Diabetes Research and Clinical Practice, 15 (1992) 0
1992 Elsevier
Science Publishers
57-62
57
B.V. All rights reserved 0168-8227/92/$05.00
DIABET 00586
Islet amyloid polypeptide (IAPP/amylin) causes insulin resistance in perfused rat hindlimb muscle Hiromichi
Tabata, Junji Hirayama, Ryoichi Sowa, Hiroto Furuta, Toshiro Negoro, Tokio Sanke and Kishio Nanjo
The First Department of Medicine,
Wakyvama
University of Medical Science,
Wakavama, Japan
Summary It has been reported that islet amyloid polypeptide (IAPP) has insulin antagonistic effects in vivo and in vitro. To determine whether IAPP affects glucose metabolism in skeletal muscle, we performed in situ rat hindlimb perfusion which is a near-physiological system. Forty min after the beginning of insulin infusion at 1000 pU/ml, the synthesized rat amide form of IAPP was infused at 1 nM or 10 nM for 50 min and glucose concentration in the effluent was measured to calculate glucose uptake (GU). The GU did not change during the 1 nM IAPP infusion, but significantly decreased during 10 nM IAPP infusion (554 k 24 to 445 k 29 nmol/g/min, P < 0.01). Rat calcitonin gene-related peptide (CGRP), which has sequence homology with IAPP and has been reported to inhibit insulin action, was also administered. Similar to the effect of IAPP, the GU did not change during 1 nM CGRP infusion but significantly decreased during 10 nM CGRP infusion (507 k 7 to 323 & 15 nmol/g/min, P < 0.01). In the experiments without insulin infusion, the GU was not changed even by 10 nM IAPP infusion. Therefore, IAPP directly reduced only the insulin-mediated GU in the skeletal muscle, and this effect of IAPP occurred at the same dose as that of CGRP. These data suggest that both IAPP and CGRP may cause insulin resistance in skeletal muscle not through a CGRP receptor but a yet unknown receptor, which has similar binding affinity for both IAPP and CGRP. Key words: Islet amyloid polypeptide (IAPP); Amylin; Calcitonin gene-related resistance; Hindlimb perfusion
Introduction Islet amyloid polypeptide (IAPP) is a major protein component of islet amyloid deposits [ 1,2] which has been found in patients with nonCorrespondence to: H. Tabata, The First Dept. of Medicine, Wakayama University of Medical Science, 27-Nanaban-cho, Wakayama 640. Japan.
peptide (CGRP);
Insulin
insulin-dependent diabetes mellitus (NIDDM). Sanke et al. have shown that human IAPP is derived from an 89-amino acid precursor by proteolytic processing in islet P-cell [3] and co-secreted with insulin in response to glucose and other secretagogues [4-61. Therefore, IAPP may have some important role(s) in the pathogenesis and/or development of NIDDM. Several investigators have reported that IAPP inhibits insulin
58
effects invivo [7-131 and invitro [8,9,14,15], particularly in skeletal muscle. Since calcitonin gene-related peptide (CGRP), which has a sequence structural homology with IAPP, has been also reported to cause insulin resistance [ 12-151, the relationship of the effects between IAPP and CGRP is very interesting. In this study, the effects of IAPP and CGRP on glucose metabolism in skeletal muscle were studied using an in situ rat hindlimb perfusion system with which glucose utilization can be observed more directly and near-physiologically than in an in vitro system.
buffer
Fig. 1. Scheme of the in situ rat hindlimb perfusion system. Perfusion was conducted in a temperature-controlled chamber at 37°C. Perfusate was sufficiently oxygenated through 3 m plastic tube.
