Xenoimplantation using a diffusion chamber with a B-cell line (MIN6) as a bioartificial endocrine pancreas (Bio-AEP).

This experiment was designed to investigate whether MIN6-cells, which are derived from transgenic mouse insulinoma cells, could be a useful tool for transplantation. An implantable diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) was constructed by placing a pancreatic B-cell line (MIN6) in a mixed matrix in the center of a ring holder sandwiched between two nuclepore membranes (pore size 0.2 microns), which were held in place by a silicon seal. Nine streptozotocin (STZ)-induced diabetic rats were each implanted with the diffusion chamber containing mouse insulinoma cells (xenograft-implantation) as a Bio-AEP, but without any immunosuppressant. In three of the STZ-diabetic rats with a Bio-AEP, a return to normoglycemia was observed up to 20 week after implantation (good control). Four of the nine STZ-diabetic rats, which had received diffusion chambers with MIN6, also showed a return to normoglycemia for up to 10 wk (fair control). In two of the rats, blood glucose levels showed poor control. The results indicate that MIN6 cells should be useful for the implantation of xenographic cells in diabetic animals.

Absence of the Gly40-ser mutation in the glucagon receptor gene in Japanese subjects with NIDDM.

Recent studies have shown both association and linkage between a Gly40-Ser mutation in the glucagon receptor gene and NIDDM in French patients with familial NIDDM. This mutation was present in heterozygous form in 4.6% of diabetic probands but only 1% of the French population, suggesting that it was an important risk factor in the development of NIDDM. A total of 348 unrelated Japanese subjects (220 with NIDDM, 53 with impaired glucose tolerance (IGT) and 75 normal subjects) were screened for the presence of the Gly40-Ser mutation. Seventy-two percent of the NIDDM patients and 52% of IGT subjects had a positive family history of NIDDM. The Gly40-Ser mutation, which could be readily detected in a positive control subject, was not found in any of the 348 Japanese subjects studied. Thus, the Gly40-Ser mutation does not play an important role in the pathogenesis of NIDDM in Japanese patients.

Identification of microsatellite markers near the human genes encoding the beta-cell ATP-sensitive K+ channel and linkage studies with NIDDM in Japanese.

ATP-sensitive K+ (KATP) channels play a key role in stimulus-secretion coupling in pancreatic beta-cells. Recent studies have shown that the beta-cell KATP channel comprises two subunits: a novel member of the inwardly rectifying K+ channel family, designated BIR and expressed at highest levels in pancreatic islets, and the sulfonylurea receptor (SUR). Moreover, the genes encoding these two proteins are adjacent to one another on human chromosome 11. Genetic factors contribute to the development of NIDDM, and it seems likely that mutations in genes encoding proteins involved in insulin secretion or action may contribute to NIDDM susceptibility. The present study examined the contribution of the linked BIR and SUR genes to the development of NIDDM. These genes were localized to the same yeast artificial chromosome as two microsatellite DNA polymorphisms, D11S902 and D11S921. These microsatellite DNA polymorphisms were typed in 140 Japanese NIDDM-affected sib pairs. There was no evidence for linkage between these markers and NIDDM, suggesting that genetic variation in the BIR and SUR genes does not play a major role in susceptibility to NIDDM in Japanese.

Embedded-culture of pancreatic beta-cells derived from transgenic mouse insulinoma as a potential source for xenotransplantation using a diffusion chamber.

Embedded-culture of pancreatic beta-cells derived from a transgenic mouse insulinoma (MIN6 cells) was studied in vitro and in vivo. The MIN6 cells were enmeshed in an agarose-PVMA-collagen matrix for long-term maintenance. The cells formed islet-like cell clusters (ICCs) in the mixed matrix. When 10 mmol/L nicotinamide was added to these cultures the cells secreted insulin in response to various concentrations of glucose, whereas the untreated control cells were unresponsive. Both control and nicotinamide-treated MIN6 cells exhibited normal beta-cell function for up to 35 days in the mixed matrix, and the cells were much better preserved with nicotinamide than without it. MIN6 cells were suspended in the mixed matrix with nicotinamide and transferred into diffusion chambers to create a bio-artificial endocrine pancreas (Bio-AEP). In streptozotocine-induced diabetic rats with implanted Bio-AEP but without any immunosuppressants, a return to normoglycaemia was observed for up to 12 wk or more after transplantation. Our results indicate that nicotinamide-treated MIN6 cells embedded in a mixed matrix should be useful for the study of xenotransplantation and the development of a bioartificial endocrine pancreas.

