358
M. Pierce, L. Bukowiecki, K. Asplund and N. Freinkel
Malaisse, W.J.: Hormonal and environmental modifications of islet activity. In: Steiner, O.F. and N. Freinkel (eds.): Handbook of Physiology, Section 7, Volume 1. Endocrine Pancreas pp. 237-260. American Physiological Society, Washington O.C. 1972 Matschinsky, F.M., J. Ellemllln: Dissociation of the insulin releasing and the metabolic functions of hexoses in islets of Langerhans. Biochem.Biophys.Res.Comm. 50: 193-199 (1973) Matschinsky, F.M., R. Landgraf, J. Ellerman, J. Kotler·Brajt· burg: Glucoreceptor mechanisms in islets of Langerhans. Diabetes 21 (Suppl. 2): 555-569 (1972) Montague, W., K. W. Taylor: Islet cell metabolism during insulin release. Biochem.J. 115: 257·262 (1969)
Pace, C.S., J. Ellerman, B.A. Ho ver, S.N. Stillings, F.M. Mat· chinsky: Multiple metabolie functions of glucose in rat pancreatic islets. Diabetes 24: 476-488 (1975) Randle, P.J., S.J.H. Ashcroft, J.R. GiII: Carbohydrate metabolism and release of hormones. In: Dickens, F., P.J. Randle and W.J. Whelan (eds.): Carbohydrate metabolism and its disorders. Vol. 1,427-447, Academic Press, London 1968 Warburg, 0., W. Christian: Isolierung und Kristallisation des Gärungsferments Enolase. Biochem.Z. 310: 384-421 (942) Webb, J.L.: lodoacetate and iodoacetamide. Enzyme and Metabolie Inhibitors 3: 1-283 (1966)
Requests for reprints should be addressed to: Or. B.J. Lin, Dept. Physiology, Medical Sciences Building, Univ. Toronto, Toronto, Ontario (Canada, M5S IA8)
[32P] Orthophosphate Efflux from Pancreatic Islets: Graded Response to Glucose Stimulation* M. Pierce, l. Bukowiecki, K. Asplund** and N. Freinkel Center for Endocrinology, Metabolism and Nutrition, and the Departments of Medicine and Biochemistry, Northwestern University Medical School, Chicago, Illinois
Downloaded by: Universite Laval. Copyrighted material.
Horm. Metab. Res. 8 (1976) 358-361
© Georg Thieme Verlag Stuttgart
Summary
Introduction
The dose-response relationships of the glucose-induced rapid transient efflux of [32p] orthophosphate from prelabeled pancreatic islets ("phosphate flush") have been investigated. Threshold levels for eliciting a "phosphate flush" were between 0.5 and 1.0 mg/mI glucose, the apparent "Km" for this event was 1.0-1.5 mg per ml and the apparent "V max " was reached at ambient glucose concentrations between 1.5 and 2.0 mg per mI. This dose-response curve is somewhat shifted to the left in comparison with previously published data for glucose-induced insulin release.
Stimulation with nutrient secretagogues may trigger a rapid, transient efflux of [32 P] orthophosphate from prelabeled pancreatic islets, the "phosphate flush" (Freinkel, EI Younsi, Bonnar and Dawson 1974a). The phenomenon is highly specific for sugars and amino acids that are capable of promoting insulin release (Freinkel et al. 1974a, Freinkel, EI Younsi. Bonnar and Dawson 1974b, Freinkel, EI Younsi, Christensen and Dawson 1974c, Pierce and Freinkel 1975). On the basis of the available data, we have suggested that the "phosphate flush" may represent one of the ear· lier compunents in the sequence of events that constitutes stimulus-secretion coupling in the ß-cell (Freinkel et al. 1974b, Pierce and Freinkel 1975). To evaluate this proposal further, we have now characterized the precise dose-response relationships between ambien glucose and the net outflow of orthophosphate- 32 P from isolated islets.
