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Size-related and size-unrelated functional heterogeneity among pancreatic islets
Life Sciences 69 (2001) 2627–2639
Size-related and size-unrelated functional heterogeneity among pancreatic islets Toru Aizawaa,*, Tsuyoshi Kanekoa, Keishi Yamauchia, Hiroki Yajimaa, Tomoko Nishizawab, Toshihiko Yadac, Hiroshi Matsukawad, Minoru Nagaia, Satoko Yamadaa, Yoshihiko Satoa, Mitsuhisa Komatsua, Nobuo Itohb, Hiroya Hidakae, Yoshitaka Kajimotof, Kiyoshi Hashizumea a
Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto, Japan b Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan c Department of Physiology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi, Japan d Department of Physiology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan e Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan f Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan Received 12 September 2000; accepted 11 May 2001
Abstract Functional heterogeneity of pancreatic islets was systematically analyzed for the first time using freshly isolated single rat pancreatic islets. First, 60 islets were sequentially exposed to 3, 9.4, 15.6, and 24.1 mM glucose for 30 min each in incubation experiments: 36 (60%) responded in a concentrationdependent and 19 (32%) in an all-or-none manner, and 5 (8%) islets did not respond to high glucose. As a group, the larger the islet, the higher the b cell glucose sensitivity. However, glucose-stimulated elevation of [Ca21]i in the b cell, insulin/glucagon ratio in the islet, and expression of glucose transporter 2, glucokinase, and pancreatic duodenal homeobox factor-1 in the b cell were not significantly related to islet size. Second, 50 islets were stimulated with 16.7 mM glucose in perifusion. A biphasic insulin release was found in 39 (78%), and no or little first phase response in 11 (22%) islets, irrespective of the islet size. Nevertheless, when the response was plotted as a group, it was clearly biphasic. Islet size, insulin content and the amount of insulin release were positively correlated with each other. In conclusion, there are size-related and size-unrelated functional diversity among pancreatic islets. The reason for such heterogeneity remained to be determined. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Pancreatic b cell; Insulin; Glucose sensitivity; Islet architecture
* Corresponding author. Department of Geriatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. Tel.: 181-263-37-2686; fax: 181-263-37-2710. E-mail address: [email protected] (T. Aizawa) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 3 2 -7
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Introduction A high concentration of glucose elicits a concentration-dependent, biphasic insulin secretion from the pancreas, and impairment of insulin secretion in patients with diabetes is characterized by reduced b cell glucose sensitivity and disappearance or diminution of the brisk, initial response to glucose [1,2]. These salient features of insulin secretion in health and disease have been interpreted in relation to the physiology and pathophysiology of stimulussecretion coupling at the level of b cell [1,2]. An assumption here is that the kinetics of insulin output from the pancreas faithfully reflect those from individual pancreatic islets and/or individual b cells. However, evidence supporting this assumption does not exist. There are more than several thousand islets in rat pancreas, each islet contains approximately several to ten thousand b cells, and functional heterogeneity at the level of individual b cells has long been recognized [3,4]. Therefore, functional heterogeneity among individual islets is not unexpected. As appropriately discussed for individual b cells [5], even if each islet responds to high glucose in an all-or-none manner, concentration-dependent insulin output from the pancreas would be observed if there is a Gaussian distribution of the threshold for glucose among individual islets. Since islets are the smallest units of coordinated insulin secretion [6], data on the function of individual islets have been expanding. But functional homogeneity among pancreatic islets has never been seriously questioned except in 1 study in which groups of ventral and dorsal islets were compared [7]. Based on these considerations, we systematically analyzed insulin release by individual islets, and found significant functional heterogeneity among them. Methods Pancreatic islets were obtained from adult male Wistar rats by collagenase dispersion, and insulin release was measured in incubation and perifusion experiments at 378C as reported [8–11]. Krebs-Ringer bicarbonate (KRB) buffer supplemented with 0.1% bovine serum albumin (BSA) containing (in mM) 118.4 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.9 CaCl2, and 25 NaHCO3 (equilibrated with 5% CO2-95% O2, pH 7.4) was used for isolation of the islets and the KRB buffer containing 0.2% BSA was used in experiments for determination of insulin release as reported [8–11]. KRB buffer used for measurement of cytosolic free Ca21 concentration ([Ca21]i) was composed of (in mM) 121.7 NaCl, 4.4 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.0 CaCl2, 5 NaHCO3, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) at pH 7.4 with NaOH supplemented with 0.1% BSA as reported [12,13]. Size of the islets was individually determined under an operating microscope immediately before the experiments. In this study, beautifully isolated, round islets with minimum exocrine tissue were selected. Small, medium, and large islets were defined as the diameter being ,200 mm, $200 and ,300 mm, and $300 mm, respectively. In static incubation experiments, 1 islet/tube was first incubated in KRB buffer containing 3 mM glucose for 30 min (preincubation). At the end of preincubation, the buffer was aspirated and 200 ml fresh KRB buffer containing 3 mM glucose was introduced for the 1st experimental incubation. Thirty minutes later, 100 ml of the buffer was removed for insulin assay, 100 ml fresh KRB buffer containing 15 mM glucose was added, and the incubation was carried out for another 30 min (2nd experimental incubation). The same procedure was
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repeated for 2 more times using KRB buffer with 20 mM and 35 mM glucose, successively, as a refill. At the end of the 4th experimental incubation, buffer was simply aspirated for insulin assay. Thus, insulin concentration in the buffer obtained at the end of the 4 successive experimental incubations was determined, and the release in response to each concentration of glucose during the respective 30 min-incubations was calculated. Glucose concentration of each media sample was later directly determined by the glucose oxidase method and the mean values were 3, 9.4, 15.6, and 24.1 mM in the buffer used for the 1st, 2nd, 3rd and 4th experimental incubations, respectively. The perifusion system used in the present study was a modification of the previously described multiple parallel perifusion system [8–11]. The modification is primarily a down-sizing, so that insulin release from a single islet is reliably determined. After a 30 min-prewash with KRB buffer containing 3 mM glucose, glucose concentration was increased to 16.7 mM. Perifusate was collected at 1, 2 or 5 min intervals, as needed, and stored at 2208C. The lag period, from switching to the high glucose buffer to its appearance in the collection tube, was approximately 2.5 min, which was not corrected for the data presentation. In rat islets, a glucose-induced elevation of [Ca21]i cannot be consistently demonstrated. Therefore, measurement of [Ca21]i was made in single b cells by dual wavelength fura-2 microfluorometry combined with digital imaging [12,13]. The cells were loaded with fura-2 by incubation with 1 mM fura-2 acetoxymethylester (Molecular Probes, Eugene, OR) for 30 min at 378C; the coverslip (with the cells on it) was then mounted in a chamber and superfused with