- Proliferation and Differentiation of Pancreatic beta Cells in L-type Calcium Channel alpha(1D) Subunit (Ca(v)1.3) Heterozygous Knock Out Mice After Partial Pancreatectomy.
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Yoon Hee Choi, Il Hee Yun, Sun Hee Suh, Dong Jun Lim, Jae Hyuung Cho, Hyuk Sang Kwon, Bong Yun Cha, Ho Young Son, Chung Gyu Park, Kun Ho Yoon
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Korean Diabetes J. 2007;31(3):208-219. Published online May 1, 2007
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DOI: https://doi.org/10.4093/jkda.2007.31.3.208
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S: L-type voltage-dependent calcium channel (LTCC) plays a crucial role in insulin secretion from pancreatic beta cells through Ca2+ influx. In the recent report, LTCC Ca(v)1.3 subtype homozygous knock out mice showed impairment of postnatal pancreatic beta cell development as well as insulin secretion. METHODS: We performed 90% partial pancreatectomy in heterozygous Ca(v)1.3 knock out mice to investigate the effect of partial deficiency of Ca(v)1.3 gene on beta cell regeneration in the adult. Glucose homeostasis, metabolic profiles including serum insulin and lipid levels and morphologic changes of pancreatic islets were studied. RESULTS: 90% Partial pancreatectomy induced glucose intolerance only in the heterozygous knock out mice at 8 weeks after surgery. Distribution of islet size was significantly different between two groups after partial pancreatectomy; median value of islet size of heterozygote was larger than that of wild type (642.8 micrometer2 vs 1459.8 micrometer2, P < 0.01). The frequency of single beta cell unit, considered as a unit of beta cell neogenesis, was much lower in heterozygote than that of wild type (41% vs 23.3%, P < 0.05). CONCLUSION: These data suggest that Ca(v)1.3 gene deficiency is specifically associated with impairment of beta cell regeneration, especially neogensis and eventual glucose intolerance in the 90% partial pancreatectomized mice.
- Characterization of Preadipocyte factor-1 (Pref-1) Expressing Pancreatic Cells.
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Marie Rhee, Sun Hee Suh, Youn Joo Yang, Ji Won Kim, Sung Yoon Jeon, Oak Kee Hong, Seung Hyun Ko, Yoon Hee Choi, Bong Yun Cha, Ho Yong Son, Kun Ho Yoon
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Korean Diabetes J. 2005;29(6):507-516. Published online November 1, 2005
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Preadipocyte factor-1/Delta-like 1(Pref-1/Dlk1) is a type I membrane protein that has six epidermal growth factor (EGF)-like repeats in its extracellular and a short cytoplasmic domain. It is widely expressed in embryonic tissues, whereas its expressions were limited in adult and postnatal stage. To characterize the Pref-1 expressing cells during pancreas development and regeneration after birth, we analyzed Pref-1 expression in embryonic and adult partial pancreatectomized rat pancreas, and primary cultured neonatal pig pancreatic cells. METHODS: Whole fetuses or pieces of rat pancreas were obtained at E20. 90% partial pancreatectomy (Px) and sham operation were done using 5 week-old Sprague-Dawley rats. Experimental animals were divided into 11 groups by time of killing after surgery; 0, 1, 3, 6 and 12 hours, 1, 2, 3, 5, 7, and 14 days. All tissues were immunostained with Pref-1 and analysed by reverse transcriptase (RT)-PCR. Porcine neonatal pancreas cell clusters (NPCCs) were prepared from neonatal pigs aged 1-2 days. Cells were harvested on day 0, 3, 4, 5, 6, and 7 after dispersion. All cells were immunostained with Pref-1 and other specific cell markers such as Pan-cytokeratin (Pan-CK), vimentin (VT) and islet hormones, and confirmed by Western blot, RT-PCR and fluorescence activated cell sorting (FACS) analysis. RESULTS: In the rat embryonic pancreas at E20, Pref-1 expression was restricted only in the small branching ductules. In adult rat pancreas, Pref-1 was not expressed at all. Whereas, Pref-1 transiently expressed in the small regenerating duct cells located in foci of regeneration in Px model, then completely disappeared at day 7. The Pref-1 mRNA measured by RT-PCR was peaked at day 3 after Px and then gradually disappeared. Pref-1 expression pattern was also reproduced in monolayer cultured NPCCs. In NPCCs, protein levels of Pref-1 were peaked at day 0 to day 4 then gradually disappeared until day 7 by western blot. Most of Pref-1 expressing cells were co-stained with cytokeratin. The proportion of Pref-1 expressing cells in dispersed NPCCs were counted and isolated by FACS at 3 days after culture were 25% and then decreased over time during 7 days culture period. CONCLUSIONS: Pref-1 expression was regained in adult pancreatic cells during regeneration in vivo and in vitro and Pref-1 might be a useful marker for the pancreatic protodifferentiated cells.