Materials and Methods Right hindlimb perfusion Male Wistar rats (180-250 g body weight) were fed a commercial diet with water ad libitum. After an overnight fast, rats were anesthetized intrasodium with pentobarbital peritoneally (5 mg/lOO g body weight). Surgical treatment was partially modified as in the method described by Ruderman et al. [ 161. The left common iliac artery and vein were tightly ligated and only the right hindlimb muscle was used for the perfusion. The perfusate consisted of a Krebs-Ringer bicarbonate buffer containing 7 mM glucose, 0.5% bovine serum albumin and 4.5% Dextran T-70 (Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The perfusate was sufficiently gassed with 95 y0 0, and 5 y0 CO, through a 3 m plastic tube starting 60 min before the beginning of the perfusion. Perfusion was conducted in a temperature-controlled chamber at 37°C. The perfusate was infused at the rate of 10 ml/min/leg through the abdominal aorta and was collected through a cannula inserted in the inferior vena cava. The perfusion system is schematically shown in Fig. 1. Infusion of insulin, IAPP and CGRP A synthesized amide form of rat IAPP(rIAPP) and rat CGRP(rCGRP) was obtained from the Protein Research Foundation Peptide Institute Inc., Osaka, Japan. After 40 min of pre-perfusion,
semi-synthesized human insulin (Novo Industry, Copenhagen, Denmark) in a concentration of 1000 pU/ml was infused for 140 min. Forty min after the beginning of the insulin infusion, 1 nM or 10 nM rIAPP or rCGRP was infused for 50 min. To determine whether IAPP affects basal (noninsulin-mediated) glucose uptake, 10 nM rIAPP was infused without insulin infusion. Perfusion was performed 50 min after the discontinuation of rIAPP or rCGRP infusion. Calculation of glucose uptake (GU) Glucose concentrations in the effluent were measured every 10 min with a glucose analyzer (Yellow Springs Instruments, Yellow Springs, U.S.A.). The weight of the right hindlimb muscle was calculated as 5 y0 of body weight according to the data of measurements in another experiment. The glucose uptake (GU) was calculated as follows: GU (nmol/g/min) = (A glucose x perfusion rate)/(weight of right hindlimb muscle). Statistical analysis For statistical comparison with GU, the average GU for the last 20 min of every interval (before, during and after rIAPP or rCGRP infusion) was calculated. All values were expressed as mean 2 SE. Statistical analysis was done with
59
paired Student’s r-test, and a P value less than 0.05 was considered significant.
TABLE
1
Comparison of glucose uptake rIAPP or rCGRP infusion
The perfusate was sufficiently oxygenated at approximately 400-500 mmHg during 180 min of the perfusion. In the control experiments, GU increased gradually during pre-perfusion and reached a plateau (about 250 nmol/g/min) at around 30 or 40 min after the beginning of the pre-perfusion, and the constant level continued without insulin infusion throughout the experiment (for at least 180 min). When insulin was infused after the 40 min pre-perfusion, GU increased again and reached a plateau (about 500-600 nmol/g/min) 30 min after the beginning of insulin infusion, and the constant level continued without rIAPP or rCGRP infusion for at least 100 min. Changes in the GU in the experiments with rIAPP infusion are shown in Fig. 2. No significant changes in GU were seen during the 1 nM rIAPP infusion. However, the GU began to fall 20 min after the beginning of the 10 nM rIAPP infusion and a significant decline of the GU was observed compared with that before the rIAPP infusion (Fig. 2). The decreased GU was gra-
:f
5
800
during
and after
12 Glucose uptake (nmol/g/min)
Results
c
before,
1 rat IAPP 1 insulin 1OOO_fflJ/ml
r
30
60
90
120
150
180 min
Fig. 2. Changes in glucose uptake during rat hindlimb perfusion. Forty min after the beginning of the insulin infusion at 1000 pU/ml, 1 nM rIAPP (0) or 10 nM rIAPP (0) was administered for 50 min. Values are means + SE.
Before
During
After
Insulin ( - ) rIAPP 10nM
4
264+38
264+38
211* 59
Insulin (1000 pU/ml) rIAPP 1 nM 10 nM rCGRP 1 nM 10 nM
4 4 4 4
524+53 554+24 538+59 507k I
511+44 445*29* 549567 323?15*
552 + 31 616+22 550+ 51 428+26
Before, During, After: Averages of GUS in every last 20 min before, during and after rIAPP or rCGRP infusion. Values are means + SE. *P < 0.01 vs before.
dually restored after discontinuation of the 10 nM rIAPP infusion. The averages of the GUS for the last 20 min of every period (before, during and after rIAPP or rCGRP infusion) are summarized in Table 1. Although 1 nM rIAPP did not affect GU, the GU during 10 nM rIAPP infusion was significantly reduced compared with those before and after the rIAPP infusion (Fig. 3 and Table 1). However, the GU was not affected by the 10 nM rIAPP infusion without insulin infusion (Fig. 4 and Table 1). Similar to the experiment with IAPP infusion, the GU during 10 nM rCGRP infusion significantly decreased compared with that before
Before
During
After
Before
During
After
Fig. 3. Effect of rat IAPP on glucose uptake during hindlimb perfusion. Before, During, After: averages of GUS in every last 20 min. before, during and after rIAPP infusion, respectively. Values are means + SE.