KRB buffer. After a 30 min-prewash with KRB buffer containing 3 mM glucose, glucose in the buffer was increased to 16.7 mM. b cells were identified based on the larger diameter (12.5–17.5 mm) and the Ca21 response to 300 mM tolbutamide, a sulfonylurea compound. b cells from the large and small islets (as defined above) were separately examined. The islet insulin and glucagon content were determined after extraction with 0.15 N HCl/70% ethanol [14]. Western blotting of glucokinase (GCK) and glucose transporter 2 (GLUT2) was performed using lysates of the islets as described [15]. In brief, immediately after the isolation, the islets were hand-picked into ice-cold BSA-free KRB buffer. Then, small and large islets were selectively pooled and lysed in the solution containing (in mM) 20 Hepes (pH 7.4), 2 EDTA, 2 EGTA, and 1 phenylmethylsulfonyl fluoride, with 1% Triton X-100, 0.1% SDS, 10 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin A. Usually, 300 small islets were lysed in 300 ml lysis solution and 50 large islets were lysed in 250 ml lysis solution. The lysates (protein-adjusted) were subjected to SDS-PAGE, and immunoblotted with the antibodies. For the morphological study, the whole rat pancreas and the isolated islets were fixed with 10% formaldehyde, and 4 mm-thickness serial sections were made from the paraffin-embedded tissue. Sections (1 out of every 20 consecutive sections) were initially stained with hematoxylin and eosin (H-E) to ascertain the diameter of the islets. After identification of large and small islets, immunostaining with anti-GCK, -GLUT2 and -pancreatic duodenal homeobox factor-1 (PDX-1/IDX-1/SPF-1/IPF-1) antibodies was performed using selected sections. Insulin and glucagon were determined by radioimmunoassay using commercially available kits (Linco Research Inc., St. Louis, MO, for insulin and Daiichi Radioisotope Laboratories, Tokyo, for glucagon). The minimum detectable amount of insulin was 1.0 pg/tube. Antibodies against GLUT2 and GCK were obtained from Transformation Research Inc. (Framingham, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA) respectively. The antibody for
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Fig. 1. Concentration-dependency of glucose-induced insulin release. A, Individual data. [Insulin release with the highest concentration (24.1 mM) of glucose]-[insulin release with basal (3 mM) glucose] was calculated for each islet and taken as 100%. Responses to the intermediate concentrations of glucose were plotted as % values. Minus values are due to assay variation (see Text for the experimental procedure). Upper panel, large islets; middle panel, medium islets; lower panel, small islets. The data from 4 small islets and 1 medium islet, which did not show a significant response to high glucose (,20% of the basal), was omitted. B, Grouped data. All data from large, medium, and small islets were plotted as a group. Values are mean6SEM.
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Fig. 1 (continued).
PDX-1 was generated as described [16]. Statistical analysis was performed by one way ANOVA with Fisher’s protected least significance difference test and Wilcoxon’s signed rank test (SAS Institute Inc., Cary, NC). P,0.05 was considered significant. Results Analysis of insulin release in static incubation experiments Sixty islets (23 small, 22 medium and 15 large) were individually stimulated with a sequential exposure (30 min each) to 3, 9.4, 15.6 and 24.1 mM glucose. Thirty-six (60%) responded to high glucose in a concentration-dependent manner, and 19 (32%) responded in all-or-none fashion (Fig. 1A). Five islets (8%) did not respond to high glucose (,20% increase from the baseline). The response was regarded all-or-none fashion when the following criteria are fulfilled: 1) the greatest response with 9.4 mM glucose, 2) ,10% increase from the baseline with 9.4 mM and the greatest response with 15.6 mM, or 3) ,10% increase from the baseline up to 15.6 mM. When individual data from each islet were summed and treated as a group, typical concentration-dependency curves were obtained irrespective of the islet size (Fig. 1B). b cell glucose sensitivity was highest, intermediate, and lowest in the large, medium and small islets, respectively (Fig. 1B). There was a positive correlation between the islet insulin content and the amount of released insulin, both at the basal and stimulated state (data not shown). In a separate experiment, the insulin content of non-stimulated islets was found to be positively correlated with islet size (data not shown). Despite the fact that b cell glucose sensitivity is lower in the small islets, glucose-stimulated insulin release by the small islets was totally suppressed by 10 mM nifedipine, a blocker of the L-type voltage-dependent Ca21 channels (VDCC) (data not shown), indicating that glucose-
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stimulated insulin release from the b cell in small islets is normally dependent upon Ca21 influx through the L-type VDCC. Insulin release by the single islets in perifusion A total of 50 (25 small, 13 medium, and 12 large) islets were individually stimulated with a square wave application of 16.7 mM glucose. The mean insulin release from each category was clearly biphasic, and there was a positive correlation between islet size and insulin release (Fig. 2A). However, when the values were individually analyzed, there was considerable heterogeneity in the temporal profile (Fig. 2B). To facilitate a qualitative comparison, percent change from the baseline insulin release was calculated for each islet and the response was classified based on the amplitude of the first phase response. The maximum increase during the initial 5 min of glucose stimulation was employed as an index of the first phase response; Group A, B and C were defined as a maximum response .650%, ,650% and .220%, and ,220% respectively (Fig. 2B). Group A thus has a large first phase, Group B a small first phase, and Group C little or no first phase. Thirty-eight percent of islets were in Group A, 40% in Group B, and 22% in Group C. A biphasic response was seen in 39 (78%) islets (Groups A and B combined) (Fig. 2B). [Ca21]i in the b cells [Ca21]i in 112 b cells from the large islets and 87 b cells from the small islets was unselectively determined. As shown in Fig. 3A, the change in [Ca21]i upon exposure to 16.7 mM glucose could be grouped into 4 patterns: a, a transient decrease of [Ca21]i followed by a sharp rise and subsequent regular oscillations; b, an initial dip and sharp rise followed by irregular oscillations; c, an initial dip and sharp rise alone; d, little or no response to glucose. Even in the cells with little or no response to glucose, the [Ca21]i response to tolbutamide was retained (data not shown). The response to high glucose was not significantly different between the b cells from the large and small islets, in terms of incidence of response patterns (Fig. 3B) and the average values (data not shown). In the b cells from large islets, the decrease in initial peak with glucose stimulation was generally associated with the decreased response to depolarizing concentration of K1. This phenomenon was not generally found in the b cells from small islets (Fig. 3A, the bottom panel). Analysis of glucagon to insulin ratio, expression of GCK and GLUT2 in the isolated islets There was no significant difference in glucagon to insulin ratio (pg/ng) based on islet size (30.869.5, 36.764.2, and 36.964.3, in the small, medium and large islets, respectively). Also, by Western blotting, there was no significant difference in the expression of GCK and GLUT2 based on islet size, as indexed by intensity of Western blotting, of GCK and GLUT2 based on islet size (data not shown). Histological studies Immunostaining with anti-GCK, -GLUT2 and -PDX-1 antibodies revealed no significant difference in the staining between the b cells in the large and small islets in situ in the pancreas (Fig. 4A) or after isolation (Fig. 4B).
