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Chick Pancreatic B Islets as an Alternative In Vitro Model for Screening Insulin Secretagogues
Research Notes Chick Pancreatic B Islets as an Alternative In Vitro Model for Screening Insulin Secretagogues S. P. Datar,* D. S. Suryavanshi,† and R. R. Bhonde‡1 *Sir Parshurambhau College, Pune 411 030, Maharashtra, India; †Bombay Veterinary Science College, Parel, Mumbai 400 012, Maharashtra, India; and ‡National Centre for Cell Science, Pune 411 007, Maharashtra, India insulin secretory response of chick B islets against the tolbutamide and glucose challenge was comparable to that of normal mouse islets. However, diabetic mouse islets did not respond to glucose challenge, indicating impaired functionality. We have identified a critical window that lies within 5 to 6 d posthatching for isolating chick B islets showing maximum glucose responsiveness and insulin secretion. The previous reports on chicken pancreatic islets involve the use of 4- to 6-wk-old chicks in which islets were found to be nonresponsive to glucose and, hence, could not be used for testing insulin secretory activity. However, our data on B islets from 5- to 6-d-old chick pancreata is highly promising, as islets are responsive to insulin secretagogues. The present data thus indicates that chick B islets can be used as an alternative in vitro model for screening insulin secretagogue and hypoglycemics.
Key words: chick pancreata, B islet, hypoglycemic screening, insulin secretagogue, in vitro model 2006 Poultry Science 85:2260–2264
INTRODUCTION Diabetes mellitus is a metabolic syndrome that represents complex pathophysiological interactions among hyperglycemia, β cell dysfunction, insulin resistance, dyslipidemia, and endothelial cell dysfunction. Ability of glucose to stimulate insulin secretion is also hampered. To understand the pathophysiology of diabetes, experimental diabetic animals have been routinely used for in vivo studies (Tatarkiewicz et al., 1997; Farzami et al., 2003; Vessal et al., 2003). Isolated islets from different mammals also provide a good source for in vitro studies on insulin secretagogues and screening hypoglycemics (Tsukuda et al., 1998; Bhonde et al., 1999; Paty et al., 2002; Esmaeili and Yazdanparast, 2004). However, isolated islets from other vertebrates have not been tested for their ability to respond to insulin secretagogue and hypoglycemic agents. Recently, we have shown that developing chick
2006 Poultry Science Association Inc. Received May 4, 2006. Accepted July 5, 2006. 1 Corresponding author: [email protected]
embryo in a shell-less culture system could be used to depict glucose-induced malformations similar to those obtained in fetuses and embryos of diabetic mothers (Datar and Bhonde, 2005). In this study, we used chick pancreatic B islets as an alternative to mammalian islets for screening hypoglycemics. Organ culture studies on chick endocrine pancreata have revealed that chick B cells are sensitive to very high concentrations of glucose and are incapable of a sustained increase in insulin release in response to prolonged or repetitive insulinotropic stimuli. The effect of glucose, glucagons, and tolbutamide stimulation of chicken pancreas organ cultures has been studied, and dependence of insulin secretion on extracellular Ca has also been reported (King and Hazelwood, 1976). All these reports suggest that avian and mammalian β cell insulin secretory mechanisms are similar and that birds may be different from mammals in their pattern of insulin release (Naber and Hazelwood, 1977; Foltzer et al., 1982). Isolation of functional A islets from the splenic lobe of the chicken pancreas has been reported to understand the A cell function of avian islets (Ruffier et al., 1998). We previously reported a simple technique for isolation of viable and functional B islets from the dorsal
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ABSTRACT Previously, we reported a simple technique to isolate functional B islets from chick pancreata with retention of their insulin secretory ability in response to glucose challenge. To test the hypothesis that chick B islets are equally good candidates as mammalian islets for screening hypoglycemics and insulin secretagogues, we compared the structural and functional status of chick B islets with those of normal and diabetic mouse islets. Pancreata from chick, normal (nondiabetic) mice, and diabetic mice were collected, fixed, and processed for histological analysis using Gomori stain to distinguish A and B cells from islets. Similarly isolated islets from these animals were treated with different concentrations of tolbutamide, a known insulin secretagogue, and glucose to study insulin release. Histological analysis of pancreata from chicks and normal mice revealed intact B cells, whereas those from diabetic mice were destroyed. The
RESEARCH NOTE 1
Table 1. Record of blood glucose level in chick before and after hatching Chick embryos or hatched chicks 10-d incubation (6) 17-d incubation (5) Newly hatched (4) 2 d after hatching (6) 5 d old (15) 6 d old (10) 8 d old (10) 11 d old (12) 13 d old (10) 15 d old (12)
Blood glucose level (mg/100 mL) 94 115 150 200 250 262 280 295 325 400
± ± ± ± ± ± ± ± ± ±
2.0a 3.1b 2.3c 1.0d 2e 1f 1.5g 1.543h 3.411i 3.833j
lobe of the chick pancreas (Datar and Bhonde, 2006). The present study was undertaken to examine the suitability of chick B islets for screening insulin secretagogues as an alternative model for replacing mammalian islets for hypoglycemic testing.
MATERIALS AND METHODS Animals Four- to six-week-old Balb/c mice of both sexes were obtained from the inbred colony maintained at the Animal House Facility of the National Centre for Cell Science. One-day-old Rhode Island Red chicks of both sexes were obtained from Central Hatchery, Kirkee, Pune, India. These were maintained for 4 to 5 d in cardboard boxes in a room under the 60-W yellow bulb, as per the requirement of warmth to the birds, as indicated by their behavior. Chicks were fed on commercial feed available from Central Hatchery. They were allowed free access to fresh water for 24 h. Water was supplemented with the antimicrobial antibiotic Enrocin (Emil Pharmaceuticals Industries Ltd., Tarapur, Thane, India) and multivitamin drops of Vimeral (Sundar Chemicals Pvt. Ltd., Chennai, India) at the recommended veterinary doses.