60
-T-
I
Before
During
After
Fig. 4. Effect of rat IAPP (10 nM) on non-insulin mediated glucose uptake. Before, During, After: averages of GUS in every last 20 min, before, during and after rIAPP infusion, respectively. Values are means + SE.
1nHCCRP
1OnMCGRP
Fig. 5. Effect of rat CGRP on glucose uptake during hindlimb perfusion. Before, During, After: averages of GUS in every last 20 min, before, during and after rCGRP infusion, respectively. Values are means f SE.
and after the rCGRP infusion although 1 nM rCGRP did not affect the GU (Fig. 5 and Table 1).
Discussion Several investigators have reported that IAPP inhibits insulin effects in vivo [7-131 and in vitro [ 8,9,14,15]. We have also recognized similar effects of human IAPP during hyperinsulinemic euglycemic glucose clamp study in dogs [ 111. We have considered that IAPP may cause peripheral insulin resistance in vivo from other evidence that IAPP did not stimulate hepatic glucose output in perfused rat liver [ 171, although there were some reports that IAPP inhibits insulin action in the
liver [ 9,12,18]. In the present study, we examined the direct effects of IAPP on skeletal muscle using an in situ rat hindlimb perfusion system, with which glucose metabolism in skeletal muscle can be studied directly and near-physiologically. As we expected, rat IAPP (10 nM) inhibited GU in this rat hindlimb perfusion system. Consistent with other reports in vitro [ 7,151, this inhibitory effect of IAPP on glucose uptake should be an insulin-mediated action because the same dose (10 nM) of IAPP did not affect the GU without insulin. On the other hand, it has been reported that CGRP also inhibits insulin action [ 12-151. Because of sequence similarity between IAPP and CGRP, it is interesting to study the relationship between their similar effects. Several investigators [ 19-211 have suggested that IAPP may act through CGRP receptors from a specific binding study using ‘251-labeled CGRP. However, the same dose of CGRP as that of IAPP was required to exhibit the inhibitory effect on the GU in the present study. This finding indicates that both IAPP and CGRP may act on the glucose metabolism in skeletal muscle through a yet unknown receptor which has a similar binding affinity for both IAPP and CGRP. Young et al. [9] reported that the inhibitory effects of IAPP on insulin-stimulated glucose metabolism in skeletal muscle are due to inhibition of glucose phosphorylation. The precise mechanism of the insulin antagonistic effect of IAPP and/or CGRP in skeletal muscle is still unclear, but IAPP may alfect the glucose metabolism at a post-receptor site of insulin via an unknown mechanism because the present study was performed at maximal insulin concentration (1000 pU/ml). Recently it has been reported that the peripheral plasma concentrations of IAPP in nondiabetic subjects and patients with NIDDM were 1 to 10 pM [6]. Although 10 nM IAPP inhibited the GU, IAPP in the concentration of 1 nM did not affect the GU. Taking account of the peripheral concentration of the IAPP, this finding suggests that the insulin antagonistic effects of the IAPP observed in skeletal muscle should not be
61
due to a physiological but to a pharmacological effect. This concept also supports our speculation that a considerably high dose (10 nM) of IAPP acts via an unknown receptor different from IAPP or CGRP receptors to exhibit its action.
Acknowledgements
We are indebted to Dr. K. Nakajima, Protein Research Foundation Peptide Institute Inc., Osaka, Japan for supplying the synthesized amide form of rat IAPP.
