Miriam Pipeleers-Marichal

Effects of Chronically Elevated Glucose Levels on the Functional Properties of Rat Pancreatic β-Cells

This study examines the effects of chronically elevated glucose levels on the survival and function of purified rat β-cells. Prolonged exposure (9 days) of β-cell aggregates to 20 mmol/l glucose did not lead to cell losses, but reduced the amount of insulin secreted in response to glucose. This decrease was not caused by cellular desensitization but resulted from the lower cellular insulin content after a prolonged imbalance between stimulated rates of insulin synthesis and release. Virtually all β-cells exhibited a state of metabolic and biosynthetic activation, which was maintained for at least 2 h in glucose-depleted media. Their rates of protein and insulin synthesis were amplified by glucose, reaching (half-) maximal stimulation at lower glucose concentrations (2 and 5 mmol/l, respectively) than control cells cultured at 10 mmol/l glucose (5 and 10 mmol/l, respectively). As for insulin release, the net glucose effect on insulin synthesis was markedly reduced as compared with that in control cells. This was also the case after culture at 6 mmol/l glucose. In the latter condition, the lower glucose-inducible activities were caused by cellular desensitization, with 50% of the β-cells unresponsive to glucose and the other 50% responding with a lower sensitivity (half-maximal stimulation at 7 mmol/l glucose). Comparison of β-cells cultured at the three glucose concentrations indicated that prolonged exposure to elevated glucose levels increases the number of degranulated cells, of cells with a high proportion of immature insulin granules, and of cells with glycogen deposition-morphologic features previously described in conditions of hyperglycemia. It is concluded that chronic exposure (9 days) of rat β-cells to elevated glucose levels induces a prolonged state of β-cell activation and glucose hypersensitivity rather than a glucotoxicity or glucose desensitization. This shift in the functional state of the β-cell population is responsible for a reduced insulin release in response to glucose, as observed in other conditions of prolonged exposure to high glucose levels.

Differences in Baseline Lymphocyte Counts and Autoreactivity Are Associated With Differences in Outcome of Islet Cell Transplantation in Type 1 Diabetic Patients

OBJECTIVE The metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function. RESEARCH DESIGN AND METHODS Thirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model. RESULTS Patients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability. CONCLUSIONS Higher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

Transplantation of Purified Islet Cells in Diabetic Rats: I. Standardization of Islet Cell Grafts

A standardized procedure was developed for the preparation of rat islet cell grafts with selected cell number and composition. After collagenase digestion of pancreases and elutriation of tissue fragments, islets were isolated and dissociated, and cells were purified by autofluorescence-activated cell sorting. Approximately 30% of the initial β-cell mass was lost during digestion and elimination of small mostly exocrine particles. Fifty percent was recovered in isolated islet preparations and 30% in the purified β-cell suspensions of >95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet β-cells from islet endocrine non–beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 106 pure β-cells with or without 8 × 105 pure endocrine non–²-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure β-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified β-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Metabolic and morphologic studies in intraportal-islet-transplanted rats.

The intraportal injection of 350 to 1,000 isolated islets into streptozotocin-diabetic rats immediately normalized (∼ 24 hours) fasting plasma glucose and insulin levels. Polyuria, polydipsia, and hyperglucagonemia disappeared more gradually over a 2-to-12-week period—the time required for normalization varying with the severity of the diabetes and the number of islets transplanted. In long-term islet-transplanted rats (> five months), the hepatic insulin and glucagon reserves averaged 50 per cent and 25 per cent, respectively, of the corresponding normal pancreatic hormone content. Glucagon was increased slightly in the pancreas of streptozotocin-diabetic rats and decreased considerably in transplanted animals. However, total pancreatic glucagon (i.e. pancreatic and hepatic reserves) in transplanted animals was the same as the pancreatic content of normal control rats, indicating the presence of feedback control mechanism(s) in the regulation of pancreatic glucagon reserves. Long-term transplanted islets demonstrated well-granulated A-, B-, and D-cell movement out of the vascular space and the formation of narrow intercellular spaces and junctional complexes with surrounding hepatocytes.