Measurement of Blood Glucose Levels in Chicks Chick embryos were cultured using the shell-less chick embryo culture system, developed and reported earlier (Datar and Bhonde, 2005). Blood was collected from the heart and large blood vessels of the developing embryo and the wing vein of the hatched-out chicks with the help of an insulin syringe. Blood glucose level was measured using a blood glucose meter (Accu-Chek Sensor Comfort, Roche Diagnostics Corp., Indianapolis, IN).
Induction of Experimental Diabetes in Mice by Streptozotocin Injection After overnight fasting, mice were injected with freshly prepared streptozotocin (STZ; 200 mg/kg of BW, pre-
pared in a chilled citrate buffer, pH 4.5, Sigma-Aldrich, St. Louis, MO). Diabetic status was confirmed by measurement of blood glucose (Accutrend Sensor Comfort blood glucose meter, Roche Diagnostics Corp.) after 1 wk of STZ injection. Mice with blood glucose levels of 250 to 300 mg/dL were considered frank diabetic and were used for the isolation of islets and the histology of diabetic pancreata (Fernandez et al., 1997).
Isolation of Chick B Islets Islets were isolated from 5- to 6-d-old Rhode Island Red chicks by using protocol described earlier (Datar and Bhonde, 2006). In short, aseptically removed dorsal ventral lobes of pancreata were subjected to sequential digestion. The dissociation medium consisted of Hanks’ Balanced Salt Solution, containing Ca and supplemented with 1.5 mg/mL of Collagenase Type V (Sigma-Aldrich). The digestion was followed by centrifugation at 1,500 rpm for 5 min. After 2 washings, the pellet was seeded in culture flasks (25 cm2, Nunc A/S, Roskilde, Denmark) containing Dulbecco’s modified Eagle’s medium:Ham’s F-12 (1:1) (Gibco Laboratories, Gaithersburg, MD), pH 7.4, supplemented with 10% (vol/vol) fetal calf serum (Gibco Laboratories). Islets were ready after 48 h of incubation at 37°C in a CO2 incubator (Forma Scientific Inc., Marietta, OH).
Isolation of Mouse Islets Pancreatic islets were isolated from normal and diabetic mice by using the protocol of Shewade et al. (1999). In short, aseptically removed pancreata were digested using digestion medium with BSA, soybean trypsin inhibitor, and type IV collagenase. The digestion was followed by centrifugation at 200 × g for 10 min. The vertexed pellet was seeded in the bottles containing RPMI 1640 (Gibco Laboratories) with 10% fetal calf serum. Islets were ready for experimentation after 48 h of incubation at 37°C in a CO2 incubator (Forma Forma Scientific Inc.).
Histology of Pancreata Dorsal lobes of pancreata from 5- to 6-d-old chicks and pancreata from normal and diabetic mice were dissected out and fixed in 10% formal saline. Tissues were then processed for routine paraffin sectioning and Gomori staining for pancreatic A and B cells (Gomori, 1941, 1950).
Assessment of Islet Viability Using Trypan Blue Dye Exclusion Test The viability of the islets was checked by Trypan blue dye exclusion test (Warburton and James, 1995), using 0.4% (wt/vol) Trypan blue (ICN Pharmaceuticals Inc., Costa Mesa, CA). Blue stained islets were scored as nonviable, and the unstained were scored as viable islets.
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a–j Superscripts denote the groups that are significantly different from one another (P < 0.001). 1 Numbers in parentheses indicate the number of chicks used. Values of blood glucose levels are the mean ± SEM. Statistical analysis was carried out by using 1-way ANOVA.
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Figure 1. Transverse sections of pancreas. Chick pancreas showing normal β cells despite high levels of blood glucose (panel A). Normal mouse pancreas showing A and B cells (panel B). Diabetic mouse pancreas showing necrotic B cells due to hyperglycemia (panel C).
Specificity of islets was determined by dithiocarbazone (DTZ) staining (Samual et al., 1994). Dithiocarbazone staining was carried out by adding 10 L of DTZ stock to islets suspended in 1 mL of Krebs-Ringer bicarbonate buffer (pH 7.4) with N-2-hydroxyethylpiperazine-N1-2ethanesulfonic acid (10 mM; HEPES) and incubated at 37°C for 10 to 15 min. The stained islets looked bright red under the inverted microscope (Olympus, Tokyo, Japan). Nonislet tissue remained unstained.
Assessment of Islet Functionality (Insulin Release Assay)
RESULTS Measurement of Blood Glucose Level Table 1 indicates the level of blood glucose in chicks before and after hatching. It was observed that there was a gradual increase in blood glucose level from embryonic chick to 15 d after hatching.
Histology of Pancreata Chick pancreata showed nonnecrotic and healthy β cells in B islets, despite maintaining elevated blood sugar levels (250 to 300 mg/dL) comparable to that of a diabetic mammal (Figure 1, panel A). Nondiabetic mouse pancreata showed normal histological structure, depicting islets containing both A cells (20%) and B cells (80%;
Triplicate groups of 150 to 200 islets each were placed in a single well of a 24-well plate (Nunc A/S) each containing 1 mL of Krebs-Ringer bicarbonate HEPES (10 mM) buffer (pH = 7.4), 0.1% BSA or 0.59% BSA (mouse or chick islets, respectively), and basal glucose concentrations of 5.5 or 16 mM (mouse or chick islets, respectively). The plates were then incubated at 37°C in a CO2 incubator for 1 h. The supernatant was collected and stored at −20°C. These islets were then challenged with Krebs-Ringer bicarbonate HEPES (10 mM) buffer (pH = 7.4) containing elevated glucose (16 mM for mouse islets and 42 mM for chick islets) and 2 concentrations of tolbutamide (3.1 and 12.2 mg/dL) for chick islets. Islets were then incubated for a further period of 1 h. The supernatants were collected and stored at −20°C. The insulin concentrations of all the stored samples were determined by enzyme amplified sensitivity immunoassay using BioSource INSEASIA (BioSource Europe SA, Nivelles, Belgium) performed on microtiter plates.