References Westermark, P., Wernstedt, C., Wilander, E., Hayden, D.W., O’Brien, T.D. and Johnson, K.H. (1987) Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. U.S.A. 84, 3881-3885. Cooper, G.J.S., Willis, AC., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. U.S.A. 84, 8628-8632. Sanke, T., Bell, G.I., Sample, C., Rubenstein, A.H. and Steiner, D.F. (1988) An islet amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing. J. Biol. Chem. 263, 17243-17246. Ogawa, A., Harris, V., McCorkle, S.K., Unger, R.H. and Luskey, R.H. (1990) Amylin secretion from the rat pancreas and its selective loss after streptozotocin treatment. J. Clin. Invest. 85, 973-976. Mitsukawa, T., Takemura, J., Asai, J. et al. (1990) Islet amyloid polypeptide response to glucose, insulin and somatostatin analogue administration. Diabetes 39, 639-642. Sanke, T., Hanabusa, T., Nakano, Y. et al. (1991) Plasma islet amyloid polypeptide (amylin) levels and their responses to oral glucose in Type 2 (non-insulin dependent) diabetic patients. Diabetologia 34, 129-132. Cooper, G.J.S., Leighton, B., Dimitriadis, G.D. et al. (1988) Amylin found in amyloid deposits in human type 2 diabetes melhtus may be a hormone that regulates glycogen metabolism in skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 85, 7763-7766. Leighton, B. and Foot, E. (1990) The effects of amylin on
carbohydrate metabolism in skeletal muscle in vitro and in vivo. Biochem. J. 269, 19-23. 9 Young, D.A., Deems, R.O., Deacon, R.W., McIntosh, R.H. and Foley, J.E. (1990) Effects of amylin on glucose metabolism and glycogenolysis in vivo and in vitro. Am. J. Phvsiol. 259, E457-E461. 10 Johnson, K.H., O’Brien, T.D., Jordan, K., Betsholtz, C. and Westermark, P. (1990) The putative hormone islet amyloid polypeptide (IAPP) induces impaired glucose tolerance in cats. Biochem. Biophys. Res. Commun. 167, 507-513. 11 Sowa, R., Sanke, T., Hirayama, J. et al. (1990) Islet amyloid polypeptide amide causes peripheral insulin resistance in vivo in dogs. Diabetologia 33. 118-120. 12 Molina, J.M., Cooper, G.J.S., Leighton, B. and Olefsky, J.M. (1990) Induction of insulin resistance in vivo by amyhn and calcitonin gene-related peptide. Diabetes 39, 260-265. 13 Yamaguchi, A., Chiba, T., Morishita, T. et al. (1990) Calcitonin gene-related peptide and induction of hyperglycemia in conscious rats in vivo. Diabetes 39, 168-174. 14 Leighton, B. and Cooper, G.J.S. (1988) Pancreatic amylin and caicitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335, 632-635. 15 Hothersall, J.S., Muirhead, R.P. and Wimalawansa, S. (1990) The effect of amylin and calcitonin gene-related peptide on insulin-stimulated glucose transport in the diaphragm. Biochem. Biophys. Res. Commun. 169, 45 l-454. 16 Ruderman, N.B., Houghton, C.R.S. and Hems, R. (1971) Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism. Biochem. J. 124, 639-65 1. 17 Nishimura, S., Sanke, T., Machida, K. et al. (1990) Effect of islet amyloid polypeptide on perfused rat liver - dissociation of cyclic AMP production and glucose output. J. Jpn. Diabetes Sot. 33, 883-887. 18 Fronti, S., Choi, S.B., Banduch, D. and Rosseti, L. (1991) In vivo insulin resistance induced by amylin primarily through inhibition of insulin-stimulated glycogen synthesis in skeletal muscle. Diabetes 40, 568-573. 19 Morishita, T., Yamaguchi, A., Fujita. T. and Chiba, T. (1990) Activation of adenylate cyclase by islet amyloid polypeptide with COOH-terminal amide via calcitonin gene-related peptide receptors on rat liver plasma membranes. Diabetes 39, 875-877. 20 Galeazza, M.T., O’Brien, T.D., Johnson, K.H. and Seybold, V.S. (1991) Islet amyloid polypeptide (IAPP) competes for two binding sites of CGRP. Peptides 12, 585-591. 21 Zhu, G., Dudly, D.T. and Saltiel. A.R. (1991) Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors. Biochem. Biophys. Res. Commun. 177, 771-776.