- The Effects of Dexamethasone on the Expansion and Transdifferentiation of Transplanted Porcine Neonatal Pancreas Cell Clusters into beta-cells in Normal Nude Mice.
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Ji Hun Yang, Sun Hee Suh, Sung Yoon Jeon, Oak Kee Hong, Kun Ho Yoon
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Korean Diabetes J. 2004;28(5):356-366. Published online October 1, 2004
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Several studies have suggested that glucocorticoid has an influence on the development and function of the -cells. Thus, we undertook this study to determine whether exposure to dexamethasone (Dx) has an influence on the expansion or transdifferentiation of transplanted porcine NPCCs. METHODS: After transplantation (Tx) of 4,000 islet equivalents (IEQs) of porcine NPCCs into normal nude mice, Dx (1mg/kg) or the control vehicle were injected daily for 10 weeks. To clarify the effects of timing and duration of the Dx, one group was treated by Dx at the first 2 weeks (n=10) and the other group was treated later 8 weeks (n=10) during the 10 weeks treatment period. Thr total graft and beta-cell masses were determined by morphometric analysis. We preformed semi-quantitative RT-PCR for evaluating the pancreas transcription factors. RESULTS: The relative volume and absolute mass of the beta-cells and the total graft were significantly decreased by 10 weeks Dx treatment. Moreover, Dx treatment at thr first 2 weeks (n=10) also significantly decreased the total graft mass and absolute mass of the beta-cells. The relative volume of the beta-cells was negatively correlated and the area of the duct cysts was positively correlated with the duration of the Dx treatment. Pancreas transcription factors including PDX1, Ngn 3, ISL1 and NKx6.1 were decreased in the graft by 2 days treatment of Dx. CONCLUSION: These results suggest that Dx treatment suppresses the expansion and transdifferentiation of transplanted pancreas precursor cells into beta-cell.
- Development of Adult Porcine Islet Isolation Method for Xenotransplantation.
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Sung Rae Kim, Kun Ho Yoon, Hyuk Sang Kwon, Sun Hee Suh, Seung Hyun Ko, Jung Min Lee, Soon Jib Yoo, Yoo Bae Ahn, Ki Ho Song, Hyun Shik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2004;28(2):75-87. Published online April 1, 2004
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AND PURPOSE: Xenotransplantation using porcine islet cells might be an alternative to allotransplantation, which has been limited due to the lack of donors. Various researches using porcine islet cells have been performed in foreign countries; however, they have never been studies in Korea. Therefore, the purpose of this study was to explore the possibility of thise new treatment for cases of diabetes by establishing of improved islet isolation skill. METHODS: The pancreas and islets were extracted from pigs weighing around 100kg. To establish an islet isolation method, the islet yield, purity and the distribution size of the isolated islets were step wise compared in various ways, and then the superior method adopted. To determine the conveyance method after organ extraction, the conveyance method of pouring collagenase P was compared with the conveyance method of injecting Custidol. For digestion, the mechanical shaking and static incubation methods were also compared. To isolate islets from the digested pancreata, isolation methods were analyzed using 3 and 4 layers' Ficoll. The islet yield was appraised after their isolation using the optimized islet isolation method. To assess the results of the islet isolation, appraised the purity and the survival rates of cells, the insulin secretion resulting from the glucose stimulation test was examined. RESULTS: The method of injecting 4degrees C Custidol was effective for the conveyance and storage of the isolated pancreas in comparison with an injection of collagenase P(3465+/-1488 IEQ/g pancreas vs. 48+/-1.7 IEQ/g pancreas, p<0.01). The digestion method was superior to the mechanical shaking method at keeping a stable condition(3465+/-1488 IEQ/g pancreas vs. 1265+/-141.4 IEQ/g pancreas, p<0.01). Ficoll isolation using 3 layers gave the same results as using 4 layers. The average weights of the isolate Pancreatic islets was 23.8+/-3.3g. The numbers of islets per gram was 3465+/-1488.2(IEQ), with a the purity of 86.3+/-2.0%, and a survival rate of over 95%. The insulin secretion caused by glucose stimulation substantially increased in concentration from 24 to 72 hours(24hr: 5mM 3.12mU/mL --< 20mM 6.79mU/mL(2.17 fold), 72hr: 5mM 2.38mU/mL --< 9.93mU/mL(4.17fold))
- 3-Dimensional Long Term Culture of Monolayer Cultured Dispersed Neonatal Porcine Pancreas Cells (NPCC).