Development of a method for embedded-culture of pig pancreatic islet-like cell clusters in agarose containing maltose-carrying polystyrene (HEVM) and nicotinamide.

A method for embedded-culture of islet-like cell clusters (ICCs) from neonatal pig pancreas is described. The procedure is based on a sequential treatment of the pancreas with a proteolytic enzyme and ethylenediaminetetraacetate (EDTA). During the exposure to EDTA-Dispase, small pieces of pancreas were gradually digested, with gentle stirring. Under- and over-digested pancreatic cell clusters were separated from the pancreatic fragments by filtration, and formed whole islet-like cell clusters in RPMI 1640 containing 11.0 mM D-glucose and 10% fetal bovine serum (FBS). The ICCs were embedded in agarose with or without a gel containing a random copolymer hydroxethylmethacrylate-vinylbenzyl maltonamide (HEVM) of hydroxethylmethacrylate-(HEMA)-vinylbenzyl maltonamide (VMA) and nicotinamide. They remained morphologically intact and a physiological response to acute stimulation with glucose was obtained when they were embedded in the agarose containing HEVM and nicotinamide. These findings suggest that ICCs in agarose containing HEVM and nicotinamide could be a useful tool for morphological, biochemical and molecular biological studies, and also as a potential source of material for transplantation.

Reversal of glucose insensitivity of pancreatic B-cells due to prolonged exposure to high glucose in culture: effect of nicotinamide on pancreatic B-cells.

Prolonged in vitro exposure to high glucose has been shown to induce a decrease in pancreatic B-cell sensitivity to glucose stimulation. This experiment was designed to study whether nicotinamide affects the B-cells' response to regulatory stimuli of glucose after prolonged culture with high glucose. Neonatal pig pancreatic islet-like cell clusters (ICCs) formed from single cells were embedded in agarose-gel. Some of them were maintained in RPMI 1640 containing a high glucose concentration (16.7 mM) and 10% fetal bovine serum (FBS) with or without nicotinamide. Then, the embedded-ICCs were stimulated by glucose at concentrations of 5.5 mM and 16.7 mM. After the prolonged culture with high glucose, the ICCs showed no response to acute glucose stimulation. There was a significant increase in glucose-mediated insulin secretion when the ICCs were maintained with the medium containing nicotinamide. We conclude that nicotinamide could protect B-cell desensitization to glucose after prolonged exposure to high glucose.

Survival and B-cell function of neonatal pig pancreatic islet-like cell clusters in an extracellular matrix.

Islet-like cell clusters (ICCs), formed from single cells of pig pancreas in suspension culture, were embedded in an extracellular matrix. It was recently reported that nicotinamide prevented dissolution of the extracellular matrix by the ICCs. In this experiment, various conditions for embedded culture of ICCs in an extracellular matrix were studied, in an attempt to maintain the function as well as the extracted insulin content of culture specimens. The ICCs in the matrix were refed with RPMI 1640, containing 10 mM nicotinamide, 10% fetal bovine serum (FBS), 11 mM D-glucose and with or without 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) or 1.0 micrograms/ml caerulein. A comparison between the different culture media showed that embedded ICCs, maintained in RPMI 1640 with caerulein, in the presence of nicotinamide, had higher insulin content accumulation than when maintained in medium containing nicotinamide alone, but had impaired glucose-stimulated insulin secretion. In the medium containing IBMX and nicotinamide, embedded ICCs showed higher insulin accumulation but lower insulin content, compared to ICCs maintained in the presence of caerulein, and also showed impaired glucose-stimulated insulin release. Thus, the effect of nicotinamide on the survival and function of B-cells is amplified by the presence of caerulein or IBMX.