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A recently published study by Butler et al. concluded that incretin treatment had adverse effects on the human type 2 diabetic pancreas including 'a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by α-cell hyperplasia with the potential for evolution into neuroendocrine tumours'. Incretin therapy has become widely used for type 2 diabetes, so these conclusions have instigated major concerns with regard to patient safety. We reassessed both the clinical case information and virtual microscopy images of the same 34 cases that were used in the Butler study as well as Network for Pancreatic Organ Donation (nPOD) cases that were not included. Whereas we would like to stress that it is important to investigate in depth any indication that incretin treatment may lead to inflammation or dysplasia in the pancreas, we find that the data presented in the Butler paper have serious methodological deficiencies that preclude any meaningful conclusions., (© 2014 John Wiley & Sons Ltd.)

Background: Rapid revascularization of islet cell implants is important for engraftment and subsequent survival and function. Development of an adequate vascular network is expected to allow adaptive growth of the β-cell mass. The present study compares omentum and kidney capsule as sites for growth and differentiation of immature β-cell grafts., Methods: Perinatal porcine islet cell grafts were implanted in omentum or under kidney capsule of nondiabetic nude rats. Implants were compared over 10 weeks for their respective growth, cellular composition, number and size of β cells, their proliferative activity, and implant blood vessel density., Results: In both sites, the β-cell volume increased fourfold between weeks 1 and 10 reflecting a rise in β-cell number. In the omental implants, however, the cellular insulin reserves and the percent of proliferating cells were twofold higher than in kidney implants. In parallel, the blood vessel density in omental implants increased twofold, reaching a density comparable with islets in adult pig pancreas. A positive correlation was found between the percent bromodeoxyuridine-positive β cells and the vessel density., Conclusions: Growth of the β-cell volume proceeds similarly in the omentum and under the kidney capsule. However, the omentum leads to higher insulin reserves and an increased pool of proliferating cells, which might be related to a more extended vascular network. