0013-7227/90/1274-1609$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 4 Printed in U.S.A.
Secretion and Degradation of Glucagon by HIT Cells* PETER DIEMf, TIMOTHY F. WALSETH, HUI-JIAN ZHANG, AND R. PAUL ROBERTSON The Diabetes Center and Division of Endocrinology, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
ulated by arginine for 60 min and measured by RIA. Thirty-six percent of immunoreactive glucagon was found in the fractions representing authentic glucagon, whereas the remaining 64% eluted earlier. Experiments examining the fate of radiolabeled glucagon exposed to HIT cells revealed time-dependent degradation of the radioisotope to earlier eluting forms, which accounted for approximately 50% of the radioactivity by 60 min and was complete by 18 h, indicating that the early peak detected by RIA represented a metabolite of glucagon. Radioisotopic insulin was degraded more slowly with an apparent half-life of approximately 36 h. We conclude that HIT cells are not only able to synthesize, secrete, and degrade insulin, but also much smaller amounts of glucagon. {Endocrinology 127: 1609-1612, 1990)
ABSTRACT. HIT cells have been widely used to study synthesis and secretion of insulin. It has been assumed that this cell line secretes no other islet hormones. To ascertain whether HIT cells synthesize, secrete, and degrade glucagon, we examined cell extracts for this peptide and compared secretion and degradation of glucagon and insulin during stimulation of the cells by arginine. Glucagon levels in acid extracts of HIT cells were found to be 0.72 ± 0.15 pmol/mg protein. Both glucagon and insulin were maximally stimulated in a glucagon/insulin molar ratio of 0.029 by arginine concentrations of 25-50 nM, and the concentration of arginine that provided half-maximum responses for both hormones was approximately 3 tnM. Diminution of arginine-induced glucagon secretion was caused by somatostatin, a physiological inhibitor of pancreatic islet a-cell function. HPLC was used to authenticate the glucagon levels stim-
T
HE HIT cell is a clonal line of pancreatic islet /3cells derived from Simian virus transformation of Syrian hamster pancreatic islets (1). Subclone HIT-T15 has been very useful in studying regulation of insulin secretion, because it is responsive to both glucose and nonglucose secretagogues, and because glucose-induced insulin secretion is suppressed by physiologic concentrations of epinephrine, somatostatin (SRIF), and prostaglandin E2 (2-13). Although investigators using this cell line have assumed that it secretes no other islet hormones, there is virtually no published information describing measurements of islet hormones other than insulin. To investigate this assumption, we have examined glucagon content, secretion, and degradation by HIT cells.
serum and 11.1 mM glucose as previously described (8). Before each study was conducted, cells were subcultured in the absence of experimental drugs for 2 days by plating ~10 6 cells per well
in 12-well plates. The medium was replaced 16 h before the actual use of the cells to ensure that the cells were metabolically active. Cells were monitored for bacterial contamination, and no antibiotics were used. Studies were performed with culture passages 71-78 unless otherwise stated. Glucagon content, secretion, and degradation
Materials and Methods HIT cell cultures HIT cells were grown in 5% CO2/95% air at 37 C and maintained at RPMI-1640 medium containing 10% fetal calf Received April 30, 1990. Address all correspondence and requests for reprints to: Dr. R. Paul Robertson, Diabetes Center, Box 101 UMHC, University of Minnesota, Minneapolis, Minnesota 55455. * This work was supported by NIH Grant RO-AM-33974 and a grant from the Minnesota Medical Foundation. t Current address: Diabetes Station, Medizinische Universitatsklinik, Inselspital, 3010 Bern, Switzerland. Supported in part by a grant from the Swiss National Science Foundation.
