Zhidong Ling

β Cells Can Be Generated from Endogenous Progenitors in Injured Adult Mouse Pancreas

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in todays diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Expression and Functional Activity of Glucagon, Glucagon-Like Peptide I, and Glucose-Dependent Insulinotropic Peptide Receptors in Rat Pancreatic Islet Cells

Rat pancreatic α- and β-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in β-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in β-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet α-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in β-cells and not in α-cells. Receptor activity, measured as cellular cAMP production after exposing islet β-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified α-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in β-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat β-cells causes cAMP production required for insulin release, while adenylate cyclase in α-cells is positively regulated by GIP.

Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation.

Human beta cells can be maintained in serum-free culture at 6 mmol/liter glucose, with 80% cell recovery and preserved glucose-inducible functions after 1 wk. Between 0 and 10 mmol/liter, glucose dose-dependently increases the number of beta cells in active protein synthesis (15% at 0 mmol/liter glucose, 60% at 5 mmol/liter, and 82% at 10 mmol/liter), while lacking such an effect in islet non-beta cells (> 75% activated irrespective of glucose concentrations). As in rat beta cells, this intercellular difference in glucose sensitivity determines the dose-response curves during acute glucose stimulation of human beta cells. During 2-h incubations, human beta cells synthesize 7 fmol insulin/10(3) cells at 0 mmol/liter glucose, 20 fmol at 5 mmol/liter, and 31 fmol at 10 mmol/liter. Culture at higher (10 or 20 mmol/liter) glucose does not affect beta cell recovery but decreases by 50-85% the net effect of glucose upon insulin synthesis and release. These reduced responses to glucose are not caused by diminished cellular activities but are the consequence of a shift of beta cells to a state of sustained activation. The presence of more activated cells at low glucose eliminates glucose-dependent cell recruitment as a mechanism for adjusting beta cell responses to acute variations in glucose concentration. It leads to elevated basal biosynthetic (3-fold) and secretory (10-fold) activities, and, hence, to a 4-fold reduction in the beta cell insulin content and the amount of insulin released at maximal glucose stimulation. Prolonged exposure of human beta cells to high glucose can thus lead to a loss of their glucose regulation as a consequence of sustained cellular activation, without signs of glucose-induced toxicity or desensitization.

Screening for Insulitis in Adult Autoantibody-Positive Organ Donors

Antibodies against islet cell antigens are used as predictive markers of type 1 diabetes, but it is unknown whether they reflect an ongoing autoimmune process in islet tissue. We investigated whether organs from adult donors that are positive for autoantibodies (aAbs) against islet cell antigens exhibit insulitis and/or a reduced β-cell mass. Serum from 1,507 organ donors (age 25–60 years) was analyzed for islet cell antibodies (ICAs), glutamate decarboxylase aAbs (GADAs), insulinoma-associated protein 2 aAbs (IA-2As), and insulin aAbs. Tissue from the 62 aAb+ donors (4.1%) and from matched controls was examined for the presence of insulitis and for the relative area of insulin+ cells. Insulitis was detected in two cases; it was found in 3 and 9% of the islets and consisted of CD3+/CD8+ T-cells and CD68+ macrophages; in one case, it was associated with insulin+ cells that expressed the proliferation marker Ki67. Both subjects belonged to the subgroup of three donors with positivity for ICA, GADA, and IA-2-Ab and for the susceptible HLA-DQ genotype. Comparison of relative β-cell area in aAb+ and aAb− donors did not show a significant difference. Insulitis was found in two of the three cases that presented at least three aAbs but in none of the other 59 antibody+ subjects or 62 matched controls. It was only detected in <10% of the islets, some of which presented signs of β-cell proliferation. No decrease in β-cell mass was detected in cases with insulitis or in the group of antibody+ subjects.

Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipient characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft

Summary Islet allografts in insulin-dependent diabetic (IDDM) patients exhibit variable survival lengths and low rates of insulin-independence despite treatment with anti-T-cell antibodies and maintenance immunosuppression. Use of poorly characterized freshly isolated preparations makes it difficult to determine whether failures are caused by variations in donor tissue. This study assesses survival of standardized beta-cell allografts in C-peptide negative IDDM patients on maintenance immunosuppression following kidney transplantation and without receiving anti-T-cell antibodies or additional immunosuppression. Human islets were isolated from pancreatic segments after maximal 20 h cold-preservation. During culture, preparations were selected according to quality control tests and combined with grafts with standardized cell composition (≥ 50 % beta cells), viability ( ≥ 90 % ), total beta-cell number (1 to 2 · 106/kg body weight) and insulin-producing capacity (2 to 4 nmol · graft–1· h–1). Grafts were injected in a liver segment through the repermeabilized umbilical vein. After 2 weeks C-peptide positivity, four out of seven recipients became C-peptide negative; two of them were initially GAD65-antibody positive and exhibited a rise in titre during graft destruction. The other three patients remained C-peptide positive for more than 1 year, two of them becoming insulin-independent with near-normal fasting glycaemia and HbA1 c; they remained GAD65- and islet cell antibody negative. The three patients with surviving grafts presented a history of anti-thymocyte globulin therapy at kidney transplantation. Long-term surviving grafts increased C-peptide release following intravenous glucagon or oral glucose but not following intravenous glucose. Thus, cultured human beta-cells can survive for more than 1 year in IDDM patients on maintenance anti-rejection therapy for a prior kidney graft and without the need for an increased immunosuppression at the time of implantation. The use of functionally standardized beta-cell grafts helps to identify recipient and graft factors which influence their survival and metabolic effects. Insulin-independence can be achieved by injection of 1.5 million beta-cells per kg body weight in a liver segment. These beta-cell implants respond well to adenylcyclase activators but poorly to glucose. [Diabetologia (1998) 41: 452–459]

Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft.