1992 Elsevier
Science Publishers
57-62
57
B.V. All rights reserved 0168-8227/92/$05.00
DIABET 00586
Islet amyloid polypeptide (IAPP/amylin) causes insulin resistance in perfused rat hindlimb muscle Hiromichi
Tabata, Junji Hirayama, Ryoichi Sowa, Hiroto Furuta, Toshiro Negoro, Tokio Sanke and Kishio Nanjo
The First Department of Medicine,
Wakyvama
University of Medical Science,
Wakavama, Japan
Summary It has been reported that islet amyloid polypeptide (IAPP) has insulin antagonistic effects in vivo and in vitro. To determine whether IAPP affects glucose metabolism in skeletal muscle, we performed in situ rat hindlimb perfusion which is a near-physiological system. Forty min after the beginning of insulin infusion at 1000 pU/ml, the synthesized rat amide form of IAPP was infused at 1 nM or 10 nM for 50 min and glucose concentration in the effluent was measured to calculate glucose uptake (GU). The GU did not change during the 1 nM IAPP infusion, but significantly decreased during 10 nM IAPP infusion (554 k 24 to 445 k 29 nmol/g/min, P < 0.01). Rat calcitonin gene-related peptide (CGRP), which has sequence homology with IAPP and has been reported to inhibit insulin action, was also administered. Similar to the effect of IAPP, the GU did not change during 1 nM CGRP infusion but significantly decreased during 10 nM CGRP infusion (507 k 7 to 323 & 15 nmol/g/min, P < 0.01). In the experiments without insulin infusion, the GU was not changed even by 10 nM IAPP infusion. Therefore, IAPP directly reduced only the insulin-mediated GU in the skeletal muscle, and this effect of IAPP occurred at the same dose as that of CGRP. These data suggest that both IAPP and CGRP may cause insulin resistance in skeletal muscle not through a CGRP receptor but a yet unknown receptor, which has similar binding affinity for both IAPP and CGRP. Key words: Islet amyloid polypeptide (IAPP); Amylin; Calcitonin gene-related resistance; Hindlimb perfusion
Introduction Islet amyloid polypeptide (IAPP) is a major protein component of islet amyloid deposits [ 1,2] which has been found in patients with nonCorrespondence to: H. Tabata, The First Dept. of Medicine, Wakayama University of Medical Science, 27-Nanaban-cho, Wakayama 640. Japan.
peptide (CGRP);
Insulin
insulin-dependent diabetes mellitus (NIDDM). Sanke et al. have shown that human IAPP is derived from an 89-amino acid precursor by proteolytic processing in islet P-cell [3] and co-secreted with insulin in response to glucose and other secretagogues [4-61. Therefore, IAPP may have some important role(s) in the pathogenesis and/or development of NIDDM. Several investigators have reported that IAPP inhibits insulin
58
effects invivo [7-131 and invitro [8,9,14,15], particularly in skeletal muscle. Since calcitonin gene-related peptide (CGRP), which has a sequence structural homology with IAPP, has been also reported to cause insulin resistance [ 12-151, the relationship of the effects between IAPP and CGRP is very interesting. In this study, the effects of IAPP and CGRP on glucose metabolism in skeletal muscle were studied using an in situ rat hindlimb perfusion system with which glucose utilization can be observed more directly and near-physiologically than in an in vitro system.
buffer
Fig. 1. Scheme of the in situ rat hindlimb perfusion system. Perfusion was conducted in a temperature-controlled chamber at 37°C. Perfusate was sufficiently oxygenated through 3 m plastic tube.
Materials and Methods Right hindlimb perfusion Male Wistar rats (180-250 g body weight) were fed a commercial diet with water ad libitum. After an overnight fast, rats were anesthetized intrasodium with pentobarbital peritoneally (5 mg/lOO g body weight). Surgical treatment was partially modified as in the method described by Ruderman et al. [ 161. The left common iliac artery and vein were tightly ligated and only the right hindlimb muscle was used for the perfusion. The perfusate consisted of a Krebs-Ringer bicarbonate buffer containing 7 mM glucose, 0.5% bovine serum albumin and 4.5% Dextran T-70 (Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The perfusate was sufficiently gassed with 95 y0 0, and 5 y0 CO, through a 3 m plastic tube starting 60 min before the beginning of the perfusion. Perfusion was conducted in a temperature-controlled chamber at 37°C. The perfusate was infused at the rate of 10 ml/min/leg through the abdominal aorta and was collected through a cannula inserted in the inferior vena cava. The perfusion system is schematically shown in Fig. 1. Infusion of insulin, IAPP and CGRP A synthesized amide form of rat IAPP(rIAPP) and rat CGRP(rCGRP) was obtained from the Protein Research Foundation Peptide Institute Inc., Osaka, Japan. After 40 min of pre-perfusion,
semi-synthesized human insulin (Novo Industry, Copenhagen, Denmark) in a concentration of 1000 pU/ml was infused for 140 min. Forty min after the beginning of the insulin infusion, 1 nM or 10 nM rIAPP or rCGRP was infused for 50 min. To determine whether IAPP affects basal (noninsulin-mediated) glucose uptake, 10 nM rIAPP was infused without insulin infusion. Perfusion was performed 50 min after the discontinuation of rIAPP or rCGRP infusion. Calculation of glucose uptake (GU) Glucose concentrations in the effluent were measured every 10 min with a glucose analyzer (Yellow Springs Instruments, Yellow Springs, U.S.A.). The weight of the right hindlimb muscle was calculated as 5 y0 of body weight according to the data of measurements in another experiment. The glucose uptake (GU) was calculated as follows: GU (nmol/g/min) = (A glucose x perfusion rate)/(weight of right hindlimb muscle). Statistical analysis For statistical comparison with GU, the average GU for the last 20 min of every interval (before, during and after rIAPP or rCGRP infusion) was calculated. All values were expressed as mean 2 SE. Statistical analysis was done with
59
paired Student’s r-test, and a P value less than 0.05 was considered significant.