Statistical Analysis Results are expressed as mean ± SEM. The statistical significance of differences among groups was analyzed using 1-way ANOVA.
Figure 2. Isolated B islets of 5- to 6-d-old chicks stained with diathizone (100×).
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Assessment of Islet Specificity Using Diphenylthiocarbazone Staining
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Table 2. Insulin secretory response of islets to glucose challenge (expressed as mIU/mL)
Stimulation
Normal mouse islets
Diabetic mouse islets
Chick islets
Basal glucose Elevated glucose
49.310 ± 0.35b 99.321 ± 0.23d
22.25 ± 0.20a 21.90 ± 0.30a
80.123 ± 0.40c 162.30 ± 0.30e
Serial no. 1 2
Superscripts denote the groups that are significantly different from one another (P ≤ 0.001). Basal glucose concentrations were 5.5 mM for mouse and 16 mM for chick islets. Stimulated glucose concentrations were 16 mM for mouse and 42 mM for chick islets. Quantitative analysis of insulin release was performed by ELISA. Data are represented as mean ± SEM. Statistical analysis was carried out using 1-way ANOVA. a–e 1
Viability and DTZ Specificity of Islets Nondiabetic mouse, diabetic mouse, and normal chick pancreatic islets showed 85 to 90% viability after 48 h of incubation. Nondiabetic mouse, diabetic mouse, and chick (Figure 2) pancreatic islets were stained brick red with DTZ, thus confirming their identity as B islets.
Insulin Release Assay Table 2 shows the functionality of islets in terms of insulin secretion, and Table 3 shows the responsiveness of chick islets to tolbutamide in terms of stimulation index. It was observed that there was a 2-fold increase in insulin secretion on stimulation with glucose by islets of nondiabetic mice as well as chicks, whereas diabetic mouse islets did not respond to glucose stimulation. Nondiabetic mouse islets and chick islets also responded to tolbutamide stimulation, indicating similarity in behavior.
DISCUSSION In the present investigation, we have shown that the chicken pancreatic B islets retain structural integrity and functionality, despite the high blood glucose level maintained physiologically. Unlike diabetic mouse pancreata, which showed necrotic B cells, chick pancreatic β cells were structurally intact. Because the chicken pancreas contains 2 types of islets (A and B), we isolated islets from the dorsal and ventral lobes of the chicken pancreas to obtain predominantly insulin-producing B islets (Weir et al., 1976).
It is evident from the literature that aves maintain an elevated level of glucose in the blood. A measurement of blood glucose level from embryonic chick to 15 d after hatching made in the present study (Table 1) revealed that the increase in the blood glucose level was gradual. This is in agreement with the previous reports indicating the presence of small amounts of insulin with rapid buildup in insulin biosynthesis and stores from d 13 of incubation (Swenne and Lundqvist, 1980). Marked increase in the responsiveness to elevated glucose concentrations in the form of increased insulin secretion from 12- to 16-d-old chick embryos and newly hatched chicks has been reported (Leibson et al., 1976). Insulin secretory activity has been demonstrated to be maximum from embryonic d 15 to 9 d posthatching in organ culture studies of the chick pancreas (Foltzer et al., 1982). These reports and our glucose measurement data depicting an age-dependent hike in blood glucose level enabled us to identify a critical window for isolating chick B islets from appropriate age of chick. We found that the critical window for isolation of chick B islets showing maximum glucose responsiveness and insulin secretion lies within 5 to 6 d after hatching. Hence, we selected 5- to 6-d-old chicks for isolation of pancreatic B islets and insulin secretion studies. Increases in plasma insulin concentrations up to 7 d posthatching and high sensitivity to insulin during this age have been demonstrated (Tokushima et al., 2003), which also supports our choice of the age of chick for islet isolation. Moreover, blood glucose levels of diabetic mice and 5- to 6-d-old chicks are comparable, making chick B islets of this age more appropriate to study their suitability for screening insulin secretagogue. In addition, our present data on the similarity of nondiabetic mouse islets and chick islets to tolbutamide stimulation further strengthens age selection for islet study.
Table 3. Responsiveness of islets to insulin secretagogue in terms of stimulation index1 Serial no. 1 2
Tolbutamide stimulation (mg/dL)
Normal mouse islets
Diabetic mouse islets
Chick islets
3.1 12.2
1.75 ± 0.01b 1.89 ± 0.01d
0.87 ± 0.02a 0.90 ± 0.02a
1.83 ± 0.01c 1.98 ± 0.01e
Superscripts denote the groups that are significantly different from one another (P ≤ 0.001). Stimulation index = a ratio of the amount of insulin produced on tolbutamide stimulation to the amount of insulin produced at basal glucose concentrations. Statistical analysis was carried out by using 1-way ANOVA. a–e 1
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Figure 1, panel B). In addition, the histological structure of the pancreata of diabetic mice showed necrotic B cells due to STZ treatment (Figure 1, panel C).
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ACKNOWLEDGMENTS We thank the director of the National Centre for Cell Science for providing facilities to carry out the present work. The financial support provided by the University Grants Commission to S. P. Datur is also acknowledged. The technical help provided by Malati Umrani, technician, National Centre for Cell Science, is highly appreciated.
