Research fund of King Gustaf V, the Nordic Insulin Fund, and Louis-Hansen's Memorial Fund.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Westermark, P., Wernstedt, C., Wilander, E., Sletteen, K. (1986) Biochem. Biophys. Res. Commun. 140, 827-831. Westermark, P., Wernstedt, C., Wilander, E. Hayden, D.W., O'Brien, T.D., Johnson, K.H. (1987) Proc. Natl. Acad. Sci. USA 84, 3881-3885. Westermark, P., Wernstedt, C. O'Brien. T.D., Hayden. D.W., Johnson, K.H. (1987) Am. J. Pathol. 127, 414-417. Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B., Reid, K.B.M. (1987) Proc. Natl. Acad. Sci. USA 84, 86288632. Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M. (1982) Nature 298, 240-244. Steenbergh; P.H., Hoppener, J.W.M., Zandberg, J., Lips, C.J.M., Jansz, H.S. (1985) FEBS Lett. 183, 403-407. Johnson, K.H., O'Brien, T.D., Hayden, D.W., Jordan, K., Ghobrial, H.K.G., Mahoney, W.C., Westermark, P. (1988) Am. J. Pathol. 130, l-8. Westermark, P., Wilander, E., Westermark, G.T., Johnson, K.H. (1987) Diabetologia 30, 887-892. Cooper, G.J.S., Willis, A.C., Reid, K.B.M., Clark, A., Baker, C.A., Turner, R.C., Lewis, C.E., Morris, J.F., Howland, K., Rothbard, J.B. (1987) Lancet ii, 966. Betsholtz, C., Svensson, V., Rorsman, F., Engstrom, U., Westermark, G.T., Wilander, E., Johnson, K.H., Westermark, P. (1989) Exp. Cell Res. 183, 484-493. 512
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Lukinius, A., Wilander, E., Westermark, G-T., Engtrom, U., Westermark, P. (1989) Diabetologia 32, 240-244. Clark, A., Edwards, C.A., Ostle, L.R., Sutton, R., Rothbard, J.B., Morris, J.F., Turner, R.C. (1989) Cell Tissue Res. 257, 179-185. Sanke, T., Bell, G.I., Sample, C., Rubenstein, A-H., Steiner, D.F. (1988) J. Biol. Chem. 263, 17243-17246. Mosselman, S., Hdppener, J.W.M., Lips, C.J.M., Jansz, H.S. (1989) FEBS Lett. 247, 154-158. Mosselman, S., Hoppener, J.W.M., Zandberg, J., van Mansfield, A.D.M., Geurts van Kessel, A.H.M., Lips. C.J.M., Jansz, H.S. (1988) FEBS Lett. 239, 227-232. Glenner, G.G. (1988) Biochem. Biophys. Res. Commun. 155, 608614. Betsholtz, C., Christmansson, L., Engstrom, U., Rorsman, F., Svensson, V., Johnson, K.H., Westermark, P. (1989) FEBS Lett. 251, 261-264. Nishi, M., Chan, S.J., Nagamatsu, S., Bell, G.I., Steiner, D.F. (1989) Proc. Natl. Acad. Sci. USA 86, 5738-5742. Leighton, B., Cooper, G.J.S. (1988) Nature 335, 632-635. Cooper, G.J.S., Leighton, B., Dimitriadis, G.D., ParryBillings, M., Kowalchuk, J-M., Howland, K., Rothbard, J-B., Willis, A-C., Reid, K.B.M. (1988) Proc. Natl. Acad. Sci. USA 85, 7763-7766. Ohsawa, H., Kanatsuka, A., Yamaguchi, T., Makino, H. Yoshida, S. (1989) Biochem. Biophys. Res. Commun. 160, 961-967. Johnson, K-H., O'Brien, T-D., Jordan, K., Westermark, P. (1989) Am. J. Pathal. 135, 245-250. O'Brien, T-D., Hayden, D-W., Johnson, K-H., Stevens, J-B. (1985) Vet. Pathol. 22, 250-261. Johnson, K.H., Stevens, J.B. (1973) Diabetes 22, 81-90. Johnson, K-H., Hayden, D-W., O'Brien, T-D., Westermark, P. (1986) Am. J. Pathol. 125, 416-419. Betsholtz, C., Christmansson, L., Engstrom, U., Rorsman, F., Jordan, K., Murtaugh, M., Johnson, K-H., Westermark, P. (1989) Diabetes. In press.
513
167,
March
No.
16,
2, 1990
BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
507-513
THE PUTATIVE HORMONE ISLET AMYLOID POLYPEPTIDE (IAPP) INDUCES IMPAIRED GLUCOSE TOLERANCE IN CATS K.H.
Johnson*,
*Department Medicine,
T.D.
of Veterinary Pathobiology, University of Minnesota, St.
