Porcine Pancreatic Endocrine Cells Research Articles

Promotion of β-cell differentiation by the alkaloid conophylline in porcine pancreatic endocrine cells

We previously found that conophylline, an alkaloid isolated from the leaves of Ervatamia microphylla, induced β-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured fetal rat pancreatic tissue and that it also decreased the blood glucose level in streptozotocin-treated fetal rats. In the present research, we looked into the effect of conophylline on the differentiation of newborn pig pancreatic endocrine cells into insulin-secreting cells. Conophylline potentiated the differentiation of monolayer cells into insulin-producing cells in the presence of nicotinamide in 3 weeks. Next we prepared islet-like cell clusters (ICC). Cononophylline together with nicotinamide also increased the number of insulin-producing cells and the insulin content in ICC in 3–6 weeks. The ICC thus prepared were sensitive to the glucose concentration for the insulin secretion. Conophylline increased the mRNA expression of PDX-1, neurogenin3, neuroD/Beta2, and insulin in ICC. Thus, the vinca alkaloid conophylline potentiated β-cell differentiation in porcine pancreatic endocrine-rich cells in cluster cultures. Pig pancreatic cells are practical candidate for use in transplantation therapy. Conophylline may thus be useful for the large-scale preparation of porcine insulin-producing cells for the regeneration therapy of type-1 diabetes mellitus.

Effect of long‐term culture on the expression of antigens and adhesion molecule in single porcine pancreatic endocrine cells

Changes in the expression of galactose (Gal) alpha1-3Gal, swine lymphocyte antigen (SLA) class II and intracellular adhesion molecule (ICAM)-1 of single porcine pancreatic endocrine (PE) cells during the culture period were investigated. Cultured porcine PE-cells were fixed in 10% buffered formalin for histological evaluation. At 1, 3, 6, 9 and 12 weeks of culture, mRNA was obtained from porcine PE-cells so that the expression of SLA class II and ICAM-1 genes could be examined by reverse transcriptase-polymerase chain reaction. The rates of Galalpha1-3Gal and SLA class II-positive cells did not decrease during the culture period, but the rates of Galalpha1-3Gal and SLA class II strongly positive cells significantly decreased. ICAM-1-positive cells were scarcely observed during the culture period. SLA class II and ICAM-1 mRNAs were detected at 1 and 3 weeks of culture, but were not detected after 6 weeks of culture. These results suggest that partial reduction in the expression of these antigens could be obtained by a long-term culture.

Effect of GLP-1 (glucagon-like peptide 1:7-36 amide) on porcine pancreatic endocrine cell proliferation and insulin secretion.

We have established a method of isolation and primary monolayer culture of porcine pancreatic endocrine (PE) cells using nicotinamide. Recently, several insulin-stimulating factors have been recognized as modifying the function of endocrine cells. Glucagon-like peptide 1 (GLP-1), derived from intestinal cells, is one of the substances that may regulate insulin secretion and cell proliferation. The aim of the current study was to examine the effect of GLP-1 on PE cell proliferation and insulin secretion in our culture system. The PE cells were prepared by nonenzymatic digestion and cultured in media with or without 10 nmol/L GLP-1 and maintained for 15 days. Basal insulin secretion and the glucose stimulated-response were observed by enzyme assay. PE-cell proliferation was assessed using 5-bromo 2' deoxyuridine (BrdU). beta-Cell specificity was confirmed by double staining with anti-BrdU and insulin antibody. mRNA expression in PE cells for insulin and pancreatic and duodenal homeobox gene 1 (PDX-1), a transcription factor, was semiquantitated by a real-time PCR method. Neither baseline nor glucose-stimulated insulin secretion differed significantly with or without the addition of 10 nmol/L GLP-1. However, significant changes of insulin secretion were observed in doses up to 100 nmol/L of GLP-1. BrdU incorporation increased with 10 nmol/L GLP-1 from 2.35+/- 0.16% to 9.11+/- 0.53% as compared with the control (from 2.17 +/- 0.57% to 6.13 +/- 0.35%). This increase was observed after a 12-day culture period. Double staining identified well-preserved insulin-containing cells, but neither the number of positive cells nor staining intensity differed significantly between the 2 groups during the 15-day culture period. The level of mRNA expression for both insulin and PDX-1 increased slightly but significantly. GLP-1 has a proliferative effect on PE cells and affects gene expression in short-term culture.