REFERENCES Bhonde, R. R., P. B. Parab, and V. S. Sheorin. 1999. An in vitro model for screening oral hypoglycemics. In Vitro Cell. Dev. Biol. Anim. 35:366–368. Datar, S. P., and R. R. Bhonde. 2005. Shell-less chick embryo culture as an alternative in vitro model to investigate glucoseinduced malformations in mammalian embryos. Rev. Diabet. Stud. 2:221–227.
Datar, S. P., and R. R. Bhonde. 2006. A simple technique for isolation of functional B islets from chick pancreas. J. Cell Tissue Res. 6:605–608. Esmaeili, M. A., and R. Yazdanparast. 2004. Hypoglycaemic effect of Teucrium polium: Studies with rat pancreatic islets. J. Ethnopharmacol. 95:27–30. Farzami, B., D. Ahmadvand, S. Vardasbi, F. J. Majin, and S. H. Khaghani. 2003. Induction of insulin secretion by a component of Urtica dioica leave extract in perifused islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats. J. Ethnopharmacol. 89:47–53. Fernandez, A., L. C. King, Y. Guz, R. Stein, C. V. Wright, and G. Teitman. 1997. Differentiation of new insulin producing cells is induced by injury in adult pancreatic islets. Endocrinology 138:1750–1762. Foltzer, C., K. Haffen, M. Kedinger, and P. Mialhe. 1982. Stimulation of insulin and glucagon secretion in organ culture of chick endocrine pancreas during embryonic life and after hatching. Gen. Comp. Endocrinol. 47:213–220. Gomori, G. L. 1941. Gomori’s chromium haemotoxylin phloxine stain. Am. J. Pathol. 17:398–399. Gomori, G. L. 1950. Gomori’s aldehyde fuschin stain (special stain for β cells). Am. J. Clin. Pathol. 20:665–666. King, D. L., and R. I. Hazelwood. 1976. Regulation of avian insulin secretion by isolated perfused chicken pancreas. Am. J. Physiol. 231:1830–1839. Leibson, L., V. Bondareva, and L. Soltitshza. 1976. The secretion and the role of insulin in chick embryos and chickens. Pages 69–79 in The Evolution of Pancreatic Islets. T. A. I. Grillo, L. Leibson, A. Epple, ed. Pergamon, Oxford, UK. Naber, S. P., and R. L. Hazelwood. 1977. In vitro insulin release from chicken pancreas. Gen. Comp. Endocrinol. 32:495–504. Paty, B. W., J. S. Harmon, C. L. Marsh, and R. P. Robertson. 2002. Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets. Transplantation 73:353–357. Ruffier, L., J. Simon, and N. Rideau. 1998. Isolation of functional glucagon islets of Langerhans from the chicken pancreas. Gen. Comp. Endocrinol. 112:153–162. Samual, A. C., M. B. Kermit, S. D. Sandra, C. R. Thelma, and L. L. William. 1994. Staining and in vitro toxicity of dithiozone with canine porcine and bovine islets. Cell Transplant. 3:299–306. Shewade, Y. M., M. Umrani, and R. R. Bhonde. 1999. Large-scale isolation of islets by tissue culture of adult mouse pancreas. Transplant. Proc. 31:1721–1723. Swenne, I., and G. Lundqvist. 1980. Islet structure and pancreatic hormone content of the developing chick embryo. Gen. Comp. Endocrinol. 4:190–198. Tatarkiewicz, K., E. Sitarek, M. Sabat, and T. Orlowski. 1997. Reversal of hyperglycemia in streptozotocin diabetic mice by xenotransplantation of microencapsulated rat islets. Ann. Transplant. 2:20–30. Tokushima, Y., B. Sulistiyanto, K. Takahashi, and Y. Akiba. 2003. Insulin-glucose interactions characterised in newly hatched broiler chicks. Br. Poult. Sci. 44:746–751. Tsukuda, K., M. Sakurada, I. Niki, Y. Oka, and M. Kikuchi. 1998. Insulin secretion from isolated rat islets induced by the novel hypoglycemic agent A-4166, a derivative of Dphenylalanine. Horm. Metab. Res. 30:42–49. Vessal, M., M. Hemmati, and M. Vasei. 2003. Antidiabetic effects of quercetin in streptozocin-induced diabetic rats. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 3:357–364. Warburton, S., and R. James. 1995. Hemocytometer cell counts and viability studies. Pages 1.1–1.5 in Cell and Tissue Culture: Laboratory Procedures. A. Doyle, J. B. Griffiths, and D. G. Newell. John Wiley & Sons, New York. Weir, G. C., P. C. Goltos, E. P. Steinberg, and Y. C. Patel. 1976. High concentration of somatostatin immunoreactivity in chicken pancreas. Diabetologia 12:129–132.
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Our present results further demonstrate that pure B islets could be isolated from dorsal and ventral lobes of 5- to 6-d-old chick pancreata and maintained in culture. The islet yield also is higher when the 7,000 islets per dorsal lobe of the 5- to 6-d-old chick pancreas are compared with the 2,500 islets per mouse pancreas (Shewade et al., 1999; Datar and Bhonde, 2006). Because these isolated islets are insulin secretory, viable, and responsive to insulin secretagogues like glucose and tolbutamide, they represent an attractive alternative model for screening hypoglycemics. Employment of chick islets for this purpose will also minimize the use of mammals for experimentation, thus supporting the “3R” principle of reduction, refinement, and replacement of laboratory animals. In the present investigation, we have demonstrated for the first time that isolated pancreatic B islets from the chick are responsive to insulin secretagogues like glucose and tolbutamide. The previous reports on chicken pancreatic islets used 4- to 6-wk-old chicks in which islets were found to be nonresponsive to glucose, making them unsuitable for testing insulin secretagogue activity. On this background, our data on B islets from 5- to 6-d-old chick pancreata is highly promising, because islets are responsive to insulin secretagogues. With the inclusion of biotechnology courses for graduate and postgraduate students and in the light of present restrictions on the usage of mammals for experimentation as well as the expenditure on their maintenance, our investigations on the suitability of chick islets for screening hypoglycemic agents would offer a simple, economical, and yet effective system. The present study represents a first attempt to evaluate isolated chick B islets from 5- to 6-d-old chicks for screening insulin secretagogues. It is interesting to note that although chick islets remain in a hyperglycemic environment comparable to a diabetic mouse, they remain intact and retain functional β cell mass as revealed by their insulin secretory activity and, hence, seem to be more appropriate for secretagogues studies.
