Islet Exhaustion Research Articles

Long-term Effect of Islet Transplantation on Glycemic Variability

Islet transplantation has been reported to restore normoglycemia and the overall metabolic control in type 1 diabetes mellitus (DM). In the most experienced centers, islet transplantation clinical outcome is similar to that of the whole pancreas transplantation. Long-term islet transplantation function remains a very interesting matter worth discussing. A progressive islet function decrease was reported, probably due to islet exhaustion. In 5 islet-transplanted patients with at least 3-yr follow-up and still insulin independent, their glycemic control was characterized by a blinded retrospective continuous glucose monitoring system (CGMS). Islet transplantation restored glycemic control and glucose variability. Data were compared with patients in the waiting list. All the parameters of glycemic variability tested had improved significantly in patients who had islet transplantation compared with those patients who were on the waiting list. In conclusion, islet transplantation is able to maintain a proper glucose control and normalize glycemic variability in selected patients. A blinded retrospective CGMS is a useful method to characterize glucose homeostasis deeply in vivo in islet-transplanted patients.

Hepatic steatosis after islet transplantation: Can ultrasound predict the clinical outcome? A longitudinal study in 108 patients

Percutaneous intra-portal islet transplantation (PIPIT) is a less invasive, safer, and repeatable therapeutic option for brittle type 1 diabetes, compared to surgical pancreas transplantation. Hepatic steatosis is a consequence of the islet engraftment but it is curiously present in a limited number of patients and its meaning is controversial. The aims of this study were to assess hepatic steatosis at ultrasound (US) after PIPIT investigating its relationship with graft function and its role in predicting the clinical outcome.From 1996 to 2012, 108 patients underwent PIPIT: 83 type-1 diabetic patients underwent allo-transplantation, 25 auto-transplantation. US was performed at baseline, 6, 12, and 24 months, recording steatosis prevalence, first detection, duration, and distribution. Contemporaneously, steatotic and non-steatotic patients were compared for the following parameters: infused islet mass, insulin independence rate, β-score, C-peptide, glycated hemoglobin, exogenous insulin requirement, and fasting plasma glucose.Steatosis at US was detected in 21/108 patients, 20/83 allo-transplanted and 1/25 auto-transplanted, mostly at 6 and 12 months. Infused islet mass was significantly higher in steatotic than non-steatotic patients (IE/kg: S=10.822; NS=6138; p=0.001). Metabolically, steatotic patients had worse basal conditions, but better islet function when steatosis was first detected, after which progressive islet exhaustion, along with steatosis disappearance, was observed. Conversely, in non-steatotic patients these parameters remained stable in time. Number of re-transplantations was significantly higher in steatotic than in non-steatotic patients (1.8 vs 1.1; p=0.001).Steatosis at US seems to be related to the islet mass and local overworking activity. It precedes metabolic alterations and can predict graft dysfunction addressing to therapeutic decisions before islet exhaustion. If steatosis does not appear, no conclusion can be drawn.

Early endocrine and molecular changes in metabolic syndrome models

The twenty-first century arrived in the middle of a global epidemic of metabolic syndrome (MS) and type 2 diabetes mellitus (DM2). It is generally accepted that an excess of nutrients linked to a low physical activity triggers the problem. However, the molecular features that interact to develop the MS are not clear. In an effort to understand and control them, they have been extensively studied, but this goal has not been achieved yet. Nonhuman animal models have been used to explore diet and genetic factors in which experimental conditions are controlled. For example, only one factor in the diet, such as fats or carbohydrates can be modified to better understand a single change that would be impossible in humans. Most of the studies have been done in rodents. However, it is difficult to directly compare them, because experiments are different in more than one variable; genetic strains, amount, and the type of fat used in the diet and sex. Thus, the only possible criteria of comparison are the relevance of the observed changes. We review different animal models and add some original observations on short-term changes in metabolism and beta cells in our own model of adult Wistar rats that are not especially prone to get fat or develop DM2, treated with 20% sucrose in drinking water. One early change observed in pancreatic beta cells is the increase in GLUT2 expression that is located to the membrane of the cells. This change could partially explain the presence of insulin hypersecretion and hyperinsulinemia in these rats. Understanding early changes that lead to MS and in time to pancreatic islet exhaustion is an important biomedical problem that may contribute to learn how to prevent or even reverse MS, before developing DM2.

