It has previously been observed that the insulin-producing cells of human pancreatic islets are more resistant to alloxan-, streptozotocin-, nitroprusside-, or cytokine-induced injury than those of ...mouse and rat islets.
Human pancreatic islets were obtained from heart-beating organ donors. The expression of the stress proteins heat shock protein 70 (hsp70) and heme oxygenase and the anti-apoptosis gene bcl-2 was determined in isolated rat, mouse, and human islets, either cultured in vitro or transplanted under the kidney capsule of nude mice, using immunoblot analysis. Rat and human islet sensitive hydrogen peroxide was assess by glucose oxidation measurements. Isolated islets were also analyzed for their catalase and superoxide dismutase activities, and the islet cell levels of reduced glutathione were determined in response to hydrogen peroxide and nitroprusside. Programmed cell death in human and rat islets in response to streptozotocin was evaluated using TUNEL staining.
Cultured human islets expressed higher contents of hsp70 than mouse and rat islets at basal conditions. Also after 4 weeks under the kidney capsule of normoglycemic mice, the hsp70 levels were higher in human islets than in rat islets. The expression of another stress protein, heme oxygenase (HO), was strongly increased in cultured rat islets, but was not affected in human islets. Expression of the bcl-2 gene could not be detected in human islets. In spite of this, 0.5 mM streptozotocin induced apotosis in rat but not in human islet cells. Hydrogen peroxide (0.1 and 0.4 mM) decreased glucose oxidation rates in rat but not in human islets. The levels of reduced glutathione were moderately decreased in human and rat islet cells and sharply decreased in mouse islet cells in response to hydrogen peroxide. Moreover, the activities of catalase and superoxide dismutase (SOD) were markedly lower in mouse islets than in human islets. The activity of catalase was lower in rat islets than in human islets.
Human islets differ clearly from mouse and rat islets in their increased expression of hsp70, catalase, and SOD, which may explain the increased resistance of human islets to beta cell toxins.
Islet transplantation can restore insulin production in type 1 diabetes patients. However, survival of the islet allografts will face rejection or recurrence of autoimmunity or a combination of both. ...In a study on islet-after-kidney transplants, we previously reported that islet cell recipients presented low T-cell alloresponses for HLA mismatches that were shared by the islet cell graft and the prior kidney graft, that is, repeated mismatch, while vigorous responses were measured against novel HLA mismatches.
We now investigated T-cell alloreactivity to repeated HLA-mismatches in three non-uremic type 1 diabetic patients each receiving three sequential islet cell implants.
These islet-after-islet recipients patients exhibited low or absent responses to repeated mismatches to the first graft which was accompanied by sustained graft function, and reduced responsiveness towards subsequent grafts. In one patient, T-cell responses towards these mismatches were noticed following new mismatches in subsequent grafts, with loss of graft function.
These case reports further support the view that subsequent islet implantations can reduce alloreactivity for repeated HLA mismatches. They demonstrate the usefulness of monitoring T-cell reactivity against islet allografts to correlate immune function with graft survival and to identify conditions for preservation of beta-cell function.
Rat pancreatic beta cells differ in their individual sensitivity to glucose-inducible metabolic changes. The present study examines whether beta cells with a higher metabolic threshold require higher ...glucose levels for stimulation of their secretory activity. Purified beta cells were distributed according to their metabolic redox state at 7.5 mM glucose; the metabolically responsive (high responsive) and unresponsive (low responsive) subpopulations of comparable size and viability were reaggregated in the presence of 3Htyrosine and then perfused at 2.8 mM glucose with 10-min pulses of increasing glucose concentration. Glucose elicited first-phase insulin release in both high and low responsive subpopulations from, respectively, 4.2 and 8.3 mM on. The amplitude of both secretory responses increased dose dependently, the rates in the high responsive subpopulation being 2-fold higher than in the low responsive one. At all stimulating glucose levels, fractional release of 3H-labeled insulin was 3- to 4-fold higher than that of immunoreactive insulin. Preferential release of newly formed insulin was already maximally stimulated at 4.2 mM glucose in the high responsive subpopulation, whereas it increased dose-dependently in the low responsive one. These results indicate the existence of intercellular differences in the secretory activity of glucose-exposed beta cells, both in terms of glucose sensitivity and of amplitude. This heterogeneity in beta cell secretory responsiveness parallels that which has been previously described for the cellular metabolic and biosynthetic functions. It is concluded that glucose dose-dependently recruits beta cells into both biosynthetic and secretory activities. Co-existence of inactive and activated cells can explain preferential release of newly synthesized over preformed hormone during glucose stimulation.