Key words: chick pancreata, B islet, hypoglycemic screening, insulin secretagogue, in vitro model 2006 Poultry Science 85:2260–2264
INTRODUCTION Diabetes mellitus is a metabolic syndrome that represents complex pathophysiological interactions among hyperglycemia, β cell dysfunction, insulin resistance, dyslipidemia, and endothelial cell dysfunction. Ability of glucose to stimulate insulin secretion is also hampered. To understand the pathophysiology of diabetes, experimental diabetic animals have been routinely used for in vivo studies (Tatarkiewicz et al., 1997; Farzami et al., 2003; Vessal et al., 2003). Isolated islets from different mammals also provide a good source for in vitro studies on insulin secretagogues and screening hypoglycemics (Tsukuda et al., 1998; Bhonde et al., 1999; Paty et al., 2002; Esmaeili and Yazdanparast, 2004). However, isolated islets from other vertebrates have not been tested for their ability to respond to insulin secretagogue and hypoglycemic agents. Recently, we have shown that developing chick
2006 Poultry Science Association Inc. Received May 4, 2006. Accepted July 5, 2006. 1 Corresponding author: [email protected]
embryo in a shell-less culture system could be used to depict glucose-induced malformations similar to those obtained in fetuses and embryos of diabetic mothers (Datar and Bhonde, 2005). In this study, we used chick pancreatic B islets as an alternative to mammalian islets for screening hypoglycemics. Organ culture studies on chick endocrine pancreata have revealed that chick B cells are sensitive to very high concentrations of glucose and are incapable of a sustained increase in insulin release in response to prolonged or repetitive insulinotropic stimuli. The effect of glucose, glucagons, and tolbutamide stimulation of chicken pancreas organ cultures has been studied, and dependence of insulin secretion on extracellular Ca has also been reported (King and Hazelwood, 1976). All these reports suggest that avian and mammalian β cell insulin secretory mechanisms are similar and that birds may be different from mammals in their pattern of insulin release (Naber and Hazelwood, 1977; Foltzer et al., 1982). Isolation of functional A islets from the splenic lobe of the chicken pancreas has been reported to understand the A cell function of avian islets (Ruffier et al., 1998). We previously reported a simple technique for isolation of viable and functional B islets from the dorsal
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ABSTRACT Previously, we reported a simple technique to isolate functional B islets from chick pancreata with retention of their insulin secretory ability in response to glucose challenge. To test the hypothesis that chick B islets are equally good candidates as mammalian islets for screening hypoglycemics and insulin secretagogues, we compared the structural and functional status of chick B islets with those of normal and diabetic mouse islets. Pancreata from chick, normal (nondiabetic) mice, and diabetic mice were collected, fixed, and processed for histological analysis using Gomori stain to distinguish A and B cells from islets. Similarly isolated islets from these animals were treated with different concentrations of tolbutamide, a known insulin secretagogue, and glucose to study insulin release. Histological analysis of pancreata from chicks and normal mice revealed intact B cells, whereas those from diabetic mice were destroyed. The
RESEARCH NOTE 1
Table 1. Record of blood glucose level in chick before and after hatching Chick embryos or hatched chicks 10-d incubation (6) 17-d incubation (5) Newly hatched (4) 2 d after hatching (6) 5 d old (15) 6 d old (10) 8 d old (10) 11 d old (12) 13 d old (10) 15 d old (12)
Blood glucose level (mg/100 mL) 94 115 150 200 250 262 280 295 325 400
± ± ± ± ± ± ± ± ± ±
2.0a 3.1b 2.3c 1.0d 2e 1f 1.5g 1.543h 3.411i 3.833j
lobe of the chick pancreas (Datar and Bhonde, 2006). The present study was undertaken to examine the suitability of chick B islets for screening insulin secretagogues as an alternative model for replacing mammalian islets for hypoglycemic testing.
MATERIALS AND METHODS Animals Four- to six-week-old Balb/c mice of both sexes were obtained from the inbred colony maintained at the Animal House Facility of the National Centre for Cell Science. One-day-old Rhode Island Red chicks of both sexes were obtained from Central Hatchery, Kirkee, Pune, India. These were maintained for 4 to 5 d in cardboard boxes in a room under the 60-W yellow bulb, as per the requirement of warmth to the birds, as indicated by their behavior. Chicks were fed on commercial feed available from Central Hatchery. They were allowed free access to fresh water for 24 h. Water was supplemented with the antimicrobial antibiotic Enrocin (Emil Pharmaceuticals Industries Ltd., Tarapur, Thane, India) and multivitamin drops of Vimeral (Sundar Chemicals Pvt. Ltd., Chennai, India) at the recommended veterinary doses.