Subcutaneous transplantation of macroencapsulated porcine pancreatic endocrine cells normalizes hyperglycemia in diabetic mice.

The ultimate goal of islet transplantation is the unlimited availability of insulin-secreting cells to be transplanted in a simple procedure that requires no use of immunosuppressive drugs. Immunoisolation of xenogeneic pig islets for transplantation has great potential therapeutic benefits for treatment of diabetes. Approximately 4 x 10(6) porcine pancreatic endocrine cells (PEC) isolated from 6-month-old pigs were macroencapsulated in agarose-poly(styrene sulfonic acid) mixed gel and implanted into a prevascularized subcutaneous site in streptozotocin-induced C57BL/6 diabetic mice. Animals receiving an equal number of free porcine PEC were used as controls. After transplantation, nonfasting blood glucose, body weight, intraperitoneal glucose tolerance test, and immunohistologic evaluations were processed. All 10 animals receiving the subcutaneous xenografts of the macroencapsulated porcine PEC normalized hyperglycemia within 5 days after transplantation, maintained the duration of normoglycemia for 24 to 76 days, and gradually gained weight. The subcutaneous xenografts of free porcine PEC could not reverse hyperglycemia. The recipient became hyperglycemic again when the implanted graft was retrieved at day 45 after transplantation. The glucose clearances were significantly ameliorated at day 21 and day 45 after transplantation when compared with those in diabetic mice. The immunohistochemical results revealed an inherent intact structure of the macroencapsulated porcine PEC and positive double-immunofluorescence staining for insulin and glucagon. Subcutaneous transplantation of macroencapsulated porcine PEC normalized hyperglycemia in diabetic mice. Our results identified a potential for a favorable development of subcutaneous transplantation of porcine PEC as a cure for diabetes.

Effect of adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness in the cultured adult porcine endocrine pancreas: a preliminary study.

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules--laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel--were used. Approximately 2.0 x 10(5) cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3-4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

Analysis of gene expression and insulin secretion by monolayer-forming adult porcine pancreatic endocrine cells.

We recently established a method of isolation and primary monolayer culture of porcine pancreatic endocrine cells that involves the use of nicotinamide. To obtain genetic information on cultured porcine endocrine cells and to examine cell function in relation to insulin secretion during long-term culture. Gene expression of insulin and several transcription factors, including PDX-1, Beta2/NeuroD, Pax6, and Nkx6.1, was assessed by reverse transcription-polymerase chain reaction analysis, and the insulin protein level was estimated by immunohistochemistry and enzyme assay during a 12-week period. During the culture period, insulin accumulation in the medium at 5 weeks had decreased by almost half the level of accumulation in the first week. In contrast to the alteration of secretory function, insulin gene expression was maintained for at least 12 weeks, and regulatory transcription factors were expressed at the same levels until 9 weeks. These observations suggest that gene expression is not involved in the cause of decreased baseline insulin secretion. Moreover, although the insulin response to high glucose and potassium loading was maintained, the magnitude of the responses to both stimuli was attenuated in the late period of culture. Insulin secretion tended to decrease in our culture system, and the secretory response to pharmacological stimulation was attenuated despite maintenance of messenger RNA expression of insulin and other islet-specific genes for at least 9 weeks in vitro. These findings indicate that cell integrity is maintained and that the alteration in insulin secretion must be explained by another mechanism.

Effect of stimulators such as GLP-1, PACAP, and nicotinamide on glucose-stimulated insulin secretion from porcine pancreatic endocrine cells in long-term culture.