Transplantation of Long-term Cultured Porcine Islets in the Rat: Prolonged Graft Survival and Recipient Growth on Reduced Immunosuppression

To evaluate whether further improvement in porcine islet xenotransplantation is feasible, a number of questions were addressed. Earlier we showed significant improvement in the nude mouse of the porcine islets by selection through long-term culture. Now these islets were tested in the stringent pig-to-rat model. Islets were isolated from adult pigs, cultured for 1.5-3 weeks and transplanted to rats. Possible rejection mechanisms were assessed by interference of the cellular response with cyclosporine A (CsA), blocking macrophages with gadolinium chloride (GdCl), and suppressing the humoral response with cyclophosphamide. Modifications in graft size and condition were analyzed. Untreated control recipients showed primary nonfunction (PNF). CsA treatment could fully overcome PNF and resulted in graft survival from 10 to over 134 days. Rejection was the main cause of function loss. Although rejection could not be prevented by intensifying the induction therapy, increased maintenance immunosuppression effectively blocked rejection, albeit at the expense of toxicity. Blocking the humoral response was ineffective; all grafts showed PNF. In contrast, depletion of macrophages fully prevented PNF. Combination of GdCl and CsA gave no additional effect, and grafts were rejected between 57 and 162 days. Generally, graft survivals were similar to those reported in the literature; however, long-term cultured islets required much less maintenance immunosuppression. Cessation of graft function was not always due to rejection; in some cases "islet exhaustion" was found, possibly caused by discrepancy between the graft size and the rapidly growing recipient. Neither the presence of damaged islet tissue in the graft nor the size of the graft exerted any influence on graft survival. On rejection, no real infiltration of the graft was seen; destruction gradually processed from the outside. The good functional capability of the cultured islets was illustrated by disappearance of the clinical symptoms and increase in body weight, which almost doubled in the long-term survivors.

Pancreatic islet expression profiling in diabetes-prone C57BLKS/J mice reveals transcriptional differences contributed by DBA loci, including Plagl1 and Nnt

BackgroundC57BLKS/J (BLKS) mice are susceptible to islet exhaustion in insulin-resistant states as compared with C57BL6/J (B6) mice, as observed by the presence of the leptin receptor (Lepr) allele, Leprdb/db. Furthermore, DBA2/J (DBA) mice are also susceptible to β-cell failure and share 25% of their genome with BLKS; thus the DBA genome may contribute to β-cell dysfunction in BLKS mice.ResultsHere we show that BLKS mice exhibit elevated insulin secretion, as evidenced by improved glucose tolerance and increased islet insulin secretion compared with B6 mice, and describe interstrain transcriptional differences in glucose response. Transcriptional differences between BLKS and B6 mice were identified by expression profiling of isolated islets from both strains. Genomic mapping of gene expression differences demonstrated a significant association of expression differences with DBA loci in BLKS mice (P = 4×10-27).ConclusionTwo genes, Nicotinamide nucleotide transhydrogenase (Nnt) and Pleiomorphic adenoma gene like 1 (Plagl1), were 4 and 7.2-fold higher respectively in BLKS islets, and may be major contributors to increased insulin secretion by BLKS islets. Contrary to reports for B6 mice, BLKS mice do not harbor a mutant Nnt gene. We detected 16 synonymous polymorphisms and a two-amino acid deletion in the Plagl1 gene in BLKS mice. Several inflammatory glucose-responsive genes are expressed at a higher level in BLKS, suggesting an inflammatory component to BLKS islet dysfunction. This study describes physiological differences between BLKS and B6 mice, and provides evidence for a causative role of the DBA genome in β-cell dysfunction in BLKS mice.