Formation of insulin-positive cells in implants of human pancreatic duct cell preparations from young donors

Aims/hypothesisPancreatic ducts are considered as potential sites for neogenesis of beta cells. In vitro studies have reported formation of islets from postnatal human and rodent duct tissue. We examined whether postnatal human duct-cell preparations can generate new beta cells after transplantation.MethodsPancreatic duct cells were prepared from the non-endocrine fraction of human donor pancreases that were processed for islet-cell isolation. Grafts containing 0.5 million duct cells with 1% contaminating insulin-positive cells were implanted under the kidney capsule of normoglycaemic nude mice. At 0.5 and 10 weeks post-transplantation, implants were examined for their cellular composition and for the volumes of their composing cell populations, i.e. cytokeratin 19-positive duct cells, synaptophysin-, insulin- and glucagon-positive endocrine cells.ResultsBetween week 0.5 and 10, duct-cell volume decreased by at least 90% whereas the change in insulin-positive cell volume depended on donor age. Implants from donors over10 years had a threefold decrease in their insulin-positive cell volume, while those from donors under 10 years had a 2.5-fold increase. After 10 weeks, the implants from the younger donors consisted of 19% insulin-positive cells occurring as single units or small cell clusters. Three percent of these insulin-positive cells also expressed the ductal marker CK 19 and were consistently found in conjunction with ductal epithelia; up to 1% was positive for the proliferation marker BrdU and located in small endocrine cell clusters.Conclusions/interpretationThese data indicate that duct cell preparations from donors under 10 years can generate insulin-positive cells. This process might involve differentiation of CK 19-positive-insulin cells that are formed at the duct epithelia as well as proliferation of insulin-positive cells within endocrine cell aggregates.

Transplantation of Purified Islet Cells in Diabetic Rats: II. Immunogenicity of Allografted Islet β-Cells

This study examines whether the survival of allografted rat islet β-cells is influenced by the presence of other pancreatic donor cells. Grafts (RT1u/l) of different cellular composition were intraportally transplanted in streptozocin-induced diabetic rats (RT1n/n). All grafts corrected the diabetic state within 3 days. Implants of freshly isolated islets contained various endocrine and nonendocrine cell types; they became diffusely infiltrated within 1 wk and were completely destroyed within 2 wk. A 4-day culture period did not lead to major changes in the cellular composition of the islets or in their survival as allograft. Islet cell aggregates prepared after islet dissociation and cell purification were less acutely infiltrated and less rapidly rejected. Aggregates composed of sorted MHC class II–negative cells maintained basal normoglycemia in 3 of 5 recipients for 5 wk but only in 1 of 5 for 20 wk. Aggregates of purified islet β-cells remained relatively free of diffuse infiltrations during the 1st wk and preserved the normalized state in 7 of 13 recipients for 5 wk; after 20 wk, 6 of 13 were still aglucosuric, but 40% of the implants were diffusely infiltrated and depleted of insulin. Reaggregation of purified islet β-cells with purified islet endocrine non–²-cells promoted their long-term survival as allograft: 11 of 13 recipients of mixed islet endocrine cells maintained normal basal glycemia over 20 wk; their implants contained relatively constant insulin reserves and remained virtually devoid of diffuse infiltrations. These results demonstrate that techniques aiming at the elimination of surface MHC class II–positive cells are less successful in preparing rat islet allografts of low immunogenicity than methods of positive cell selection. Pure islet β-cells are immunogenic as an allograft but illicit a milder and less-acute immune attack than undissociated islet tissue. Nonendocrine and damaged islet cells are suspected of enhancing the rapidity and intensity of the cytotoxic reaction. Survival of allografted β-cells is markedly prolonged by the presence of islet endocrine non–²-cells within the graft. The mechanisms underlying this effect have not yet been elucidated; they may involve immune and metabolic interactions of the endocrine non–²-cells. We conclude that purification of islet endocrine cells represents a new and powerful method for preparing insulin-producing allografts that can survive in hosts without pharmacological immunosuppression.