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Sun Hee Suh, Kun Ho Yoon, Hyuk Sang Kwon, Ok Ki Hong, Jung Min Lee, Ki Ho Song, Soon Jib Yoo, Hyun Sik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2002;26(5):383-395. Published online October 1, 2002
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We have reported porcine neonatal pancreas cell clusters (NPCCs) to be useful clinical alternative due to their growth potential and convenience. However, to apply the porcine NPCCs in human islet transplantation, there is a need to achieve in vitro maturation of porcine pancreas duct cells for the immediate cure of diabetes, and to escape hyperacute rejection. We have established a long-term 3D culture system of porcine pancreas duct cells for their in vitro induction in differentiated beta-cells. METHOD: For making NPCCs, pancreata from 1~3 days old pigs were minced, digested and cultured for 8 days. After 8 days, the cells were layered with Matrigel. After 50 days, the 3 dimensional cultures, the components of the reconstructed cell clusters were confirmed by three approaches: immunofluorescent staining, mea-surement of glucose stimulated insulin secretion and semiquantitative RT-PCR. RESULT: The monolayers of epithelial cells formed three-dimensional structures of cysts from which 50~200 micro meter diameter islet-like clusters of pancreas cells budded. The insulin and DNA contents, and the ratio of insulin/DNA, did not change significantly, even after 50 days of culturinge. The levels of insulin and galactosyl transferase mRNA showed a tendency to increase in the monolayer culture of the duct cells until day 8, after which the levels significantly decreased. However, the level of glucagon mRNA was maintained until day 50. Compared with their basal secretion at 5mM glucose, the cysts/cultivated porcine islet buds exposed to stimulatory 20mM glucose did not show difference in insulin secretion. CONCLUSION: We have shown the expansion of dispersed porcine neonatal pancreas cells in vitro, and the reconstruction of a three-dimensional structure, following Matrigel overlaying, but were unable to observe the transition of duct cells to beta cells, as observed in human duct cells. Further studies will be required to elucidate this difference.
- Selective beta-Cell Loss and alpha-Cell Expansion in Islets of Type 2 Diabetic Patients.