Thus, the "phosphate flush" appears to displayamore narrow dose-response curve to ambient glucose concentrations than the actual release of insulin. It is proposed that this may constitute further evidencc that the "phosphate flush" reflects an early step in stimulus-secretion coupling and that there may be some loss of sensitivity as the glucose signal is transmitted from the site of recognition to the final site of hormone release. Key·Words: Orthophosphate Efflux - Pancreatic Islets Glucose Stimuliltion - 32p *Supported in part by Research Grant AM-10699, and Training Grant AM-05071 from the National Institute of Arthritis and Metabolie Diseases, National Institutes of Health, Bethesda, Maryland, and by agrant from the Kroc Foundation. Dr. Asplund was the recipient of a Fogarty International Research Fellowship (lF05 TW 2139) during the course of these studies. **Present address: Oepartment of Histology, Biomedicum, Uppsala University, Box 571, S-75123 Uppsala, Sweden. Received: 6 Jan. 1976
Accepted: 30 Apr. 1976
Materials and Methods Pancreatic islets were isolated by the collagenase method (Lacy and Kostionovsky 1967) from fed female Sprague-Oawley rats weighing 200-300 gm. The islets were labeled with radiophosphate during a 90 min incubation period in 1 ml of modified Krebs-Ringer bicarbonate (KRB) solution containing 150 J,Ci [32p] orthophosphate and 0.5 Ibllole Na2HP04 as described previously (Freinkel et al. 1974a). Thereafter. islets were washed to remove adherent radioactivity. The labeled islets were then utilized to assess their
Graded Response of Phosphate Efflux to Glucose
For the perifusion studies, two different systems were used: one being the modified system of Lacy, Walker and Fink (1972) which we have described elsewhere (Freinkel et al. 1974a), where the volume of the chamber and associated tubing is 2.8 ml, and the flow-through rate 0.9 ml/min; the other a slight modification of the apparatus of Asplund (1973) where the corresponding values are 0.24 ml and 0.3 ml/ min respectively. 1n both systems the islets were first perifused for 30-50 min with KRB containing no glucose or 0.5 mg glucose per ml to establish basal, constant rates of 32 P efflux (Freinkel et al. 1974a). The perifusion media were then changed to expose the islets to glucose concentrations ranging from 0.5 to 3.0 mg per ml. The eluates were monitored for radioactivity, which was shown to consist almost cntirely of [32p] orthophosphatc (Freinkel et al. 1974a, 1974b). Preliminary analyses of perifusion experiments, indicated that the peak of the "phosphate flush" (corrected for the basal values observed with 0 or 0.5 mg glucose per ml), correlated significantly with the total release of radioactivity above basal values during the entire period of the "phosphate flush" (r = 0.785; p 0.005; n = 22). Accordingly, quantitative description of the stimulatory effects of glucose upon 32p efflux in the present perifusion experiments were based solelyon the relative heights of the peaks of the "phosphate flushes". Moreover, since the dynamies of radiophosphate efflux were not entirely identical in the two perifusion systems (due to differences in dead space and flow rate), the results obtained with various concentrations of glucose in individual experiments were related to those obtained with 3 mg/mi glucose under identical experimental conditions, this laUer response being designated as "100 per cent".
M. Pierce, L. Bukowiecki, K. Asplund and N. Freinkel
Malaisse, W.J.: Hormonal and environmental modifications of islet activity. In: Steiner, O.F. and N. Freinkel (eds.): Handbook of Physiology, Section 7, Volume 1. Endocrine Pancreas pp. 237-260. American Physiological Society, Washington O.C. 1972 Matschinsky, F.M., J. Ellemllln: Dissociation of the insulin releasing and the metabolic functions of hexoses in islets of Langerhans. Biochem.Biophys.Res.Comm. 50: 193-199 (1973) Matschinsky, F.M., R. Landgraf, J. Ellerman, J. Kotler·Brajt· burg: Glucoreceptor mechanisms in islets of Langerhans. Diabetes 21 (Suppl. 2): 555-569 (1972) Montague, W., K. W. Taylor: Islet cell metabolism during insulin release. Biochem.J. 115: 257·262 (1969)
Pace, C.S., J. Ellerman, B.A. Ho ver, S.N. Stillings, F.M. Mat· chinsky: Multiple metabolie functions of glucose in rat pancreatic islets. Diabetes 24: 476-488 (1975) Randle, P.J., S.J.H. Ashcroft, J.R. GiII: Carbohydrate metabolism and release of hormones. In: Dickens, F., P.J. Randle and W.J. Whelan (eds.): Carbohydrate metabolism and its disorders. Vol. 1,427-447, Academic Press, London 1968 Warburg, 0., W. Christian: Isolierung und Kristallisation des Gärungsferments Enolase. Biochem.Z. 310: 384-421 (942) Webb, J.L.: lodoacetate and iodoacetamide. Enzyme and Metabolie Inhibitors 3: 1-283 (1966)
Requests for reprints should be addressed to: Or. B.J. Lin, Dept. Physiology, Medical Sciences Building, Univ. Toronto, Toronto, Ontario (Canada, M5S IA8)
[32P] Orthophosphate Efflux from Pancreatic Islets: Graded Response to Glucose Stimulation* M. Pierce, l. Bukowiecki, K. Asplund** and N. Freinkel Center for Endocrinology, Metabolism and Nutrition, and the Departments of Medicine and Biochemistry, Northwestern University Medical School, Chicago, Illinois
Downloaded by: Universite Laval. Copyrighted material.