Islet grafts can induce insulin independence in type 1 diabetic patients, but their function is variable with only 10% insulin indepence after 5 years. We investigated whether cultured grafts with defined β cell number help standardize metabolic outcome. Nonuremic C-peptide-negative patients received an intraportal graft with 0.5–5.0 × 106 β cells per kilogram of body weight (kgBW) under antithymocyte globulin and mycophenolate mofetil plus tacrolimus. Metabolic outcome at posttransplant (PT) month 2 was used to decide on a second graft under maintenance mycophenolate mofetil/tacrolimus. Graft function was defined by C-peptide >0.5 ng/ml and reduced insulin needs, metabolic control by reductions in HbA1c, glycemia coefficient of variation, and hypoglycemia. At PT month 2, graft function was present in 16 of 17 recipients of >2 × 106 β cells per kgBW versus 0 of 5 with lower number. The nine patients with C-peptide >1 ng/ml and glycemia coefficient of variation of <25% did not receive a second graft; five of them were insulin-independent until PT month 12. The 12 others received a second implant; it achieved insulin-independence at PT month 12 when the first and second graft contained >2 × 106 β cells per kgBW. Of the 20 recipients of at least one graft with >2 × 106 β cells per kgBW, 17 maintained graft function and metabolic control up to PT month 12. At PT month 12, β cell function in insulin-independent patients ranged around 25% of age-matched control values. Thus, 1-year metabolic control can be reproducibly achieved and standardized by cultured islet cell grafts with defined β cell number.

Physiologic relevance of heterogeneity in the pancreatic beta-cell population

SummaryIn vitro studies on purified rat beta cells have indicated a functional diversity among insulincontaining cells. Intercellular differences were found in the rates of glucose-induced insulin synthesis and release. They are attributed to differences in cellular thresholds for glucose utilization and oxidation, as can be caused by varying activities in rate limiting steps such as glucokinase-dependent phosphorylation. The percent of functionally active beta cells increases dose-dependently with the glucose concentration, making cellular heterogeneity and its regulation by glucose major determinants for the dose-response curves of the total beta-cell population. Beta cells which are already responsive to low glucose concentrations are characterized by a higher content in pale immature granules; their activated biosynthetic and secretory activity accounts for preferential release of newly-formed hormone by the total beta-cell population. At any glucose level, the amplitude of insulin release depends on the percent glucose-activated cells and their cyclic AMP content, an integrator of (neuro)hormonal influences. The in vitro described heterogeneity in beta-cell functions may bear physiological relevance as several of its characteristics are also detectable in intact pancreatic tissue; furthermore, in vitro signs of heterogeneity can be altered by prior in vivo treatment indicating that they express properties of the cells in their in situ configuration. Elevated basal levels of (pro)insulin may reflect the existence of an increased number of beta cells that are activated at low physiologic glucose concentrations. Reductions in stimulated insulin levels can be caused by decreased numbers of beta cells that are activated at the prevailing glucose concentration or by insufficient cyclic AMP levels in beta cells, possibly as a result of inadequate signalling from hormones of local or distal origin. Only few markers are currently available with which to explore these mechanisms in vivo. Additional markers and tests should help assess the possible role of variations in beta-cell heterogeneity in the pathogenesis of diabetes mellitus.

Effect of nutrients, hormones and serum on survival of rat islet beta cells in culture.

SummaryThis study quantifies the survival of purified single rat beta cells under different culture conditions. Less than 10% of the cells survive 9 days of culture in Hams F10 medium without supplements. Addition of fetal calf serum (5%) increases cell survival to 54% in the absence and to 78% in the presence of isobutylmethylxanthine (50 μmol/l). The effect of serum is explained, at least partly, by the presence of albumin and of low molecular weight constituents. In serum-free Hams F10 with 50 μmol/l isobutylmethylxanthine, 75% of cells survive after the addition of bovine serum albumin (1%) and of ultroser (0.2%), a commercial serum substitute. Survival of at least 75% of cells is also maintained in Hams F10 with isobutylmethylxanthine plus albumin, and supplemented by metabolizable nutrients or by the peptides glucagon (10−8 mol/l) or growth hormone (1 μg/ml) plus insulin like growth factor-I (50 ng/ml). d-Glucose increases beta-cell survival in a dosedependent manner up to 10 mmol/l; a beneficial effect is also observed with other metabolizable compounds (leucine and glutamine) but not with non-metabolizable monosaccharides. Glucose-induced survival of islet beta cells can be attributed to its dose-dependent recruitment of cells into metabolic activities; however, a 9-day exposure to excessively high nutrient concentrations (> 20 mmol/l glucose) is deleterious to the cells. These results define culture media, with or without serum, wherein at least 75% of single rat islet beta cells can survive for a minimum of 9 days. This will allow for studies on beta-cell toxic conditions and potentially protective agents. The data also serve as basis for developing media with better survival of beta cells in cultured aggregates.

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.

Nestin Is Expressed in Vascular Endothelial Cells in the Adult Human Pancreas

In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PE-CAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.