Obesity and non-insulin-dependent diabetes mellitus in Swiss-Webster mice associated with late-onset hepatocellular carcinoma

Genetic mutations resulting in obesity and type 2 diabetes mellitus (T2D) are described for both inbred and outbred mice. However, no known mouse model completely recapitulates human T2D and its comorbidities. We identified a cohort of obese, male, outbred Swiss-Webster (SW) mice as polyuric, polydipsic, glucosuric, and hyperglycemic. Prevalence of glucosuria in the SW colony reached 60% (n=70) in males 8 weeks to 6 months of age. Despite severe obesity in some females, no females were diabetic. Pathologic findings in affected males included cachexia, dilated gastrointestinal tracts with poor muscular tone, pancreatic islet degeneration and atrophy with compensatory metaplasia and/or neogenesis, bacterial pyelonephritis, membranous glomerulopathy, and late-onset hepatic tumors with macrosteatosis, microsteatosis, and hydropic change in aged males. Serum insulin correlated with blood glucose in a nonlinear pattern, suggestive of islet exhaustion. Circulating leptin levels showed a weak inverse correlation with glucose. Diabetic males were bred with obese colony females to produce 20 male and 20 female offspring. Prevalence of diabetes in male offspring was 80% (16/20) with a median age of onset of 18 weeks. By contrast, no diabetic females were identified, despite being significantly more obese than males. Male predominance is likewise a feature of T2D in humans. To our knowledge, this is the first documentation of hepatocellular carcinoma and islet metaplasia and/or neogenesis in a spontaneous outbred mouse model of T2D. The SW availability and histopathologic features represent a promising new model for the study of T2D.

DETECTION OF CIRCULATING BETA CELLS IN THE PERIPHERAL BLOOD AFTER HUMAN ISLET TRANSPLANTION PREDICTS DETERIORATION OF METABOLIC CONTROL

O451* Aims: Recent updates of the Edmonton trial have shown that insulin independence can be progressively lost in as much as half of islet transplant recipients over the first 3 years after transplantation. This “islet exhaustion” could be due to damage to the grafts secondary to rejection, recurrence of autoimmunity or non specific insults, all 3 of which are exceedingly difficult to predict. Early prediction of islet graft injury could prompt the implementation of strategies attempting to salvage the transplanted islets. We hypothesize that islet damage could be associated with the release of beta cells in the peripheral circulation, and could be predicted by the detection of circulating insulin mRNA. Methods: Whole blood samples were prospectively taken from patients with type 1 diabetes receiving intraportal islet transplants from July 2002 to December 2003, immediately prior to transplantation and at regular time points thereafter. Only grafts with at least 50 days of follow-up were considered. Twenty islet transplant procedures performed in 12 patients were analyzed. Mean follow-up was 294 days (53-593). After RNA extraction, levels of insulin mRNA were determined by quantitative RT-PCR and expressed as number of copies/ ml, as a reflection of the amount of circulating beta cells. Results: All patients exhibited a primary peak of insulin mRNA at the time of transplantation lasting for a mean of 3.8 days (1-13), indicating a release of circulating beta cells related to the procedure. Mean amplitude of the peaks was 399 copies/ml (18-2575). Duration and amplitude of the primary peak were not correlated with graft size or outcome. Twenty-one subsequent peaks of insulin mRNA were observed during the follow-up of 14 transplantations, lasting for a mean of 5.2 days (1-22), with a mean amplitude of 90 copies/ml (15-463). Thirteen secondary peaks (62%) were simultaneous to or closely followed by events related to islet graft function (rejection episodes, increase in insulin requirements, increase in HbA1c). Duration and amplitude of peaks were higher when they heralded occurrence of an adverse event (8 vs 1 days, p=0.02; 115.5 vs 49.5 copies/ml, p=0.08). Conclusions: Insulin mRNA is always detected immediately after islet transplantation, and the duration and amplitude of the primary insulin mRNA peak is correlated neither to graft size nor to outcome. Subsequent peaks of insulin mRNA can be detected and often associated with alterations of islet graft function, especially when they are prolonged or of high amplitude. These data suggest that insulin mRNA detection in the peripheral blood is a promising method for the prediction of islet graft damage.

Islet transplantation in type 1 diabetic patients

Islet transplantation has been shown to improve overall glucose homeostasis and retard the progression of complications in type I diabetic patients. Also the percentage of recipients achieving complete insulin independence has progressively increased over recent years. An unsolved problem is whether the short-term graft function is secondary to progressive islet exhaustion or to recurrent autoimmunity despite the immunosuppressive therapy. The indications for this procedure remain limited to selected type I diabetic patients. The risks of the immunosuppressive therapy are only proposed to type I diabetic recipients with uncontrolled disease, despite all efforts of the diabetologist and the patient (brittle diabetes), or with a poor quality of life due to unawareness hypoglycemia or severe chronic and progressive complications.