At the beginning of the third day of culture, cells were washed twice for 30 min at 37 C in Krebs-Ringer buffer (KRB) containing 119 mM NaCl, 4.74 mM KC1, 2.54 mM CaCl, 1.19 mM HEPES and pH 7.4, and 0.1% albumin (Sigma, St. Louis, MO). After the second wash the cells were incubated for 60 min in KRB with varying concentrations of arginine with and without 0.1 juM SRIF (Bachem, Torrance, CA). Glucagon was measured by RIA as described previously (14) using antibody 04A obtained from Dr. Roger Unger (University of Texas, Dallas, TX). Insulin concentrations were measured by RIA using porcine standards and a guinea pig antibody directed toward porcine insulin. Acid extracts of HIT cells were performed using the method of Santerre et al. (1). HPLC analysis for glucagon and insulin were performed using an Altex Ultrapore RPSC column (0.46 ± 7.5 cm) eluted at a flow rate of 1 ml/min with an acetonitrile gradient in 0.01 M trifluoroacetic acid (TFA). The gradient program was a nonlinear acetonitrile gradient starting at 2.5% acetonitrile in 0.01 M TFA, linearly
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 01:54 For personal use only. No other uses without permission. . All rights reserved.
HIT CELLS AND GLUCAGON SECRETION AND DEGRADATION
1610
increased to 20% in 2.5 min, linearly increased to 55% in 14 min, and finally linearly increased to 100% in 2.5 min. Samples of 200 n\ were injected, and fractions were collected every 30 sec. Glucagon content in each fraction was determined by RIA after removal of acetonitrile on a Savant Speed-Vac concentrator (Savant Instruments, Hicksville, NY). [125I]glucagon and [125I] insulin contents in each fraction were determined using a Beckman Gamma 700 counter (Beckman Instruments Co., Fullerton, CA).
Endo • 1990 Vol 127 • No 4
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Data analysis
o
The figures are expressed as mean ± SEM Statistical comparisons were performed by analysis of variance. P < 0.05 was considered statistically significant. 0
10 FRACTION
20 NUMBER
FIG. 3. Glucagon concentrations of individual fractions obtained by reverse phase HPLC of HIT cell supernates after 60 min of incubation (•, HIT cell supernate; • , glucagon standard). O
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Vol. 127, No. 4 Printed in U.S.A.
Secretion and Degradation of Glucagon by HIT Cells* PETER DIEMf, TIMOTHY F. WALSETH, HUI-JIAN ZHANG, AND R. PAUL ROBERTSON The Diabetes Center and Division of Endocrinology, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
ulated by arginine for 60 min and measured by RIA. Thirty-six percent of immunoreactive glucagon was found in the fractions representing authentic glucagon, whereas the remaining 64% eluted earlier. Experiments examining the fate of radiolabeled glucagon exposed to HIT cells revealed time-dependent degradation of the radioisotope to earlier eluting forms, which accounted for approximately 50% of the radioactivity by 60 min and was complete by 18 h, indicating that the early peak detected by RIA represented a metabolite of glucagon. Radioisotopic insulin was degraded more slowly with an apparent half-life of approximately 36 h. We conclude that HIT cells are not only able to synthesize, secrete, and degrade insulin, but also much smaller amounts of glucagon. {Endocrinology 127: 1609-1612, 1990)
ABSTRACT. HIT cells have been widely used to study synthesis and secretion of insulin. It has been assumed that this cell line secretes no other islet hormones. To ascertain whether HIT cells synthesize, secrete, and degrade glucagon, we examined cell extracts for this peptide and compared secretion and degradation of glucagon and insulin during stimulation of the cells by arginine. Glucagon levels in acid extracts of HIT cells were found to be 0.72 ± 0.15 pmol/mg protein. Both glucagon and insulin were maximally stimulated in a glucagon/insulin molar ratio of 0.029 by arginine concentrations of 25-50 nM, and the concentration of arginine that provided half-maximum responses for both hormones was approximately 3 tnM. Diminution of arginine-induced glucagon secretion was caused by somatostatin, a physiological inhibitor of pancreatic islet a-cell function. HPLC was used to authenticate the glucagon levels stim-
T
HE HIT cell is a clonal line of pancreatic islet /3cells derived from Simian virus transformation of Syrian hamster pancreatic islets (1). Subclone HIT-T15 has been very useful in studying regulation of insulin secretion, because it is responsive to both glucose and nonglucose secretagogues, and because glucose-induced insulin secretion is suppressed by physiologic concentrations of epinephrine, somatostatin (SRIF), and prostaglandin E2 (2-13). Although investigators using this cell line have assumed that it secretes no other islet hormones, there is virtually no published information describing measurements of islet hormones other than insulin. To investigate this assumption, we have examined glucagon content, secretion, and degradation by HIT cells.