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Fig. 2. Insulin release elicited by 16.7 mM glucose in perifusion. A, Grouped data from the large, medium, and small islets (number of islets is indicated in the parentheses). Perifusate was switched from KRB buffer with 3 mM glucose to that with 16.7 mM glucose at time 0, after a 30 min prewash with the former solution. Values are mean6SEM. B, Individual data of the experiment shown in Fig. 2A is plotted (number of islets is indicated in the parentheses). Left panel, small islets; Right panel, large and medium islets. Percent change from the baseline is plotted. Classification is based on the initial response, with Group A, B and C being percent maximum change from the baseline during the initial 5 min of switching to high glucose buffer, .650%, ,650% and .220%, and ,220%, respectively (see Text for the details of classification).
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Fig. 3. Cytosolic free Ca21 in isolated b cells. After superfusion with 3 mM glucose for 30 min, glucose concentration was raised to 16.7 mM at time 0 and lowered to 3 mM at 30 min. A 1 min-pulse of 25 mM KCl was applied at 45 min. A, Representative recordings of b cells from large and small islets. Ca21 responses were classified into 4 patterns. Pattern a, a transient decrease of [Ca21]i followed by a sharp rise and subsequent regular oscillations; pattern b, the initial responses followed by irregular oscillations; pattern c, the initial responses alone; pattern d, little or no response to glucose (see Text for the detail). B, Incidence of response patterns.
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Fig. 3. Continued.
Discussion In the present study, we discovered considerable functional diversity of pancreatic islets, some of which were size-related and some size-unrelated. First, many islets (60%) exhibited a concentration-dependent response to high glucose, however, a significant proportion of them (32%) showed an all-or-none response. Nevertheless, when the mean values were plotted, the response was clearly concentration-dependent, and as a group, the larger the islet, the higher the b cell glucose sensitivity. It is also clear that fold increase of basal secretion is less in the small islets especially at the lower glucose concentrations (9.4 and 15.6 mM) (Fig. 1B). In order to obtain the glucose dose-response curve for each individual islet, we employed sequential incubations in increasing glucose concentrations. Similar approach was taken by others in a previous, less comprehensive study [30]. This procedure is known to produce priming phenomenon. Also, only 3 stimulating glucose concentrations were tested. Thus, one should interpret the data with some caution. Second, a high concentration of glucose elicited a biphasic response in 78% of islets, and no or little first phase response in the rest, irrespective of the islet size. Finally, the islet insulin content and insulin release correlated well each other, as reported previously [17,18]. Correlation between the size of islet and insulin release was reported in one [18] but not in the other [17] study. In the pancreas, there are more than 13105 islets, with a considerable size heterogeneity [19,20]. The relative proportion of small (,200 mm), medium ($200 mm and ,300 mm), and large ($300 mm) islets is approximately 90%, 9% and 1%, respectively [19,20]. Thus, it is obvious that insulin output from the smaller islets cannot quantitatively be dismissed. Our data imply that an alteration in the relative distribution of islet size would be expected to cause significant changes in total insulin output from the pancreas.
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Fig. 4. Immunostaining of the islets. A, Three consecutive sections of the whole pancreas. B, Three consecutive sections of isolated islets. Staining with anti-GCK (left panel), anti-GLUT2 (middle panel) and anti-PDX-1 (right panel) antibodies, and smaller islets in the upper panels and the larger ones in the lower panels both for Fig. 4A and 4B. There was no significant difference in the staining of the 3 proteins based on islet size.