'Department
of
Pathology,
#Department Received
O'Brien*, K. Jor an*, and P. Westermark c'
January
19,
University of
Pathology, Linkoping,
C. Betsholtz',
College of Veterinary Paul, Minnesota 55108
of Uppsala, University Sweden
Uppsala,
Sweden,
Hospital,
1990
Islet amyloid polypeptide (IAPP) has been implicated by in vitro studies as an inhibitor of insulin-stimulated glucose utilization by skeletal muscle cells and also as an inhibitor of insulin-stimulated insulin secretion by beta cells. Increased expression and production of IAPP by beta cells, as has been suggested to occur in cats with impaired glucose tolerance, could thus contribute substantially to the development of the insulin resistance and impaired insulin release which are the hallmarks of Type 2 diabetes mellitus. The effects of IAPP with respect to glucose metabolism in living animals, however, have not been previously reported. In the present in viva study we show that synthetic amidated IAPP induced impaired glucose tolerance in each with dramatic impairment (increases in of the 3 cats studied, glucose to T+ values of 124% and 234%) in 2 of the 3 cats. Impaired insulin responses were also evident in the 2 cats with the most dramatic states of glucose intolerance. These results provide the most direct evidence to-date that IAPP may have an important role 01990Academic Press, Inc. in the development of Type 2 diabetes mellitus. SUMMARY:
A previously undescribed protein identified as islet amyloid polypeptide (IAPP) was recently shown to be the major component of the amyloid deposits present in the pancreatic islet of Type 2 (non-insulin-dependent) diabetic humans and adult diabetic cats, and also in the amyloid deposits from a human insulinoma (l-4). IAPP is a 37 amino acid polypeptide with 43% to 46% amino acid homology with calcitonin gene-related peptide (CGRP)(5,6). IAPP and other animal species immunoreactivity in normal humans, cats, has been shown to be limited to pancreatic beta cells (2,7-lo), and ultrastructural studies in humans and cats have demonstrated that IAPE' is stored with insulin in beta cell granules (7,11,12). Nucleotide sequences of both human cDNA (10,13,14) and genomic DNA (15) clones encoding the IAPP precursor predict a prepro-IAPP 0006-291X/90 $1.50 507
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
that is 89 amino acids in length. Prepro-IAPP has an N-terminal series of hydrophobic amino acids that are characteristic of a for transport through the membrane of the signal sequence The mature 37 amino acid IAPP polypeptide endoplasmic reticulum. (represented by positions 34-70 of prepro-IAPP) is generated by proteolytic cleavage from short N- and C-terminal propeptides, and The structure of IAPP predicted C-terminal amidation (10,13,15). from cDNA sequences is identical to the structure of IAPP derived from islet amyloid deposits. The capacity for conversion of IAPP to amyloid fibrils appears limited to certain species (e.g., humans, cats, and non-human primates) that also develop maturity-onset diabetes mellitus. The fibrillogenic nature of IAPP in these several species may be most importantly related to amino acid residues in the 20-29 region in that synthetic human and cat IAPP 20-29 peptides have an intrinsic capacity of forming amyloid fibrils in vitro (10,16). Also, IAPP molecules of several rodent species (which do not develop islet amyloid) diverge substantially from the human sequence in the 2029 region (17,18). The function of IAPP is not known at this time but its conservation in multiple animal species indicates biological importance. The hormonal nature of this polypeptide [also (19,20)] is supported identified as "amylin" b y some investigators by its co-localization with insulin in pancreatic beta cells, its homology with CGRP, and its reported role as an inhibitor of insulin-stimulated glucose uptake by skeletal muscle cells in vitro (19,20). Glucose-stimulated insulin secretion by rat pancreatic islets in vitro has also been reported to be inhibited by IAPP (21) -
The implications of the latter recent in vitro studies are significant in that increased expression and production of IAPP by beta cells [as has been suggested by recent studies with cats (22)] could contribute substantially to development of the insulin resistance (i.e., insulin insensitivity) and impaired insulin response which are the hallmarks of Type 2 diabetes mellitus. However, it remains to be shown whether the in vitro effects of IAPP can also be demonstrated in living animals. In the present in viva study, blood glucose and insulin responses to synthetic human IAPP (h-IAPP) were evaluated in domestic cats using the high dose intravenous glucose tolerance test. The results of this study indicate that IAPP can induce substantial impaired glucose tolerance and impaired insulin
Vol.
167,
No.
2, 1990
BIOCHEMICAL
responses. This in vivo evidence which supports the clinical development MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
study thus provides new and more direct a potentially important role for IAPP in of Type 2 diabetes mellitus.