Assessment of intracellular insulin content during all steps of human islet isolation procedure

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification ( n = 20), and 3–4 day culture at 22°C ( n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 ± 20.8 U/pancreas (mean ± SEM) increased to 264.6 ± 22.8% ( p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput ( n = 5) was 2.4 ± 0.4 ( p < 0.05). After purification total insulin recovery was 55.3 ± 4.8% ( p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 ± 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 ± 70.6 μU/IEQ before purification to 436.4 ± 58.1 μU/IEQ ( p < 0.001) after purification. After 22°C culture ( n = 6) recovery of insulin and IEQ was 80.1 ± 8.1% ( p < 0.05) and 92.8 ± 3.5% ( p = NS), respectively. Insulin content per IEQ decreased to 85.8 ± 6.5% ( p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

NO IMPROVEMENT OF PANCREAS TRANSPLANT ENDOCRINE FUNCTION BY EXOGENOUS INSULIN INFUSION (ISLET REST) IN THE POSTOPERATIVE PERIOD

The concept of islet exhaustion maintains that exposure of pancreatic islets to hyperglycemia and other stresses leads to islet dysfunction and irreparable damage. The process of pancreatic transplantation places many stresses on islets (e.g., counter-regulatory hormones, steroids, cyclosporine toxicity). As practiced by some centers, it may be important to administer exogenous insulin in the postoperative period to provide islet rest. Using a porcine pancreas transplant model that simulates clinical transplantation, we studied 2 groups: 1 group (n = 8) received constant insulin infusion for 7 days after transplantation; the control group (n = 5) received vehicle only. The islets in the insulin infusion group were rested as evidenced by a significantly decreased mean C-peptide level (0.27 +/- 0.04 ng/ml) as compared to the control group (0.66 +/- 0.08 ng/ml) (P less than 0.05). After insulin infusion was discontinued, intravenous glucose tolerance testing found insulin, C-peptide and glucagon responses were not different between groups. Glucose clearance was also comparable; K values were -1.79 and -1.60 in the insulin infusion and control groups, respectively. In conclusion, islet rest by insulin infusion for 7 postoperative days did not improve subsequent pancreas transplant endocrine function.

Islet morphology in young, genetically diabetic Chinese hamsters during the hyperinsulinemic phase.

Plasma glucose and insulin levels were measured in genetically diabetic (subline L) hamsters from 2 1/2 weeks to over 1 year of age. This nonobese subline is known for subnormal pancreatic insulin release. Hyperglycemia was seen from 4 weeks onward. A short period of hyperinsulinemia was seen at 6 weeks of age, after which plasma insulin levels were not different from normal. During the hyperinsulinemia phase (age 6 weeks), pancreatic insulin content was subnormal, islet volume (as percent of pancreas cross-sectional area) and islet size (as islet cross-sectional area in micron2) were not different from normal, islet morphology appeared normal in electron micrographs, but granule volume was slightly but significantly below normal. Because the islets appeared morphologically normal, except for the slight degranulation during the hyperinsulinemic phase, these data suggest (a) that the islets "hypersecrete" to reduce hyperglycemia; but (b) synthesis does not increase enough to maintain a normal pancreatic insulin content; (c) that the first sign of pancreatic failure may be degranulation and decreased insulin synthesis or content; (d) that early peripheral resistance to insulin, rather than early islet failure, may cause the early hyperglycemia, and (e) that early hyperglycemia or insulin resistance contributes to the later islet exhaustion, which is seen in these diabetic animals when adult.

HPLC analysis of nucleotide profiles in glucose-stimulated perifused rat islets

Nucleotide concentrations were measured in isolated pancreatic islets from rats using HPLC. This method was used to study the role of nucleotides in islets, which were perifused for several hours with different substrates and exhibited signs of exhaustion. Using islet tissue extracts, it was possible to determine 11 nucleotides. It was hoped that alterations in islet nucleotide concentrations would provide clues for the biochemical basis for the exhaustion phenomenon; however, changes in nucleotide concentrations that were observed in exhausted islet tissue did not correlate with corresponding changes in insulin release. Therefore, they are unlikely to be directly related to islet exhaustion.