TABLE
1
Comparison of glucose uptake rIAPP or rCGRP infusion
The perfusate was sufficiently oxygenated at approximately 400-500 mmHg during 180 min of the perfusion. In the control experiments, GU increased gradually during pre-perfusion and reached a plateau (about 250 nmol/g/min) at around 30 or 40 min after the beginning of the pre-perfusion, and the constant level continued without insulin infusion throughout the experiment (for at least 180 min). When insulin was infused after the 40 min pre-perfusion, GU increased again and reached a plateau (about 500-600 nmol/g/min) 30 min after the beginning of insulin infusion, and the constant level continued without rIAPP or rCGRP infusion for at least 100 min. Changes in the GU in the experiments with rIAPP infusion are shown in Fig. 2. No significant changes in GU were seen during the 1 nM rIAPP infusion. However, the GU began to fall 20 min after the beginning of the 10 nM rIAPP infusion and a significant decline of the GU was observed compared with that before the rIAPP infusion (Fig. 2). The decreased GU was gra-
:f
5
800
during
and after
12 Glucose uptake (nmol/g/min)
Results
c
before,
1 rat IAPP 1 insulin 1OOO_fflJ/ml
r
30
60
90
120
150
180 min
Fig. 2. Changes in glucose uptake during rat hindlimb perfusion. Forty min after the beginning of the insulin infusion at 1000 pU/ml, 1 nM rIAPP (0) or 10 nM rIAPP (0) was administered for 50 min. Values are means + SE.
Before
During
After
Insulin ( - ) rIAPP 10nM
4
264+38
264+38
211* 59
Insulin (1000 pU/ml) rIAPP 1 nM 10 nM rCGRP 1 nM 10 nM
4 4 4 4
524+53 554+24 538+59 507k I
511+44 445*29* 549567 323?15*
552 + 31 616+22 550+ 51 428+26
Before, During, After: Averages of GUS in every last 20 min before, during and after rIAPP or rCGRP infusion. Values are means + SE. *P < 0.01 vs before.
dually restored after discontinuation of the 10 nM rIAPP infusion. The averages of the GUS for the last 20 min of every period (before, during and after rIAPP or rCGRP infusion) are summarized in Table 1. Although 1 nM rIAPP did not affect GU, the GU during 10 nM rIAPP infusion was significantly reduced compared with those before and after the rIAPP infusion (Fig. 3 and Table 1). However, the GU was not affected by the 10 nM rIAPP infusion without insulin infusion (Fig. 4 and Table 1). Similar to the experiment with IAPP infusion, the GU during 10 nM rCGRP infusion significantly decreased compared with that before
Before
During
After
Before
During
After
Fig. 3. Effect of rat IAPP on glucose uptake during hindlimb perfusion. Before, During, After: averages of GUS in every last 20 min. before, during and after rIAPP infusion, respectively. Values are means + SE.
60
-T-
I
Before
During
After
Fig. 4. Effect of rat IAPP (10 nM) on non-insulin mediated glucose uptake. Before, During, After: averages of GUS in every last 20 min, before, during and after rIAPP infusion, respectively. Values are means + SE.
1nHCCRP
1OnMCGRP
Fig. 5. Effect of rat CGRP on glucose uptake during hindlimb perfusion. Before, During, After: averages of GUS in every last 20 min, before, during and after rCGRP infusion, respectively. Values are means f SE.
and after the rCGRP infusion although 1 nM rCGRP did not affect the GU (Fig. 5 and Table 1).