Measurement of Blood Glucose Levels in Chicks Chick embryos were cultured using the shell-less chick embryo culture system, developed and reported earlier (Datar and Bhonde, 2005). Blood was collected from the heart and large blood vessels of the developing embryo and the wing vein of the hatched-out chicks with the help of an insulin syringe. Blood glucose level was measured using a blood glucose meter (Accu-Chek Sensor Comfort, Roche Diagnostics Corp., Indianapolis, IN).
Induction of Experimental Diabetes in Mice by Streptozotocin Injection After overnight fasting, mice were injected with freshly prepared streptozotocin (STZ; 200 mg/kg of BW, pre-
pared in a chilled citrate buffer, pH 4.5, Sigma-Aldrich, St. Louis, MO). Diabetic status was confirmed by measurement of blood glucose (Accutrend Sensor Comfort blood glucose meter, Roche Diagnostics Corp.) after 1 wk of STZ injection. Mice with blood glucose levels of 250 to 300 mg/dL were considered frank diabetic and were used for the isolation of islets and the histology of diabetic pancreata (Fernandez et al., 1997).
Isolation of Chick B Islets Islets were isolated from 5- to 6-d-old Rhode Island Red chicks by using protocol described earlier (Datar and Bhonde, 2006). In short, aseptically removed dorsal ventral lobes of pancreata were subjected to sequential digestion. The dissociation medium consisted of Hanks’ Balanced Salt Solution, containing Ca and supplemented with 1.5 mg/mL of Collagenase Type V (Sigma-Aldrich). The digestion was followed by centrifugation at 1,500 rpm for 5 min. After 2 washings, the pellet was seeded in culture flasks (25 cm2, Nunc A/S, Roskilde, Denmark) containing Dulbecco’s modified Eagle’s medium:Ham’s F-12 (1:1) (Gibco Laboratories, Gaithersburg, MD), pH 7.4, supplemented with 10% (vol/vol) fetal calf serum (Gibco Laboratories). Islets were ready after 48 h of incubation at 37°C in a CO2 incubator (Forma Scientific Inc., Marietta, OH).
Isolation of Mouse Islets Pancreatic islets were isolated from normal and diabetic mice by using the protocol of Shewade et al. (1999). In short, aseptically removed pancreata were digested using digestion medium with BSA, soybean trypsin inhibitor, and type IV collagenase. The digestion was followed by centrifugation at 200 × g for 10 min. The vertexed pellet was seeded in the bottles containing RPMI 1640 (Gibco Laboratories) with 10% fetal calf serum. Islets were ready for experimentation after 48 h of incubation at 37°C in a CO2 incubator (Forma Forma Scientific Inc.).
Histology of Pancreata Dorsal lobes of pancreata from 5- to 6-d-old chicks and pancreata from normal and diabetic mice were dissected out and fixed in 10% formal saline. Tissues were then processed for routine paraffin sectioning and Gomori staining for pancreatic A and B cells (Gomori, 1941, 1950).
Assessment of Islet Viability Using Trypan Blue Dye Exclusion Test The viability of the islets was checked by Trypan blue dye exclusion test (Warburton and James, 1995), using 0.4% (wt/vol) Trypan blue (ICN Pharmaceuticals Inc., Costa Mesa, CA). Blue stained islets were scored as nonviable, and the unstained were scored as viable islets.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
a–j Superscripts denote the groups that are significantly different from one another (P < 0.001). 1 Numbers in parentheses indicate the number of chicks used. Values of blood glucose levels are the mean ± SEM. Statistical analysis was carried out by using 1-way ANOVA.
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Figure 1. Transverse sections of pancreas. Chick pancreas showing normal β cells despite high levels of blood glucose (panel A). Normal mouse pancreas showing A and B cells (panel B). Diabetic mouse pancreas showing necrotic B cells due to hyperglycemia (panel C).
Specificity of islets was determined by dithiocarbazone (DTZ) staining (Samual et al., 1994). Dithiocarbazone staining was carried out by adding 10 L of DTZ stock to islets suspended in 1 mL of Krebs-Ringer bicarbonate buffer (pH 7.4) with N-2-hydroxyethylpiperazine-N1-2ethanesulfonic acid (10 mM; HEPES) and incubated at 37°C for 10 to 15 min. The stained islets looked bright red under the inverted microscope (Olympus, Tokyo, Japan). Nonislet tissue remained unstained.
Assessment of Islet Functionality (Insulin Release Assay)
RESULTS Measurement of Blood Glucose Level Table 1 indicates the level of blood glucose in chicks before and after hatching. It was observed that there was a gradual increase in blood glucose level from embryonic chick to 15 d after hatching.
Histology of Pancreata Chick pancreata showed nonnecrotic and healthy β cells in B islets, despite maintaining elevated blood sugar levels (250 to 300 mg/dL) comparable to that of a diabetic mammal (Figure 1, panel A). Nondiabetic mouse pancreata showed normal histological structure, depicting islets containing both A cells (20%) and B cells (80%;
Triplicate groups of 150 to 200 islets each were placed in a single well of a 24-well plate (Nunc A/S) each containing 1 mL of Krebs-Ringer bicarbonate HEPES (10 mM) buffer (pH = 7.4), 0.1% BSA or 0.59% BSA (mouse or chick islets, respectively), and basal glucose concentrations of 5.5 or 16 mM (mouse or chick islets, respectively). The plates were then incubated at 37°C in a CO2 incubator for 1 h. The supernatant was collected and stored at −20°C. These islets were then challenged with Krebs-Ringer bicarbonate HEPES (10 mM) buffer (pH = 7.4) containing elevated glucose (16 mM for mouse islets and 42 mM for chick islets) and 2 concentrations of tolbutamide (3.1 and 12.2 mg/dL) for chick islets. Islets were then incubated for a further period of 1 h. The supernatants were collected and stored at −20°C. The insulin concentrations of all the stored samples were determined by enzyme amplified sensitivity immunoassay using BioSource INSEASIA (BioSource Europe SA, Nivelles, Belgium) performed on microtiter plates.