Horm. Metab. Res. 8 (1976) 358-361
© Georg Thieme Verlag Stuttgart
Summary
Introduction
The dose-response relationships of the glucose-induced rapid transient efflux of [32p] orthophosphate from prelabeled pancreatic islets ("phosphate flush") have been investigated. Threshold levels for eliciting a "phosphate flush" were between 0.5 and 1.0 mg/mI glucose, the apparent "Km" for this event was 1.0-1.5 mg per ml and the apparent "V max " was reached at ambient glucose concentrations between 1.5 and 2.0 mg per mI. This dose-response curve is somewhat shifted to the left in comparison with previously published data for glucose-induced insulin release.
Stimulation with nutrient secretagogues may trigger a rapid, transient efflux of [32 P] orthophosphate from prelabeled pancreatic islets, the "phosphate flush" (Freinkel, EI Younsi, Bonnar and Dawson 1974a). The phenomenon is highly specific for sugars and amino acids that are capable of promoting insulin release (Freinkel et al. 1974a, Freinkel, EI Younsi. Bonnar and Dawson 1974b, Freinkel, EI Younsi, Christensen and Dawson 1974c, Pierce and Freinkel 1975). On the basis of the available data, we have suggested that the "phosphate flush" may represent one of the ear· lier compunents in the sequence of events that constitutes stimulus-secretion coupling in the ß-cell (Freinkel et al. 1974b, Pierce and Freinkel 1975). To evaluate this proposal further, we have now characterized the precise dose-response relationships between ambien glucose and the net outflow of orthophosphate- 32 P from isolated islets.
Thus, the "phosphate flush" appears to displayamore narrow dose-response curve to ambient glucose concentrations than the actual release of insulin. It is proposed that this may constitute further evidencc that the "phosphate flush" reflects an early step in stimulus-secretion coupling and that there may be some loss of sensitivity as the glucose signal is transmitted from the site of recognition to the final site of hormone release. Key·Words: Orthophosphate Efflux - Pancreatic Islets Glucose Stimuliltion - 32p *Supported in part by Research Grant AM-10699, and Training Grant AM-05071 from the National Institute of Arthritis and Metabolie Diseases, National Institutes of Health, Bethesda, Maryland, and by agrant from the Kroc Foundation. Dr. Asplund was the recipient of a Fogarty International Research Fellowship (lF05 TW 2139) during the course of these studies. **Present address: Oepartment of Histology, Biomedicum, Uppsala University, Box 571, S-75123 Uppsala, Sweden. Received: 6 Jan. 1976
Accepted: 30 Apr. 1976
Materials and Methods Pancreatic islets were isolated by the collagenase method (Lacy and Kostionovsky 1967) from fed female Sprague-Oawley rats weighing 200-300 gm. The islets were labeled with radiophosphate during a 90 min incubation period in 1 ml of modified Krebs-Ringer bicarbonate (KRB) solution containing 150 J,Ci [32p] orthophosphate and 0.5 Ibllole Na2HP04 as described previously (Freinkel et al. 1974a). Thereafter. islets were washed to remove adherent radioactivity. The labeled islets were then utilized to assess their
Graded Response of Phosphate Efflux to Glucose
For the perifusion studies, two different systems were used: one being the modified system of Lacy, Walker and Fink (1972) which we have described elsewhere (Freinkel et al. 1974a), where the volume of the chamber and associated tubing is 2.8 ml, and the flow-through rate 0.9 ml/min; the other a slight modification of the apparatus of Asplund (1973) where the corresponding values are 0.24 ml and 0.3 ml/ min respectively. 1n both systems the islets were first perifused for 30-50 min with KRB containing no glucose or 0.5 mg glucose per ml to establish basal, constant rates of 32 P efflux (Freinkel et al. 1974a). The perifusion media were then changed to expose the islets to glucose concentrations ranging from 0.5 to 3.0 mg per ml. The eluates were monitored for radioactivity, which was shown to consist almost cntirely of [32p] orthophosphatc (Freinkel et al. 1974a, 1974b). Preliminary analyses of perifusion experiments, indicated that the peak of the "phosphate flush" (corrected for the basal values observed with 0 or 0.5 mg glucose per ml), correlated significantly with the total release of radioactivity above basal values during the entire period of the "phosphate flush" (r = 0.785; p 0.005; n = 22). Accordingly, quantitative description of the stimulatory effects of glucose upon 32p efflux in the present perifusion experiments were based solelyon the relative heights of the peaks of the "phosphate flushes". Moreover, since the dynamies of radiophosphate efflux were not entirely identical in the two perifusion systems (due to differences in dead space and flow rate), the results obtained with various concentrations of glucose in individual experiments were related to those obtained with 3 mg/mi glucose under identical experimental conditions, this laUer response being designated as "100 per cent".