AND METHODS
Animals: Three young adult domestic cats (ages estimated to be 2 to 4 years) were acquired from the University of Minnesota Animal Resources Center. The cats were acclimated to the holding facilities for a minimum of one week prior to the initiation of each study. Each cat was anesthetized 20 to 24 hours prior to the experiments and, using previously described procedures (23), an indwelling catheter was placed in one of the jugular veins. wide Svnthesis, Analvsis, and Preoaration for Injection. C-amidated h-IAPP was synthesized using solid phase techniques on a Biosearch 960 peptide synthesizer with MBHA (p-methylbenzhydrylamine) resin and standard t-butoxycarbonyl(BOC) chemistry. The peptide was purified by reverse-phase high performance liquid chromatography. Amino acid composition was determined after acid hydrolysis and sequence analysis was performed using an Applied Biosystems 477A protein sequencer. Synthetic amidated h-IAPP was prepared for intravenous injection (10, 20, or 50 nM/Kg body weight) either as a suspension in 10 ml isotonic saline with 0.1% BSA (Cat 251), or in isotonic saline with 0.1% BSA after dissolving the h-IAPP in 30% acetic acid (Cats 24'7 and 249). Acetic acid-dissolved h-IAPP was adjusted to pH 7.2 before injection. Control injections consisted of the same vehicles used for injection of h-IAPP in the respective cats. Control or h-IAPP injections were given 1 minute (Cats 247 and 249) or 15 minutes (Cat 251) prior to the start of each glucose tolerance test. Glucose Tolerance Tests and Insulin Assavs. Cats with indwelling jugular catheters were fasted for 12 hours prior to administration of the high dose intravenous glucose tolerance test as previously described (23). All glucose tolerance tests were and unsedated cats between 9 and 11 AM. performed on conscious Glucose (50% dextrose) was injected via the jugular catheter at the rate of 1 g anhydrous glucose/Kg body weight over a period of 1 minute. Blood samples taken immediately prior to infection of glucose, and at 5, 15, 30, 45 and 60 minutes after glucose injection, were obtained by jugular catheter. Blood collected in NaF-containing tubes was used for determination of blood glucose concentrations by the orthotoluidine method. Serum samples were stored at -20'~ until insulin assays were performed.
Linear concentrations for glucose
regression analyses of semilogarithmic versus time were used to calculate disappearance (T%) (23).
plots of glucose the one-half time
as previously Serum insulin concentrations were measured described (23) by radioimmunoassay using a commercially available kit (RSL Insulin Kit, Catalog No. 07-160102, ICN Biomedicals, Inc., Carson, CA). 509
Vol.
167,
No.
RESULTS
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION
These in vivo studies in cats [which are known to develop a form of age-related diabetes mellitus similar to Type 2 diabetes mellitus in humans (24,25)] reveal significant effects of IAPP. An impaired glucose tolerance test response following intravenous injection of synthetic amidated h-IAPP is apparent in each of the cats when that response is compared with its own initial pre-IAPP glucose tolerance test response (Table 1). When the T?i values obtained from each cat's first injection with synthetic h-IAPP are compared with each cat's respective pre-IAPP T$ values, decreases in blood glucose clearing ability of approximately 124% (Cat 247), 13% (Cat 249), and 234% (Cat 251) are apparent. Dramatically impaired glucose tolerance test responses are therefore obvious in 2 of the 3 cats. Mean glucose T+ values increased from 33.2 minutes in control tests to 64.6 minutes after injection of h-IAPP. The impaired glucose tolerance test responses associated with injection of h-IAPP are not likely to be significantly attributable
TABLE1 Effect of Synthetic Levels in Cats
Amidated Using the
Blood
h-IAPP on Blood High Dose Intravenous
Glucose
Concentration
Hieh DAY
Fastine’
Dose
Glucose and Serum Glucose Tolerance
(ma/d11
IV
Glucose
15
5
& Serum Tolerance
Test
30
Insulin Test
Insulin Levels’ (minutes)
fulIJ/mll
45
60
Tlf2
CAT 241: PRE-IAPP IAPP IAPP
Control3 (20 (10
POST-IAPP
nM/Kg)4 nM/Kg)4
CONTROL3
88(22) 88(19) 79(20) 96(19)
526(35) 556( 16) 587(20) 655(19)
403(45) 449(25) 492(40) 545(34)
3 12(40) 398(40) 427(42) 483(40)
241(60) 351(42) 371(60) 435(60)
167(50) 333(40) 347(60) 428(42)
34.7 77.9 73.9 92.2
73(IO) 89(18) 85(12) 76(12)
559(45) 511(70) 523(33) 479(50)
441(58) 407(88) 405(74) 391(43)
352(40) 339(54) 344(43) 330(35)
293(40) 279(70) 301(37) 281(30)
231(36) 234(58) 266(33) 243(43)
44.9 50.5 59.9 58.1
80
557(130)
86(37) 96(15) 82(28)
546(30) 603(50) 494( 110)
400(190) 463(49) 442(60) 405( 120)
256(130) 398(88) 372(42) 313(72)
157(180) 344( 130) 342(66) 254( 130)
78(100) 300(130) 301(120) 199(130)
19.9 65.5 60.4 42.0
CAT 249: PRE-IAPP IAPP (20 IAPP (50 POST-IAPP
I 8 IO 11
Control3 nM/K8)4 nM/K8)4 CONTROL3
CAT251;
PRE-IAPP IAPP (50 POST-IAPP POST-IAPP ’
Control3 nM/Kg)’ Control3 CONTROL3
Serum
insulin
levels
2 Fasting
blood
glucose
3 Control
injection
3 1
IO are
in
levels
parentheses.