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Jae Hyoung Cho, In Kyu Lee, Kun Ho Yoon, Seung Hyun Ko, Sun Hee Suh, Jung Min Lee, Sung Rae Kim, Yoo Bae Ahn, Jong Min Lee, Hyun Shik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2001;25(2):164-177. Published online April 1, 2001
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It has been reported that a decrease in the beta-cell mass, may play a major role in the development of type 2 diabetes. Some stimuli that cause beta-cell loss can stimulate neogenesis from precursors as well as replication of matured beta-cells. In an animal-based studies reported that alpha-cells can also be produced in the course of alpha-cell neogenesis, after being treated with streptozotocin. Through this research, we attempted to determine the change of beta-cell mass according to the changes in alpha-cell mass and to characterize the size of the beta-cell nucleus observed in type 2 diabetes. METHOD: To estimate the relative fraction of alpha- and beta-cell mass in the pancreas, we counted beta-cells and alpha-cells by point count method. We also performed a double immunohistochemical staining with glucagon and insulin antibodies to calculate the ratio between these two cells area in the pancreas (A/B ratio). In order to measure the size of the beta-cell nucleus, an immunofluorescence staining of the nucleus and insulin was carried out. Data were gathered from type 2 diabetic subjects (n=19) and normal controls (n=8). RESULTS: Although there was no statistical difference, we observed the tendency of decrease of beta-cell mass and increase of alpha-cell mass in the pancreas of type 2 diabetic patients. The ratio of alpha-to beta-cell area in islet (A/B ratio) increased to 0.81+/-0.76 in diabetic patients compared to control with 0.26+/-0.25 (p<0.01). The mean of the A/B ratios of the islets more than 22,000 micro m2 was 1.64+/-1.10, whereas that of the islets less than 22,000 micro m2 was 0.73+/-0.67 in type 2 diabetic patients (p<0.01). The size of the beta-cell nucleus in both diabetic subjects and normal controls was bigger than that of exocrine cells (p<0.05) and 2.9% of beta-cells in type 2 diabetic subjects showed substantially enlarged nuclei more than M+5SD (M and SD means the average and standard deviation of nucleus size of exocrine cells, respectively) whereas this type of nucleus was found in only 0.5% of beta-cells in normal controls (p<0.05). CONCLUSION: The islet pathology in type 2 diabetes could be characterized by an expansion of alpha-cells associated with the selective loss of beta-cells. Some beta-cells found in diabetes showed a significant increase in size of the nucleus. Through the results from this study, we postulate that enlarged beta-cell nucleus and reverse of A/B ratio in the islets could be a marker of early senescence of beta-cells in patients with type 2 diabetes mellitus.
- The Changes of Beta Cell Mass and Islet Morphology in OLETF (Otsuka Long Evans Tokushima Fatty) Rats After Partial Pancreatectomy .
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Seung Hyun Ko, Kun Ho Yoon, Sun Hee Suh, Yu Bae Ahn, Soon Jib Yoo, Ki Ho Song, Hyun Shik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2001;25(1):50-62. Published online February 1, 2001
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Insulin resistance and incomplete beta cell compensation play a major role for development of type 2 diabetes. When insulin resistance were induced by any cause, appropriate beta-cell proliferation is a key factor for maintaining the normal glucose metabolism. Compensatory beta-cell proliferation for adapting to increased insulin resistance might be achieved by neogenesis of beta-cell from duct cells, replication of preexisting beta-cells and also inhibition of beta-cell apoptosis. Previously incomplete beta-cell compensation was observed in OLETF rat, animal model of type 2 diabetes, after partial pancreatectomy, but there were no reports about the underlying pathogenesis. Therefore, this study was designed to study on the mechanism of incomplete beta-cell compensation in OLETF rat after partial pancreatectomy especially focus on beta-cell proliferation. METHODS: 12 week-old OLETF (Otsuka Long Evans Tokushima Fatty) rats weighing 280-320 g were used. 80% partial pancreatectomy was done. Experimental animals were divided into the 4 subgroups by date of killing after surgery: 0, 3, 90 days. After glucose tolerance test, pancreas remnant was excised and immunohistochemical staining was done for insulin to quantify the beta cell mass by point-counting method and also observed the amount of fibrosis of the islets after Masson's trichrome staining of the pancreas. RESULTS: We observed that impaired glucose tolerance or diabetes were developed after 80% pancreatectomy. We observed rapidly proliferating duct cells in the adjacent area of common pancreatic duct and main duct even up to 90 days after partial pancreatectomy. In OLETF rats, beta cell mass was not increased enough compared to LETO rats and some destructive features of islet architectures were noted at 90 days after pancreatectomy. CONCLUSION: The changes of beta cell mass seems to be a dynamic process adjusting to metabolic demand. Severe hyperglycemia and islet disorganization were apparent in OLETF rats despite of existence of beta cell regeneration and renewal process. So it seemed that hyperglycemia accelerated aging process or senescence of beta cells in OLETF rats.
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