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One may argue that smaller islets are more susceptible to non-specific damage during collagenase dispersion than larger ones, so that reduced b cell glucose sensitivity in the smaller islets is an artifact. However, we think that is unlikely for the following reasons: 1) insulin release by the small islets in response to high glucose is nicely biphasic as in the larger ones, 2) insulin release per content was similar irrespective of islet size, and 3) variability of insulin release was not necessarily large in the small islets compared to larger islets. Nevertheless, damage of the islets due to collagenase dispersion, if any, which is inherent in experiments with isolated islets cannot completely be ruled out. Regarding reasons for lower b cell glucose sensitivity in the smaller islets, we first considered a possible difference in the regulation of cellular Ca21 by glucose. However, this was ruled out because glucose stimulation of insulin release was normally dependent on Ca21 influx through the L-type VDCC [21], and there was no significant difference in the regulation of [Ca21]i by glucose in the b cells based on the islet size. Next, a possible difference in the ratio of a cells to b cells based on islet size was explored because a minor functional difference was found between dorsal and ventral islets which was speculated to be due to difference in glucagon content [7]. However, we found no significant difference in the glucagon to insulin ratio in the islet extract based on islet size. We also examined the expression of several key proteins in the islet b cell, GLUT2, GCK, and PDX-1. The former 2 are critically involved in regulation of the rate of glucose metabolism in the islet b cell [1], and the latter is important for the maintenance of b cell mass in the islets and the insulin gene transcription [22–24]. A 50% reduction of GCK in the b cell significantly suppresses b cell glucose sensitivity [25]. Thus, a quantitative, not necessarily qualitative, abnormality of GCK causes impaired b cell glucose sensitivity. Contrary to our expectation, however, there was no significant difference in the expression of these molecules based on the islet size. Putative difference (both qualitative and quantitative) in the intra-islet b cell biosociology [26–28] might be the reason for the b cells in the larger islets being more glucose sensitive. In the past, size-related and size-unrelated functional heterogeneity of pancreatic islets was briefly mentioned in 2 studies [17,18]. However, in both studies, insulin release in response to a step-up of glucose concentration, from the basal to the maximum, was tested in incubation experiments so that heterogeneity in the glucose sensitivity and the temporal profile was not known. Because we obtained islets from the entire pancreas, at least a part of size-unrelated functional heterogeneity may be due to the head to tail and/or the dorsal to ventral differences in the islet cellular composition [7,29]. In the past, insulin release by freshly isolated single pancreatic islets was examined in 4 studies [30–33] using normal animals [30,31] and ob/ob mice [32,33]. In all other studies where insulin release by single islets was studied, islets cultured for a day or longer were employed [34–41]. Under such condition, central necrosis of the islet is unavoidable, especially in the larger ones. In any case, functional heterogeneity of the islets has not previously been systematically investigated. In conclusion, there are size-related and size-unrelated functional heterogeneity among pancreatic islets. The finding opens a new perspective on the kinetics of insulin secretion in health and disease.
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20. Hellman B. Actual distribution of the number and volume of the islets of Langerhans in different size classes in non-diabetic humans of varying ages. Nature 1969;184:1498–9. 21. Komatsu M, Yokokawa N, Takeda T, Nagasawa Y, Aizawa T, Yamada T. Pharmacological characterization of the voltage-dependent calcium channel of pancreatic B-cell. Endocrinology 1989;125:2008–14. 22. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H. b-cell specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the b-cell phenotype and maturity onset diabetes. Genes and Development 1998;12:1763–8. 23. Ohlsson H, Karlsson K, Edlund T. IPF1, a homeodomein-containing transactivator of the insulin gene. EMBO Journal 1993;12:4251–9. 24. Stoffers DA, Ferrer J, Clarke WL, Habener JF. Early-onset type II diabetes mellitus (MODY4) linked to IPF1. Nature Genetics 1997;17:138–9. 25. Aizawa T, Asanuma N, Terauchi Y., Suzuki N, Komatsu M, Itoh N, Nakabayashi T, Hidaka H, Ohnota H, Yamauchi K, Yasuda K, Yazaki Y, Kadowaki T, Hashizume K. Analysis of the pancreatic b cell in the mouse with targeted disruption of the pancreatic b cell-specific glucokinase gene. Biochemical and Biophysical Research Communications 1996;229:460–5. 26. Pipeleers D. The biosociology of pancreatic B cells. Diabetologia 1987;30:277–91. 27. Bosco D, Orci L, Meda P. Homologous but not heterologous contact increases the insulin secretion of individual pancreatic b-cells. Experimental Cell Research 1989;184:72–80. 28. Holz GG, Kuhtreiber WM, Habener JF. Pancreatic b-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1. Nature 1993;361:362–5. 29. Orci L, Baestens L, Ravazzola M, Stefan Y, Malaisse-Lagae F. Pancreatic polypeptide and glucagon: non-random distribution in pancreatic islets. Life Sciences 1976;19:1811–6. 30. Scott AM, Atwater I, Rojas E. A method for the simultaneous measurement of insulin release and B cell membrane potential in single mouse islets of Langerhans. Diabetologia 1981;21:470–5. 31. Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M. Widespread synchronous [Ca21]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Archives European Journal of Physiology 1991;418:417–422 32. Bergsten P, Hellman B. Glucose-induced cycles of insulin release can be resolved into distinct periods of secretory activity. Biochemical and Biophysical Resarch Communications 1993;192:1182–8. 33. Westerlund J, Hellman B, Bergren P. Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca21. Journal of Clinical Investigation 1996;97:1860–3. 34. Rosario LM, Atwater I, Scott AM. Pulsatile insulin release and electrical activity from single ob/ob mouse islets of Langerhans. Advances in Experimental and Medical Biology 1986;211:413–425 35. Martin F, Sanches-Andres JV, Soria B. Slow [Ca21]i oscillations induced by ketoisocaproate in single mouse pancreatic islets. Diabetes 1995;44:300–5. 36. Marchetti P, Scharp DW, Mclear M, Gingerich R, Finke E, Olack B, Swanson C, Gianarelli R, Navalesi R, Lacy PE. Pulsatile insulin secretion from isolated human pancreatic islets. Diabetes 1994;43:827–30. 37. Gilon P, Shepherd RM, Henquin J-C. Oscillations of secretion driven by oscillations of cytoplasmic Ca21, as evidenced in single pancreatic islets. Journal of Biological Chemistry 1993;268:22265–8. 38. Bergsten P, Grapengieser E, Gylfe E, Tengholm A, Hellman B. Synchronous oscillations of cytoplasmic Ca21 and insulin release in glucose-stimulated pancreatic islets. Journal of Biological Chemistry 1994;269:8749–53. 39. Bergsten P. Slow and fast oscillations of cytoplasmic Ca21 in pancreatic islets correspond to pulsatile insulin release. American Journal of Physiology 1995;268:E282–7. 40. Westerlund J, Gylfe E, Bergren P. Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca21. Journal of Clinical Investigation 1997;100:2547–51. 41. Zaitsev S, Efendic S, Arkhammar P, Bertorello AM, Bergren P-O. Dissociation between changes in cytoplasmic free Ca21 and insulin secretion as evidenced from measurements in mouse single pancreatic islets. Proceedings of the National Academy of Sciences, USA 1995;92:9712–6.