Statistical Analysis Results are expressed as mean ± SEM. The statistical significance of differences among groups was analyzed using 1-way ANOVA.
Figure 2. Isolated B islets of 5- to 6-d-old chicks stained with diathizone (100×).
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Assessment of Islet Specificity Using Diphenylthiocarbazone Staining
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Table 2. Insulin secretory response of islets to glucose challenge (expressed as mIU/mL)
Stimulation
Normal mouse islets
Diabetic mouse islets
Chick islets
Basal glucose Elevated glucose
49.310 ± 0.35b 99.321 ± 0.23d
22.25 ± 0.20a 21.90 ± 0.30a
80.123 ± 0.40c 162.30 ± 0.30e
Serial no. 1 2
Superscripts denote the groups that are significantly different from one another (P ≤ 0.001). Basal glucose concentrations were 5.5 mM for mouse and 16 mM for chick islets. Stimulated glucose concentrations were 16 mM for mouse and 42 mM for chick islets. Quantitative analysis of insulin release was performed by ELISA. Data are represented as mean ± SEM. Statistical analysis was carried out using 1-way ANOVA. a–e 1
Viability and DTZ Specificity of Islets Nondiabetic mouse, diabetic mouse, and normal chick pancreatic islets showed 85 to 90% viability after 48 h of incubation. Nondiabetic mouse, diabetic mouse, and chick (Figure 2) pancreatic islets were stained brick red with DTZ, thus confirming their identity as B islets.
Insulin Release Assay Table 2 shows the functionality of islets in terms of insulin secretion, and Table 3 shows the responsiveness of chick islets to tolbutamide in terms of stimulation index. It was observed that there was a 2-fold increase in insulin secretion on stimulation with glucose by islets of nondiabetic mice as well as chicks, whereas diabetic mouse islets did not respond to glucose stimulation. Nondiabetic mouse islets and chick islets also responded to tolbutamide stimulation, indicating similarity in behavior.
DISCUSSION In the present investigation, we have shown that the chicken pancreatic B islets retain structural integrity and functionality, despite the high blood glucose level maintained physiologically. Unlike diabetic mouse pancreata, which showed necrotic B cells, chick pancreatic β cells were structurally intact. Because the chicken pancreas contains 2 types of islets (A and B), we isolated islets from the dorsal and ventral lobes of the chicken pancreas to obtain predominantly insulin-producing B islets (Weir et al., 1976).
It is evident from the literature that aves maintain an elevated level of glucose in the blood. A measurement of blood glucose level from embryonic chick to 15 d after hatching made in the present study (Table 1) revealed that the increase in the blood glucose level was gradual. This is in agreement with the previous reports indicating the presence of small amounts of insulin with rapid buildup in insulin biosynthesis and stores from d 13 of incubation (Swenne and Lundqvist, 1980). Marked increase in the responsiveness to elevated glucose concentrations in the form of increased insulin secretion from 12- to 16-d-old chick embryos and newly hatched chicks has been reported (Leibson et al., 1976). Insulin secretory activity has been demonstrated to be maximum from embryonic d 15 to 9 d posthatching in organ culture studies of the chick pancreas (Foltzer et al., 1982). These reports and our glucose measurement data depicting an age-dependent hike in blood glucose level enabled us to identify a critical window for isolating chick B islets from appropriate age of chick. We found that the critical window for isolation of chick B islets showing maximum glucose responsiveness and insulin secretion lies within 5 to 6 d after hatching. Hence, we selected 5- to 6-d-old chicks for isolation of pancreatic B islets and insulin secretion studies. Increases in plasma insulin concentrations up to 7 d posthatching and high sensitivity to insulin during this age have been demonstrated (Tokushima et al., 2003), which also supports our choice of the age of chick for islet isolation. Moreover, blood glucose levels of diabetic mice and 5- to 6-d-old chicks are comparable, making chick B islets of this age more appropriate to study their suitability for screening insulin secretagogue. In addition, our present data on the similarity of nondiabetic mouse islets and chick islets to tolbutamide stimulation further strengthens age selection for islet study.
Table 3. Responsiveness of islets to insulin secretagogue in terms of stimulation index1 Serial no. 1 2
Tolbutamide stimulation (mg/dL)
Normal mouse islets
Diabetic mouse islets
Chick islets
3.1 12.2
1.75 ± 0.01b 1.89 ± 0.01d
0.87 ± 0.02a 0.90 ± 0.02a
1.83 ± 0.01c 1.98 ± 0.01e
Superscripts denote the groups that are significantly different from one another (P ≤ 0.001). Stimulation index = a ratio of the amount of insulin produced on tolbutamide stimulation to the amount of insulin produced at basal glucose concentrations. Statistical analysis was carried out by using 1-way ANOVA. a–e 1
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Figure 1, panel B). In addition, the histological structure of the pancreata of diabetic mice showed necrotic B cells due to STZ treatment (Figure 1, panel C).
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ACKNOWLEDGMENTS We thank the director of the National Centre for Cell Science for providing facilities to carry out the present work. The financial support provided by the University Grants Commission to S. P. Datur is also acknowledged. The technical help provided by Malati Umrani, technician, National Centre for Cell Science, is highly appreciated.