Somatostatin (SS)-14 and SS28 are produced by pancreatic D cells and gut mucosa and inhibit pancreatic islet insulin and glucagon release. There are five distinct SS receptor (SSTR) subtypes, namely ...SSTR1–5, which show different affinities for SS14 and SS28. In order to identify the subtype responsible for inhibition of insulin release by human B cells, SSTR-selective SS analogs were tested in isolated human islets. Glucose-stimulated insulin secretion in human islets incubated for 1 hr at 20 mM glucose, and in islets cultured for 24 hr at a near-physiological (6.1 mM) glucose concentration, was inhibited (<50% of the control) by SSTR5-specific analogs and by SS14 and SS28. SS14, SS28, and different SSTR5 preferential analogs also inhibited islet amyloid polypeptide release during the 24-hr culture. On the other hand, a group of SSTR2-selective analogs failed to inhibit insulin release. Analysis by reverse transcription-polymerase chain reaction indicated that human islets express similar amounts of SSTR2 and SSTR5 mRNAs, while human pancreatic ductal cells express much lower levels of these mRNAs. In conclusion, our data suggest that SSTR5 is an important mediator of the insulin inhibitory action of SS in cultured human islets.
Aims/hypothesis
Alginate-encapsulated human islet cell grafts have not been able to correct diabetes in humans, whereas free grafts have. This study examined in immunodeficient mice whether ...alginate-encapsulated graft function was inferior to that of free grafts of the same size and composition.
Methods
Cultured human islet cells were equally distributed over free and alginate-encapsulated grafts before implantation in, respectively, the kidney capsule and the peritoneal cavity of non-obese diabetic mice with severe combined immunodeficiency and alloxan-induced diabetes. Implants were followed for in vivo function and retrieved for analysis of cellular composition (all) and insulin secretory responsiveness (capsules).
Results
Free implants with low beta cell purity (19 ± 1%) were non-functional and underwent 90% beta cell loss. At medium purity (50 ± 1%), they were functional at post-transplant week 1, evolving to normoglycaemia (4/8) or to C-peptide negativity (4/8) depending on the degree of beta cell-specific losses. Encapsulated implants immediately and sustainably corrected diabetes, irrespective of beta cell purity (16/16). Most capsules were retrievable as single units, enriched in endocrine cells that exhibited rapid secretory responses to glucose and glucagon. Single capsules with similar properties were also retrieved from a type 1 diabetic recipient at post-transplant month 3. However, the vast majority were clustered and contained debris, explaining the poor rise in plasma C-peptide.
Conclusions/interpretation
In immunodeficient mice, i.p. implanted alginate-encapsulated human islet cells exhibited a better outcome than free implants under the kidney capsule. They did not show primary non-function at low beta cell purity and avoided beta cell-specific losses by rapidly establishing normoglycaemia. Retrieved capsules presented secretory responses to glucose, which was also observed in a type 1 diabetic recipient.
Trial registration:
ClinicalTrials.gov NCT01379729
Funding:
This study was supported by grants from the JDRF (centre grant 4-2005-1327), the Research Foundation Flanders (G.0801.10), the 6th and 7th Framework Program of the European Commission (numbers 512145 and 241883), and the Agency for Innovation by Science and Technology in Flanders (IWT-TBM7 090884).