prior
(IV)
of vehicle
to injection
of h-IAPP
or vehicle.
only.
4 Amidated
h-IAPP
was
injected
(IV)
after
solubilization
5 Amidawd
h-IAPP
was
injected
(IV)
after
sonication
in in
acetic
isotonic
acid saline
510
and
dilution
with
0.1%
with BSA.
isotonic
saline
containing
0.1%
BSA.
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to excitement-induced catecholamine release (causing subsequent inhibition of glucose-stimulated insulin release, hepatic glycogenolysis and gluconeogenesis). Use of jugular catheters, inserted in laboratory acclimated cats the day prior to the initial glucose tolerance test, virtually eliminated any discomfort or excitement during the test procedures. Additionally, a glucose tolerance test performed on each cat prior to the injection of hIAPP served as a pre-IAPP control for each respective cat. Of considerable interest is the apparent sustained effect of injected synthetic h-IAPP in maintaining a state of impaired glucose tolerance. A sustained or progressive state of impaired glucose tolerance was still observed at 24 and 48 hours after injection of h-IAPP in Cats 249 and 247, respectively. In Cat 251, a single injection of h-IAPP (50 nM/Kg body weight) produced a markedly impaired glucose tolerance test response that was still apparent at 4 days after injection of IAPP (T+ = 60.4). In the latter cat at 7 days after IAPP injection, the T$ value (42.0) was still slightly higher than the maximum normal T+ value (T% = 40) for cats. When Cat 251 was re-evaluated 11 weeks later, the glucose T+ value was within normal range prior to injection of h-IAPP. After injection of h-IAPP (50 nM/Kg body weight), a dramatically impaired glucose tolerance test (Tt = 65.5) and a markedly impaired first-phase insulin response were again observed. Comparison of glucose-stimulated insulin responses before and after injections of synthetic h-IAPP revealed markedly impaired first-phase insulin responses in the 2 cats that also had the most dramatic states of glucose intolerance (i.e., Cats 247 and 251) (Table 1). However, an impaired insulin response was not observed following injection of synthetic h-IAPP in CAT 249. The latter cat also had the most modest increase (13%) in glucose T4 value associated with injection of h-IAPP. The reason(s) for the sustained effects of synthetic h-IAPP the physiological in this study are not known. In that concentration of IAPP in serum is not known, the amounts of h-IAPP injected were selected on the basis of amounts of CGRP used for bolus to obtain in w physiological injection in a single responses in rats and mice. It is feasible that the sustained of h-IAPP noted in our study are related to serum affects concentrations that are well above physiological levels. Greater than physiological concentrations of hormone could conceivably down-regulate the glucose sensory apparatus present in IAPPskeletal muscle cells and/or pancreatic beta sensitive cells (e.g., 511
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causing sustained effects even after circulating levels of cells), IAPP have diminished. The actual physiological concentration of the amidated hormone in this study also is not known in that only a part of the synthetic h-IAPP may have achieved the conformation of the natural hormone. Also, cat IAPP is known to differ from h-IAPP at 4 positions (residues 17,18,23, and 29) (18,26). The specific mechanism(s) accounting for the impaired glucose tolerance induced by synthetic h-IAPP in cats cannot be determined on the basis of this preliminary study. However, the fact that an impaired insulin response was not evident in one of the cats suggests that the IAPP-induced glucose intolerance in the cat is not solely attributable to impairment of glucose-stimulated insulin secretion. Further studies are needed to clarify these mechanisms, but the results of this study provide the most direct evidence todate that IAPP exerts physiological changes that may play an important role in the development of Type 2 diabetes mellitus.
Acknowledgments: The authors are grateful to Ms. Kathleen Burg for valuable technical assistance. This study was supported by Grant ROl DK36734 of the National Institute of Diabetes and Digestive and Kidney Disease, the Swedish Medical Research Council (Project No. 5941) the Research fund of King Gustaf V, the Nordic Insulin Fund, and Louis-Hansen's Memorial Fund.
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