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Datar, S. P., and R. R. Bhonde. 2006. A simple technique for isolation of functional B islets from chick pancreas. J. Cell Tissue Res. 6:605–608. Esmaeili, M. A., and R. Yazdanparast. 2004. Hypoglycaemic effect of Teucrium polium: Studies with rat pancreatic islets. J. Ethnopharmacol. 95:27–30. Farzami, B., D. Ahmadvand, S. Vardasbi, F. J. Majin, and S. H. Khaghani. 2003. Induction of insulin secretion by a component of Urtica dioica leave extract in perifused islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats. J. Ethnopharmacol. 89:47–53. Fernandez, A., L. C. King, Y. Guz, R. Stein, C. V. Wright, and G. Teitman. 1997. Differentiation of new insulin producing cells is induced by injury in adult pancreatic islets. Endocrinology 138:1750–1762. Foltzer, C., K. Haffen, M. Kedinger, and P. Mialhe. 1982. Stimulation of insulin and glucagon secretion in organ culture of chick endocrine pancreas during embryonic life and after hatching. Gen. Comp. Endocrinol. 47:213–220. Gomori, G. L. 1941. Gomori’s chromium haemotoxylin phloxine stain. Am. J. Pathol. 17:398–399. Gomori, G. L. 1950. Gomori’s aldehyde fuschin stain (special stain for β cells). Am. J. Clin. Pathol. 20:665–666. King, D. L., and R. I. Hazelwood. 1976. Regulation of avian insulin secretion by isolated perfused chicken pancreas. Am. J. Physiol. 231:1830–1839. Leibson, L., V. Bondareva, and L. Soltitshza. 1976. The secretion and the role of insulin in chick embryos and chickens. Pages 69–79 in The Evolution of Pancreatic Islets. T. A. I. Grillo, L. Leibson, A. Epple, ed. Pergamon, Oxford, UK. Naber, S. P., and R. L. Hazelwood. 1977. In vitro insulin release from chicken pancreas. Gen. Comp. Endocrinol. 32:495–504. Paty, B. W., J. S. Harmon, C. L. Marsh, and R. P. Robertson. 2002. Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets. Transplantation 73:353–357. Ruffier, L., J. Simon, and N. Rideau. 1998. Isolation of functional glucagon islets of Langerhans from the chicken pancreas. Gen. Comp. Endocrinol. 112:153–162. Samual, A. C., M. B. Kermit, S. D. Sandra, C. R. Thelma, and L. L. William. 1994. Staining and in vitro toxicity of dithiozone with canine porcine and bovine islets. Cell Transplant. 3:299–306. Shewade, Y. M., M. Umrani, and R. R. Bhonde. 1999. Large-scale isolation of islets by tissue culture of adult mouse pancreas. Transplant. Proc. 31:1721–1723. Swenne, I., and G. Lundqvist. 1980. Islet structure and pancreatic hormone content of the developing chick embryo. Gen. Comp. Endocrinol. 4:190–198. Tatarkiewicz, K., E. Sitarek, M. Sabat, and T. Orlowski. 1997. Reversal of hyperglycemia in streptozotocin diabetic mice by xenotransplantation of microencapsulated rat islets. Ann. Transplant. 2:20–30. Tokushima, Y., B. Sulistiyanto, K. Takahashi, and Y. Akiba. 2003. Insulin-glucose interactions characterised in newly hatched broiler chicks. Br. Poult. Sci. 44:746–751. Tsukuda, K., M. Sakurada, I. Niki, Y. Oka, and M. Kikuchi. 1998. Insulin secretion from isolated rat islets induced by the novel hypoglycemic agent A-4166, a derivative of Dphenylalanine. Horm. Metab. Res. 30:42–49. Vessal, M., M. Hemmati, and M. Vasei. 2003. Antidiabetic effects of quercetin in streptozocin-induced diabetic rats. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 3:357–364. Warburton, S., and R. James. 1995. Hemocytometer cell counts and viability studies. Pages 1.1–1.5 in Cell and Tissue Culture: Laboratory Procedures. A. Doyle, J. B. Griffiths, and D. G. Newell. John Wiley & Sons, New York. Weir, G. C., P. C. Goltos, E. P. Steinberg, and Y. C. Patel. 1976. High concentration of somatostatin immunoreactivity in chicken pancreas. Diabetologia 12:129–132.
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Our present results further demonstrate that pure B islets could be isolated from dorsal and ventral lobes of 5- to 6-d-old chick pancreata and maintained in culture. The islet yield also is higher when the 7,000 islets per dorsal lobe of the 5- to 6-d-old chick pancreas are compared with the 2,500 islets per mouse pancreas (Shewade et al., 1999; Datar and Bhonde, 2006). Because these isolated islets are insulin secretory, viable, and responsive to insulin secretagogues like glucose and tolbutamide, they represent an attractive alternative model for screening hypoglycemics. Employment of chick islets for this purpose will also minimize the use of mammals for experimentation, thus supporting the “3R” principle of reduction, refinement, and replacement of laboratory animals. In the present investigation, we have demonstrated for the first time that isolated pancreatic B islets from the chick are responsive to insulin secretagogues like glucose and tolbutamide. The previous reports on chicken pancreatic islets used 4- to 6-wk-old chicks in which islets were found to be nonresponsive to glucose, making them unsuitable for testing insulin secretagogue activity. On this background, our data on B islets from 5- to 6-d-old chick pancreata is highly promising, because islets are responsive to insulin secretagogues. With the inclusion of biotechnology courses for graduate and postgraduate students and in the light of present restrictions on the usage of mammals for experimentation as well as the expenditure on their maintenance, our investigations on the suitability of chick islets for screening hypoglycemic agents would offer a simple, economical, and yet effective system. The present study represents a first attempt to evaluate isolated chick B islets from 5- to 6-d-old chicks for screening insulin secretagogues. It is interesting to note that although chick islets remain in a hyperglycemic environment comparable to a diabetic mouse, they remain intact and retain functional β cell mass as revealed by their insulin secretory activity and, hence, seem to be more appropriate for secretagogues studies.