Aims/Hypothesis Insulin resistance has been proposed as a risk factor for type 1 diabetes. We investigated whether adiponectin, an insulin sensitiser, can serve as an additional predictive marker for ...type 1 diabetes in first-degree relatives of known patients. Methods Adiponectin was followed in 211 persistently islet antibody-positive (Ab+) first-degree relatives of type 1 diabetic patients and in 211 age- and sex-matched persistently antibody-negative relatives, and correlated with antibody status, random proinsulin:C-peptide ratio and HLA-DQ genotype. During follow-up, 37 Ab+ relatives developed type 1 diabetes. Results In the group of 422 relatives, baseline adiponectin correlated inversely with age and BMI and was lower in male than in female participants, especially after 15 years of age (p < 0.001). There was no correlation with antibody status or later development of diabetes. In 24 Ab+ relatives sampled fasted, adiponectin levels correlated significantly with homeostasis model assessment of insulin sensitivity (p = 0.006). In Ab+ relatives (n = 211), adiponectin levels could not predict type 1 diabetes nor complement risk assessment based on islet antibodies, HLA-DQ genotype and pancreatic hormones in Cox regression analysis. Conclusions/Interpretation Adiponectin levels do not contribute to the prediction of type 1 diabetes in Ab+ relatives.
A worldwide increase in the incidence of childhood type 1 diabetes has been observed. Because in various countries the majority of new type 1 diabetic patients are diagnosed in adulthood, we ...investigated whether the rising incidence of this disorder in children reflects a global increase in the incidence of diabetes or a shift toward earlier clinical presentation.
The incidence of type 1 diabetes presenting before age 40 years was prospectively measured in the Antwerp district over a 12-year period (1989-2000). The completeness of ascertainment was evaluated by the capture-recapture method. Trends in incidence during the study period were analyzed by Poisson regression.
The incidence of type 1 diabetes diagnosed before age 40 years remained constant over the 12-year period, averaging 9.9 cases per 100,000 individuals per year. The incidence was similar in both sexes under age 15 years, but a marked male excess was noted for adult-onset disease, in particular after age 20 years, resulting in a male-to-female ratio of 0.9 under age 15 years vs. 1.6 thereafter (P = 0.001). During the 12-year observation period, there was a significant tendency toward increasing incidence under age 15 years at the expense of a decreasing incidence between ages 15 and 40 years (P = 0.025). The annual increase in incidence averaged 1.8% under age 15 years and 5.0% under age 5 years (P = 0.06).
Our results indicate that in Belgium, the increasing incidence of childhood type 1 diabetes-especially for children under age 5 years-is not attributable to a global increase in disease incidence, but rather to earlier clinical manifestation. The results suggest that an environmental factor may preferentially accelerate the subclinical disease process in young diabetes-prone subjects.
Aims/hypothesis Neogenesis of beta cells and their clustering to small aggregates is a key process in prenatal development of beta cell mass. We investigated the contribution of postnatally formed ...small aggregates to functional beta cell mass in adult rats. Methods Conditions were defined for (1) counting total beta cell number in pancreases with relative error of <10% and (2) determining their distribution over aggregates of different size and over functionally different subpopulations. Results Pancreases of 10-week-old male Wistar rats contained 2.8 ± 0.2 × 10⁶ beta cells, of which >90% was generated postnatally, involving: (1) neo-formation of 30,000 aggregates with diameter <50 μm including single cells; and (2) growth of 5,500 aggregates to larger sizes, accounting for 90% of the increase in cell number, with number of growing aggregates in the tail 50% greater than elsewhere. At 10 weeks, 86% of aggregates were <50 μm; compared with aggregates >200 μm, their beta cells exhibited a higher basal insulin content that was also resistant to glibenclamide-induced degranulation. The pool of Ki67-positive beta cells was sixfold larger than at birth and distributed over all aggregate sizes. Conclusions/interpretation We describe a method for in situ counting of beta cell numbers and subpopulations with low relative error. In adult rats, >90% of beta cells and beta cell aggregates are formed after birth. Aggregates <50 μm are more than 100-fold more abundant than aggregates >200 μm, which are selected for isolated islet studies. Their topographic and functional properties contribute to the functional heterogeneity of the beta cell population; their growth to larger aggregates with characteristic beta cell functions may serve future metabolic needs.
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