Discussion Several investigators have reported that IAPP inhibits insulin effects in vivo [7-131 and in vitro [ 8,9,14,15]. We have also recognized similar effects of human IAPP during hyperinsulinemic euglycemic glucose clamp study in dogs [ 111. We have considered that IAPP may cause peripheral insulin resistance in vivo from other evidence that IAPP did not stimulate hepatic glucose output in perfused rat liver [ 171, although there were some reports that IAPP inhibits insulin action in the
liver [ 9,12,18]. In the present study, we examined the direct effects of IAPP on skeletal muscle using an in situ rat hindlimb perfusion system, with which glucose metabolism in skeletal muscle can be studied directly and near-physiologically. As we expected, rat IAPP (10 nM) inhibited GU in this rat hindlimb perfusion system. Consistent with other reports in vitro [ 7,151, this inhibitory effect of IAPP on glucose uptake should be an insulin-mediated action because the same dose (10 nM) of IAPP did not affect the GU without insulin. On the other hand, it has been reported that CGRP also inhibits insulin action [ 12-151. Because of sequence similarity between IAPP and CGRP, it is interesting to study the relationship between their similar effects. Several investigators [ 19-211 have suggested that IAPP may act through CGRP receptors from a specific binding study using ‘251-labeled CGRP. However, the same dose of CGRP as that of IAPP was required to exhibit the inhibitory effect on the GU in the present study. This finding indicates that both IAPP and CGRP may act on the glucose metabolism in skeletal muscle through a yet unknown receptor which has a similar binding affinity for both IAPP and CGRP. Young et al. [9] reported that the inhibitory effects of IAPP on insulin-stimulated glucose metabolism in skeletal muscle are due to inhibition of glucose phosphorylation. The precise mechanism of the insulin antagonistic effect of IAPP and/or CGRP in skeletal muscle is still unclear, but IAPP may alfect the glucose metabolism at a post-receptor site of insulin via an unknown mechanism because the present study was performed at maximal insulin concentration (1000 pU/ml). Recently it has been reported that the peripheral plasma concentrations of IAPP in nondiabetic subjects and patients with NIDDM were 1 to 10 pM [6]. Although 10 nM IAPP inhibited the GU, IAPP in the concentration of 1 nM did not affect the GU. Taking account of the peripheral concentration of the IAPP, this finding suggests that the insulin antagonistic effects of the IAPP observed in skeletal muscle should not be
61
due to a physiological but to a pharmacological effect. This concept also supports our speculation that a considerably high dose (10 nM) of IAPP acts via an unknown receptor different from IAPP or CGRP receptors to exhibit its action.
Acknowledgements
We are indebted to Dr. K. Nakajima, Protein Research Foundation Peptide Institute Inc., Osaka, Japan for supplying the synthesized amide form of rat IAPP.
References Westermark, P., Wernstedt, C., Wilander, E., Hayden, D.W., O’Brien, T.D. and Johnson, K.H. (1987) Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. U.S.A. 84, 3881-3885. Cooper, G.J.S., Willis, AC., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. U.S.A. 84, 8628-8632. Sanke, T., Bell, G.I., Sample, C., Rubenstein, A.H. and Steiner, D.F. (1988) An islet amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing. J. Biol. Chem. 263, 17243-17246. Ogawa, A., Harris, V., McCorkle, S.K., Unger, R.H. and Luskey, R.H. (1990) Amylin secretion from the rat pancreas and its selective loss after streptozotocin treatment. J. Clin. Invest. 85, 973-976. Mitsukawa, T., Takemura, J., Asai, J. et al. (1990) Islet amyloid polypeptide response to glucose, insulin and somatostatin analogue administration. Diabetes 39, 639-642. Sanke, T., Hanabusa, T., Nakano, Y. et al. (1991) Plasma islet amyloid polypeptide (amylin) levels and their responses to oral glucose in Type 2 (non-insulin dependent) diabetic patients. Diabetologia 34, 129-132. Cooper, G.J.S., Leighton, B., Dimitriadis, G.D. et al. (1988) Amylin found in amyloid deposits in human type 2 diabetes melhtus may be a hormone that regulates glycogen metabolism in skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 85, 7763-7766. Leighton, B. and Foot, E. (1990) The effects of amylin on
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