Size-related and size-unrelated functional heterogeneity among pancreatic islets Toru Aizawaa,*, Tsuyoshi Kanekoa, Keishi Yamauchia, Hiroki Yajimaa, Tomoko Nishizawab, Toshihiko Yadac, Hiroshi Matsukawad, Minoru Nagaia, Satoko Yamadaa, Yoshihiko Satoa, Mitsuhisa Komatsua, Nobuo Itohb, Hiroya Hidakae, Yoshitaka Kajimotof, Kiyoshi Hashizumea a
Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto, Japan b Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan c Department of Physiology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi, Japan d Department of Physiology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan e Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan f Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan Received 12 September 2000; accepted 11 May 2001
Abstract Functional heterogeneity of pancreatic islets was systematically analyzed for the first time using freshly isolated single rat pancreatic islets. First, 60 islets were sequentially exposed to 3, 9.4, 15.6, and 24.1 mM glucose for 30 min each in incubation experiments: 36 (60%) responded in a concentrationdependent and 19 (32%) in an all-or-none manner, and 5 (8%) islets did not respond to high glucose. As a group, the larger the islet, the higher the b cell glucose sensitivity. However, glucose-stimulated elevation of [Ca21]i in the b cell, insulin/glucagon ratio in the islet, and expression of glucose transporter 2, glucokinase, and pancreatic duodenal homeobox factor-1 in the b cell were not significantly related to islet size. Second, 50 islets were stimulated with 16.7 mM glucose in perifusion. A biphasic insulin release was found in 39 (78%), and no or little first phase response in 11 (22%) islets, irrespective of the islet size. Nevertheless, when the response was plotted as a group, it was clearly biphasic. Islet size, insulin content and the amount of insulin release were positively correlated with each other. In conclusion, there are size-related and size-unrelated functional diversity among pancreatic islets. The reason for such heterogeneity remained to be determined. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Pancreatic b cell; Insulin; Glucose sensitivity; Islet architecture
* Corresponding author. Department of Geriatrics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. Tel.: 181-263-37-2686; fax: 181-263-37-2710. E-mail address: [email protected] (T. Aizawa) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 3 2 -7
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Introduction A high concentration of glucose elicits a concentration-dependent, biphasic insulin secretion from the pancreas, and impairment of insulin secretion in patients with diabetes is characterized by reduced b cell glucose sensitivity and disappearance or diminution of the brisk, initial response to glucose [1,2]. These salient features of insulin secretion in health and disease have been interpreted in relation to the physiology and pathophysiology of stimulussecretion coupling at the level of b cell [1,2]. An assumption here is that the kinetics of insulin output from the pancreas faithfully reflect those from individual pancreatic islets and/or individual b cells. However, evidence supporting this assumption does not exist. There are more than several thousand islets in rat pancreas, each islet contains approximately several to ten thousand b cells, and functional heterogeneity at the level of individual b cells has long been recognized [3,4]. Therefore, functional heterogeneity among individual islets is not unexpected. As appropriately discussed for individual b cells [5], even if each islet responds to high glucose in an all-or-none manner, concentration-dependent insulin output from the pancreas would be observed if there is a Gaussian distribution of the threshold for glucose among individual islets. Since islets are the smallest units of coordinated insulin secretion [6], data on the function of individual islets have been expanding. But functional homogeneity among pancreatic islets has never been seriously questioned except in 1 study in which groups of ventral and dorsal islets were compared [7]. Based on these considerations, we systematically analyzed insulin release by individual islets, and found significant functional heterogeneity among them. Methods Pancreatic islets were obtained from adult male Wistar rats by collagenase dispersion, and insulin release was measured in incubation and perifusion experiments at 378C as reported [8–11]. Krebs-Ringer bicarbonate (KRB) buffer supplemented with 0.1% bovine serum albumin (BSA) containing (in mM) 118.4 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.9 CaCl2, and 25 NaHCO3 (equilibrated with 5% CO2-95% O2, pH 7.4) was used for isolation of the islets and the KRB buffer containing 0.2% BSA was used in experiments for determination of insulin release as reported [8–11]. KRB buffer used for measurement of cytosolic free Ca21 concentration ([Ca21]i) was composed of (in mM) 121.7 NaCl, 4.4 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.0 CaCl2, 5 NaHCO3, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) at pH 7.4 with NaOH supplemented with 0.1% BSA as reported [12,13]. Size of the islets was individually determined under an operating microscope immediately before the experiments. In this study, beautifully isolated, round islets with minimum exocrine tissue were selected. Small, medium, and large islets were defined as the diameter being ,200 mm, $200 and ,300 mm, and $300 mm, respectively. In static incubation experiments, 1 islet/tube was first incubated in KRB buffer containing 3 mM glucose for 30 min (preincubation). At the end of preincubation, the buffer was aspirated and 200 ml fresh KRB buffer containing 3 mM glucose was introduced for the 1st experimental incubation. Thirty minutes later, 100 ml of the buffer was removed for insulin assay, 100 ml fresh KRB buffer containing 15 mM glucose was added, and the incubation was carried out for another 30 min (2nd experimental incubation). The same procedure was
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repeated for 2 more times using KRB buffer with 20 mM and 35 mM glucose, successively, as a refill. At the end of the 4th experimental incubation, buffer was simply aspirated for insulin assay. Thus, insulin concentration in the buffer obtained at the end of the 4 successive experimental incubations was determined, and the release in response to each concentration of glucose during the respective 30 min-incubations was calculated. Glucose concentration of each media sample was later directly determined by the glucose oxidase method and the mean values were 3, 9.4, 15.6, and 24.1 mM in the buffer used for the 1st, 2nd, 3rd and 4th experimental incubations, respectively. The perifusion system used in the present study was a modification of the previously described multiple parallel perifusion system [8–11]. The modification is primarily a down-sizing, so that insulin release from a single islet is reliably determined. After a 30 min-prewash with KRB buffer containing 3 mM glucose, glucose concentration was increased to 16.7 mM. Perifusate was collected at 1, 2 or 5 min intervals, as needed, and stored at 2208C. The lag period, from switching to the high glucose buffer to its appearance in the collection tube, was approximately 2.5 min, which was not corrected for the data presentation. In rat islets, a glucose-induced elevation of [Ca21]i cannot be consistently demonstrated. Therefore, measurement of [Ca21]i was made in single b cells by dual wavelength fura-2 microfluorometry combined with digital imaging [12,13]. The cells were loaded with fura-2 by incubation with 1 mM fura-2 acetoxymethylester (Molecular Probes, Eugene, OR) for 30 min at 378C; the coverslip (with the cells on it) was then mounted in a chamber and superfused with KRB buffer. After a 30 min-prewash with KRB buffer containing 3 mM glucose, glucose