Secretory Capability of Islets Transplanted Intraportally in the Diabetic Rat

Long-term islet-transplanted rats were examined for their ability to secrete insulin and glucagon and maintain normal glucose homeostasis under various experimental conditions. Normal glucose tolerance and insulin secretion were noted in nonstressed transplanted animals after oral glucose loading, whereas the intravenous administration of glucose resulted in a prompt but reduced insulin release associated with a slower return to basal glucose concentrations. Both the insulin and glucagon responses of nonstressed transplanted rats to arginine were comparable to those seen in normal animals. In transplant rats, stress severely impaired glucose homeostasis, as indicated by a marked rise in plasma glucose and a significant decrease in plasma insulin under basal conditions. These abnormalities were prevented by either alpha-adrenergic blockade or adrenalectomy. Stress also severely impaired both oral and intravenous glucose tolerance tests in transplant animals and markedly inhibited insulin secretory responses to tolbutamide and theophylline. Control and transplant rats exhibited comparable plasma epinephrine and norepinephrine values under basal conditions and in response to stress. The stress-induced impairment in glucose homeostasis of transplant rats was attributed to an increased sensitivity of the transplanted A- and B-cells to catecholamines secondary to their denervated state. Transplanted islet cells exhibited appropriate adaptive responses to chronic modulating factors such as a 48-hour fast and the administration of cortisol. However, cortisol-treated rats were unable to sustain a sufficient secretion of insulin to maintain normal carbohydrate tolerance, and consequently there was a reappearance of the diabetic state.

Presence of Pancreatic Hormones in Islet Cells With MHC-Class II Antigen Expression

In the normal rat pancreas, only few islet cells express MHC-class II antigens. Their nature and function has not yet been elucidated. We report a method for the purification of la-positive islet cells by fluorescenceactivated cell sorting. The isolated mononuclear cells appear of nonendocrine origin but contain vacuoles with partially digested secretory vesicles. Both insulinand glucagon-immunoreactive granules were identified in these vacuoles of varying size and composition. It is suggested that at least part of the rat islet cells with class II antigen expression can exhibit phagocytotic properties leading to the uptake of fragments from damaged endocrine cells. This functional characteristic may implicate this particular islet cell type in the pathology of the endocrine pancreas in type I diabetes.

Transplantation of Purified Islet Cells in Diabetic Rats: III. Immunosuppressive Effect of Cyclosporin

This study examined the effect of cyclosporin on the survival of islet β-cell allografts in streptozocin (STZ)-induced diabetic rats. At a daily oral dose of 5 mg/kg, the agent prevented the rejection of isolated islets, provided they were little contaminated by other pancreatic tissue. The immunosuppressive effect rapidly disappeared after discontinuation of the drug, except when the donor tissue had been pretreated to reduce its nonendocrine content. All recipients of cultured and selected islets maintained a normalized state for >15 wk beyond the 5-wk drug course; this was not the case for shorter periods of treatment. A long-term beneficial effect was also observed in all recipients of purified islet β-cell grafts, which reversed without treatment in half of the cases. Cyclosporin markedly reduced the mononuclear cell infiltration in each type of islet β-cell allograft; aggregates of mixed endocrine islet cells were kept virtually infiltration free. Conditions with minimal initial infiltration were associated with long-term graft survival without the need for continuous pharmacological immunosuppression. We conclude that a short-term cyclosporin treatment can induce long-term survival of allografted islet β-cells, provided the grafts are only slightly contaminated by nonendocrine elements. In rodents, sufficient immunosuppression was achieved by circulating cyclosporin levels of 100–400 ng/ml. Higher concentrations were cytotoxic for cultured islet β-cells and islet non–²-cells. A 5-wk treatment with the immunomodulator ciamexone also resulted in long-term survival of purified β-cell allografts but not of cultured islets. These findings indicate that the success of immunosuppressive treatment in islet cell transplantation can be considerably improved by preparing purified cell grafts of well-defined cellular composition.

Xenotransplantation of purified pre-natal porcine beta cells in mice normalizes diabetes when a short anti-CD4-CD8 antibody treatment is combined with transient insulin injections.

Background:  Pre‐natal porcine endocrine islet cell grafts were recently shown to contain immature beta cells with a marked potential for growth and differentiation following transplantation, and hence for a progressive and long‐term correction of diabetes in immune‐incompetent mice. The present study investigates whether these grafts are also capable of correcting hyperglycemia in immune‐competent mice receiving a short treatment with anti‐CD4–CD8 antibodies.