Transplantation of glucose-responsive insulin-secreting cells has the potential to result in a cure for diabetes. To report the development of a model of adult porcine pancreatic endocrine cells (PE cells) exhibiting glucose-stimulated insulin secretion during a long-term culture period, in vitro. The PE cells were prepared by non-enzymatic digestion and purified by modifying a technique developed in our laboratory. The cells were first cultured for 7 days in RPMI-1640 medium containing 10% fetal bovine serum and 10 mM nicotinamide. On adhesion to the culture flasks, cells were collected by trypsinization, and then cultured in tissue culture dishes in medium with or without stimulators such as glucagon-like peptide-1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), and nicotinamide. The ability of the cells to respond to glucose-stimulated insulin secretion was also observed with and without stimulators. The immunocytochemical studies demonstrated pancreatic islets with well-preserved insulin and glucagon-containing cells. The morphologic integrity of cultured porcine cells was observed for up to 5-6 weeks after the purification. At a concentration of 3.3 mM glucose, PACAP and nicotinamide did not affect glucose-dependent insulin secretion, whereas 10 nM GLP-1 stimulated insulin secretion significantly. However, when glucose concentration was increased to 20 mM, 10 nM GLP-1 had no effect on insulin secretion. We also demonstrated that GLP-1 and PACAP could maintain insulin secretion better than control in the culture up to 5 weeks. Also GLP-1 and PACAP increased the number of insulin-secreting cells in culture. These results demonstrate that GLP-1 and PACAP increased the number of pancreatic beta-cells in culture.

Effect of the Extracellular Matrix on Pancreatic Endocrine Cell Function and its Biocompatibility in Dogs

The effect of the synthetic extracellular matrix (ECM) in a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) on pancreatic endocrine cells in vitro and its biocompatibility in dogs were investigated. Two different types of ECM were used: type I collagen treated with low antigen (type I LA), and reconstituted basement membrane matrix (Matrigel) derived from Englbreth-Holm-Swarm (EHS) mouse sarcoma. Matrigel contains growth and differentiation factors and cell adhesion molecules such as laminin, heparan sulfate proteoglycan, and entactin. Purified porcine pancreatic endocrine (PE) cells were suspended in type I LA or Matrigel and then placed into a 12-well culture plate (4 x 10(7) cells/ml gel/well). The insulin accumulation from PE cells in Matrigel was significantly greater than that in type I LA (9.3 +/- 3.6 mU/well vs. 2.3 +/- 1.3 mU/well). When Bio-AEP with Matrigel and PE cells was implanted into the abdominal cavity of a pancreatectomized diabetic dog, the exogenous insulin requirement for maintaining normoglycemia was reduced for the first 4 weeks. However, after 6 weeks of implantation, fasting blood glucose levels suddenly increased. Laparotomy revealed encapsulated Bio-AEP with thick fibrous tissue. Following removal of the Bio-AEP from the abdominal cavity, another Bio-AEP containing type I LA and PE cells was implanted into the same dog. The exogenous insulin requirement was gradually decreased to almost half that of preimplantation levels. Bio-AEPs containing type I LA or Matrigel, but not PE cells, were implanted into the abdominal cavities of four healthy dogs. After 4 weeks of implantation, the Bio-AEP with Matrigel was encapsulated with fibrous tissue similar to that in the diabetic dog, but the Bio-AEP with type I LA was not. These results indicate that Matrigel may be incompatible with dogs and that the type I LA is more suitable for Bio-AEP.

Characterization of secretory and morphologic properties of primary cultured endocrine cells from porcine pancreata.

We characterized morphologic and secretory properties of porcine pancreatic endocrine cells in primary culture obtained by autolytic preparation without any exogenous proteolytic enzymes. The endocrine cells exhibited a neuron-like shape, and insulin granules were accumulated at the terminal of the processes. Thus derived endocrine cells survived in culture medium containing nicotinamide and remained sensitive to glucose for at least 6 weeks after preparations. The cells responded well to physiologic concentrations of glucose, and high K+ depolarization and the antidiabetic sulfonylureas, tolbutamide, and glibenclamide also elicited the release. With high glucose, insulin release was markedly potentiated by forskolin, glucagon, glucagon-like peptide-1, and arginine and inhibited by somatostatin, the Ca2+ channel blocker nitrendipine, and the ATP-sensitive K+ channel opener diazoxide. Epinephrine had dual effects on the release by glucose; enhanced within a low nanomolar range and inhibited at 1 micromol/L. However, the cells were unresponsive to leucine. Such secretory sensitivities to nutrients, hormones, and pharmacologic agents, and long survival rate (as long as 5-6 weeks) of these cells suggest to us therefore that derived endocrine cells may be useful for xenotransplantation of pancreatic beta cells for treatment of insulin-dependent diabetes mellitus.