serum and 11.1 mM glucose as previously described (8). Before each study was conducted, cells were subcultured in the absence of experimental drugs for 2 days by plating ~10 6 cells per well
in 12-well plates. The medium was replaced 16 h before the actual use of the cells to ensure that the cells were metabolically active. Cells were monitored for bacterial contamination, and no antibiotics were used. Studies were performed with culture passages 71-78 unless otherwise stated. Glucagon content, secretion, and degradation
Materials and Methods HIT cell cultures HIT cells were grown in 5% CO2/95% air at 37 C and maintained at RPMI-1640 medium containing 10% fetal calf Received April 30, 1990. Address all correspondence and requests for reprints to: Dr. R. Paul Robertson, Diabetes Center, Box 101 UMHC, University of Minnesota, Minneapolis, Minnesota 55455. * This work was supported by NIH Grant RO-AM-33974 and a grant from the Minnesota Medical Foundation. t Current address: Diabetes Station, Medizinische Universitatsklinik, Inselspital, 3010 Bern, Switzerland. Supported in part by a grant from the Swiss National Science Foundation.
At the beginning of the third day of culture, cells were washed twice for 30 min at 37 C in Krebs-Ringer buffer (KRB) containing 119 mM NaCl, 4.74 mM KC1, 2.54 mM CaCl, 1.19 mM HEPES and pH 7.4, and 0.1% albumin (Sigma, St. Louis, MO). After the second wash the cells were incubated for 60 min in KRB with varying concentrations of arginine with and without 0.1 juM SRIF (Bachem, Torrance, CA). Glucagon was measured by RIA as described previously (14) using antibody 04A obtained from Dr. Roger Unger (University of Texas, Dallas, TX). Insulin concentrations were measured by RIA using porcine standards and a guinea pig antibody directed toward porcine insulin. Acid extracts of HIT cells were performed using the method of Santerre et al. (1). HPLC analysis for glucagon and insulin were performed using an Altex Ultrapore RPSC column (0.46 ± 7.5 cm) eluted at a flow rate of 1 ml/min with an acetonitrile gradient in 0.01 M trifluoroacetic acid (TFA). The gradient program was a nonlinear acetonitrile gradient starting at 2.5% acetonitrile in 0.01 M TFA, linearly
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 01:54 For personal use only. No other uses without permission. . All rights reserved.
HIT CELLS AND GLUCAGON SECRETION AND DEGRADATION
1610
increased to 20% in 2.5 min, linearly increased to 55% in 14 min, and finally linearly increased to 100% in 2.5 min. Samples of 200 n\ were injected, and fractions were collected every 30 sec. Glucagon content in each fraction was determined by RIA after removal of acetonitrile on a Savant Speed-Vac concentrator (Savant Instruments, Hicksville, NY). [125I]glucagon and [125I] insulin contents in each fraction were determined using a Beckman Gamma 700 counter (Beckman Instruments Co., Fullerton, CA).
Endo • 1990 Vol 127 • No 4
S Q.
z" o (3 < O
z> _l
Data analysis
o
The figures are expressed as mean ± SEM Statistical comparisons were performed by analysis of variance. P < 0.05 was considered statistically significant. 0
10 FRACTION
20 NUMBER
FIG. 3. Glucagon concentrations of individual fractions obtained by reverse phase HPLC of HIT cell supernates after 60 min of incubation (•, HIT cell supernate; • , glucagon standard). O
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Q.
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