in the buffer was increased to 16.7 mM. b cells were identified based on the larger diameter (12.5–17.5 mm) and the Ca21 response to 300 mM tolbutamide, a sulfonylurea compound. b cells from the large and small islets (as defined above) were separately examined. The islet insulin and glucagon content were determined after extraction with 0.15 N HCl/70% ethanol [14]. Western blotting of glucokinase (GCK) and glucose transporter 2 (GLUT2) was performed using lysates of the islets as described [15]. In brief, immediately after the isolation, the islets were hand-picked into ice-cold BSA-free KRB buffer. Then, small and large islets were selectively pooled and lysed in the solution containing (in mM) 20 Hepes (pH 7.4), 2 EDTA, 2 EGTA, and 1 phenylmethylsulfonyl fluoride, with 1% Triton X-100, 0.1% SDS, 10 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin A. Usually, 300 small islets were lysed in 300 ml lysis solution and 50 large islets were lysed in 250 ml lysis solution. The lysates (protein-adjusted) were subjected to SDS-PAGE, and immunoblotted with the antibodies. For the morphological study, the whole rat pancreas and the isolated islets were fixed with 10% formaldehyde, and 4 mm-thickness serial sections were made from the paraffin-embedded tissue. Sections (1 out of every 20 consecutive sections) were initially stained with hematoxylin and eosin (H-E) to ascertain the diameter of the islets. After identification of large and small islets, immunostaining with anti-GCK, -GLUT2 and -pancreatic duodenal homeobox factor-1 (PDX-1/IDX-1/SPF-1/IPF-1) antibodies was performed using selected sections. Insulin and glucagon were determined by radioimmunoassay using commercially available kits (Linco Research Inc., St. Louis, MO, for insulin and Daiichi Radioisotope Laboratories, Tokyo, for glucagon). The minimum detectable amount of insulin was 1.0 pg/tube. Antibodies against GLUT2 and GCK were obtained from Transformation Research Inc. (Framingham, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA) respectively. The antibody for
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Fig. 1. Concentration-dependency of glucose-induced insulin release. A, Individual data. [Insulin release with the highest concentration (24.1 mM) of glucose]-[insulin release with basal (3 mM) glucose] was calculated for each islet and taken as 100%. Responses to the intermediate concentrations of glucose were plotted as % values. Minus values are due to assay variation (see Text for the experimental procedure). Upper panel, large islets; middle panel, medium islets; lower panel, small islets. The data from 4 small islets and 1 medium islet, which did not show a significant response to high glucose (,20% of the basal), was omitted. B, Grouped data. All data from large, medium, and small islets were plotted as a group. Values are mean6SEM.
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Fig. 1 (continued).
PDX-1 was generated as described [16]. Statistical analysis was performed by one way ANOVA with Fisher’s protected least significance difference test and Wilcoxon’s signed rank test (SAS Institute Inc., Cary, NC). P,0.05 was considered significant. Results Analysis of insulin release in static incubation experiments Sixty islets (23 small, 22 medium and 15 large) were individually stimulated with a sequential exposure (30 min each) to 3, 9.4, 15.6 and 24.1 mM glucose. Thirty-six (60%) responded to high glucose in a concentration-dependent manner, and 19 (32%) responded in all-or-none fashion (Fig. 1A). Five islets (8%) did not respond to high glucose (,20% increase from the baseline). The response was regarded all-or-none fashion when the following criteria are fulfilled: 1) the greatest response with 9.4 mM glucose, 2) ,10% increase from the baseline with 9.4 mM and the greatest response with 15.6 mM, or 3) ,10% increase from the baseline up to 15.6 mM. When individual data from each islet were summed and treated as a group, typical concentration-dependency curves were obtained irrespective of the islet size (Fig. 1B). b cell glucose sensitivity was highest, intermediate, and lowest in the large, medium and small islets, respectively (Fig. 1B). There was a positive correlation between the islet insulin content and the amount of released insulin, both at the basal and stimulated state (data not shown). In a separate experiment, the insulin content of non-stimulated islets was found to be positively correlated with islet size (data not shown). Despite the fact that b cell glucose sensitivity is lower in the small islets, glucose-stimulated insulin release by the small islets was totally suppressed by 10 mM nifedipine, a blocker of the L-type voltage-dependent Ca21 channels (VDCC) (data not shown), indicating that glucose-
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stimulated insulin release from the b cell in small islets is normally dependent upon Ca21 influx through the L-type VDCC. Insulin release by the single islets in perifusion A total of 50 (25 small, 13 medium, and 12 large) islets were individually stimulated with a square wave application of 16.7 mM glucose. The mean insulin release from each category was clearly biphasic, and there was a positive correlation between islet size and insulin release (Fig. 2A). However, when the values were individually analyzed, there was considerable heterogeneity in the temporal profile (Fig. 2B). To facilitate a qualitative comparison, percent change from the baseline insulin release was calculated for each islet and the response was classified based on the amplitude of the first phase response. The maximum increase during the initial 5 min of glucose stimulation was employed as an index of the first phase response; Group A, B and C were defined as a maximum response .650%, ,650% and .220%, and ,220% respectively (Fig. 2B). Group A thus has a large first phase, Group B a small first phase, and Group C little or no first phase. Thirty-eight percent of islets were in Group A, 40% in Group B, and 22% in Group C. A biphasic response was seen in 39 (78%) islets (Groups A and B combined) (Fig. 2B). [Ca21]i in the b cells [Ca21]i in 112 b cells from the large islets and 87 b cells from the small islets was unselectively determined. As shown in Fig. 3A, the change in [Ca21]i upon exposure to 16.7 mM glucose could be grouped into 4 patterns: a, a transient decrease of [Ca21]i followed by a sharp rise and subsequent regular oscillations; b, an initial dip and sharp rise followed by irregular oscillations; c, an initial dip and sharp rise alone; d, little or no response to glucose. Even in the cells with little or no response to glucose, the [Ca21]i response to tolbutamide was retained (data not shown). The response to high glucose was not significantly different between the b cells from the large and small islets, in terms of incidence of response patterns (Fig. 3B) and the average values (data not shown). In the b cells from large islets, the decrease in initial peak with glucose stimulation was generally associated with the decreased response to depolarizing concentration of K1. This phenomenon was not generally found in the b cells from small islets (Fig. 3A, the bottom panel). Analysis of glucagon to insulin ratio, expression of GCK and GLUT2 in the isolated islets There was no significant difference in glucagon to insulin ratio (pg/ng) based on islet size (30.869.5, 36.764.2, and 36.964.3, in the small, medium and large islets, respectively). Also, by Western blotting, there was no significant difference in the expression of GCK and GLUT2 based on islet size, as indexed by intensity of Western blotting, of GCK and GLUT2 based on islet size (data not shown). Histological studies Immunostaining with anti-GCK, -GLUT2 and -PDX-1 antibodies revealed no significant difference in the staining between the b cells in the large and small islets in situ in the pancreas (Fig. 4A) or after isolation (Fig. 4B).
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Fig. 2. Insulin release elicited by 16.7 mM glucose in perifusion. A, Grouped data from the large, medium, and small islets (number of islets is indicated in the parentheses). Perifusate was switched from KRB buffer with 3 mM glucose to that with 16.7 mM glucose at time 0, after a 30 min prewash with the former solution. Values are mean6SEM. B, Individual data of the experiment shown in Fig. 2A is plotted (number of islets is indicated in the parentheses). Left panel, small islets; Right panel, large and medium islets. Percent change from the baseline is plotted. Classification is based on the initial response, with Group A, B and C being percent maximum change from the baseline during the initial 5 min of switching to high glucose buffer, .650%, ,650% and .220%, and ,220%, respectively (see Text for the details of classification).
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Fig. 3. Cytosolic free Ca21 in isolated b cells. After superfusion with 3 mM glucose for 30 min, glucose concentration was raised to 16.7 mM at time 0 and lowered to 3 mM at 30 min. A 1 min-pulse of 25 mM KCl was applied at 45 min. A, Representative recordings of b cells from large and small islets. Ca21 responses were classified into 4 patterns. Pattern a, a transient decrease of [Ca21]i followed by a sharp rise and subsequent regular oscillations; pattern b, the initial responses followed by irregular oscillations; pattern c, the initial responses alone; pattern d, little or no response to glucose (see Text for the detail). B, Incidence of response patterns.
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Fig. 3. Continued.
Discussion In the present study, we discovered considerable functional diversity of pancreatic islets, some of which were size-related and some size-unrelated. First, many islets (60%) exhibited a concentration-dependent response to high glucose, however, a significant proportion of them (32%) showed an all-or-none response. Nevertheless, when the mean values were plotted, the response was clearly concentration-dependent, and as a group, the larger the islet, the higher the b cell glucose sensitivity. It is also clear that fold increase of basal secretion is less in the small islets especially at the lower glucose concentrations (9.4 and 15.6 mM) (Fig. 1B). In order to obtain the glucose dose-response curve for each individual islet, we employed sequential incubations in increasing glucose concentrations. Similar approach was taken by others in a previous, less comprehensive study [30]. This procedure is known to produce priming phenomenon. Also, only 3 stimulating glucose concentrations were tested. Thus, one should interpret the data with some caution. Second, a high concentration of glucose elicited a biphasic response in 78% of islets, and no or little first phase response in the rest, irrespective of the islet size. Finally, the islet insulin content and insulin release correlated well each other, as reported previously [17,18]. Correlation between the size of islet and insulin release was reported in one [18] but not in the other [17] study. In the pancreas, there are more than 13105 islets, with a considerable size heterogeneity [19,20]. The relative proportion of small (,200 mm), medium ($200 mm and ,300 mm), and large ($300 mm) islets is approximately 90%, 9% and 1%, respectively [19,20]. Thus, it is obvious that insulin output from the smaller islets cannot quantitatively be dismissed. Our data imply that an alteration in the relative distribution of islet size would be expected to cause significant changes in total insulin output from the pancreas.
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Fig. 4. Immunostaining of the islets. A, Three consecutive sections of the whole pancreas. B, Three consecutive sections of isolated islets. Staining with anti-GCK (left panel), anti-GLUT2 (middle panel) and anti-PDX-1 (right panel) antibodies, and smaller islets in the upper panels and the larger ones in the lower panels both for Fig. 4A and 4B. There was no significant difference in the staining of the 3 proteins based on islet size.
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One may argue that smaller islets are more susceptible to non-specific damage during collagenase dispersion than larger ones, so that reduced b cell glucose sensitivity in the smaller islets is an artifact. However, we think that is unlikely for the following reasons: 1) insulin release by the small islets in response to high glucose is nicely biphasic as in the larger ones, 2) insulin release per content was similar irrespective of islet size, and 3) variability of insulin release was not necessarily large in the small islets compared to larger islets. Nevertheless, damage of the islets due to collagenase dispersion, if any, which is inherent in experiments with isolated islets cannot completely be ruled out. Regarding reasons for lower b cell glucose sensitivity in the smaller islets, we first considered a possible difference in the regulation of cellular Ca21 by glucose. However, this was ruled out because glucose stimulation of insulin release was normally dependent on Ca21 influx through the L-type VDCC [21], and there was no significant difference in the regulation of [Ca21]i by glucose in the b cells based on the islet size. Next, a possible difference in the ratio of a cells to b cells based on islet size was explored because a minor functional difference was found between dorsal and ventral islets which was speculated to be due to difference in glucagon content [7]. However, we found no significant difference in the glucagon to insulin ratio in the islet extract based on islet size. We also examined the expression of several key proteins in the islet b cell, GLUT2, GCK, and PDX-1. The former 2 are critically involved in regulation of the rate of glucose metabolism in the islet b cell [1], and the latter is important for the maintenance of b cell mass in the islets and the insulin gene transcription [22–24]. A 50% reduction of GCK in the b cell significantly suppresses b cell glucose sensitivity [25]. Thus, a quantitative, not necessarily qualitative, abnormality of GCK causes impaired b cell glucose sensitivity. Contrary to our expectation, however, there was no significant difference in the expression of these molecules based on the islet size. Putative difference (both qualitative and quantitative) in the intra-islet b cell biosociology [26–28] might be the reason for the b cells in the larger islets being more glucose sensitive. In the past, size-related and size-unrelated functional heterogeneity of pancreatic islets was briefly mentioned in 2 studies [17,18]. However, in both studies, insulin release in response to a step-up of glucose concentration, from the basal to the maximum, was tested in incubation experiments so that heterogeneity in the glucose sensitivity and the temporal profile was not known. Because we obtained islets from the entire pancreas, at least a part of size-unrelated functional heterogeneity may be due to the head to tail and/or the dorsal to ventral differences in the islet cellular composition [7,29]. In the past, insulin release by freshly isolated single pancreatic islets was examined in 4 studies [30–33] using normal animals [30,31] and ob/ob mice [32,33]. In all other studies where insulin release by single islets was studied, islets cultured for a day or longer were employed [34–41]. Under such condition, central necrosis of the islet is unavoidable, especially in the larger ones. In any case, functional heterogeneity of the islets has not previously been systematically investigated. In conclusion, there are size-related and size-unrelated functional heterogeneity among pancreatic islets. The finding opens a new perspective on the kinetics of insulin secretion in health and disease.
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Acknowledgments This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (to T.A.). References 1. Grodsky G. Kinetics of insulin secretion. In: LeRoith D, Taylor SI, Olefsky JM, editors. Diabetes Mellitus. A Fundamental and Clinical Text. Philadelphia: Lippincott-Raven, 1996. pp. 12–20. 2. Polonsky KS. Insulin secretion in humans: Physiologic regulation and alteration in disease states. In: LeRoith D, Taylor SI, Olefsky JM, editors. Diabetes Mellitus. A Fundamental and Clinical Text. Philadelphia: Lippincott-Raven, 1996. pp. 1–11. 3. Dean PM, Matthews EK. Electrical activity in pancreatic islet cells. Nature 1968; 219:389–90. 4. Van Schravendijk CFH, Kiekens R, Pipeleers DG. Pancreatic b cell heterogeneity in glucose-induced insulin secretion. Journal of Biological Chemistry 1992;267:21344–8. 5. Grodsky GM. A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling. Journal of Clinical Investigation 1992;51:2047–59. 6. Atwater IL, Mears D, Rojas E. Electrophysiology of the pancreatic b-cell. In: LeRoith D, Taylor SI, Olefsky JM, editors. Diabetes Mellitus. A Fundamental and Clinical Text. Philadelphia: Lippincott-Raven, 1996. pp. 78–102. 7. Trimble ER, Halban PA, Wollheim CB, Renold AE. Functional differences between rat islets of ventral and dorsal pancreatic origin. Journal of Clinical Investigation 1982;69:405–13. 8. Sato Y, Aizawa T, Komatsu M, Okada N, Yamada T. Dual functional role of membrane depolarization/Ca21 influx in rat pancreatic B-cell. Diabetes 1992;41:438–43. 9. Aizawa T, Sato Y, Ishihara F, Komatsu M, Taguchi N, Hashizume K, Yamada T. ATP-sensitive K1 channelindependent glucose action in rat pancreatic b-cell. American Journal of Physiology 1994;266:C622–7. 10. Taguchi N, Aizawa T, Sato Y, Ishihara F, Hashizume K. Mechanism of glucose-induced biphasic insulin release: physiological role of adenosine triphosphate-sensitive K1 channel-independent glucose action. Endocrinology 1995;136:3942–8. 11. Asanuma N, Aizawa T, Sato Y, Schermerhorn T, Komatsu M, Sharp GWG, Hashizume K. Two signaling pathways from the upper glycolytic flux and from the mitochondria converge to potentiate insulin release. Endocrinology 1997;138:751–755 12. Yada T, Sakurada M, Ishida K, Nakata M, Murata F, Arimura A, Kikuchi M. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet b-cells. Journal of Biological Chemistry 1994;269:1290–3. 13. Yada T, Hamakawa N, Yaekura K. Two distinct modes of Ca21 signalling by ACh in rat pancreatic b-cells: concentration, glucose dependence and Ca21 origin. Journal of Physiology (Lond.) 1995;488:13–24. 14. Ishihara F, Aizawa T, Taguchi N, Sato Y, Hashizume K. Differential metabolic requirement for initiation and augmentation of insulin release by glucose: a study with rat pancreatic islets. Journal of Endocrinology 1994;143:497–503. 15. Yamauchi K, Mirarski KL, Saltiel AR, and Pessin JE. Protein-tyrosine- phosphatase, SHPTP2, is a required positive effector for insulin downstream signaling. Proceedings of the National Academy of Sciences, USA 1995;92:664–8. 16. Watada H, Kajimoto Y, Umayahara Y, Matsuoka T, Kaneto H, Fujitani Y, Kamada T, Kawamori R, Yamasaki K. The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1 (IPF1)/PDX-1 in its activation in HIT-T15 cells. Diabetes 1996;45:1478–88. 17. Steinke J, Patel TN, Ammon HPT. Relationship between glucose- and tolbutamide-induced insulin release and insulin content in single pancreatic rat islets. Metabolism 1972;21:465–70. 18. Wolters GHJ, Konijnendijk W. Relationship between insulin secretion, insulin content and dry weight of single rat pancreatic islets. Acta Endocrinologica 1980;94:365–70. 19. Hellman B. The total volume of the pancreatic islet tissue at different ages of rat. Acta Endocrinologica 1959;32:63–77.
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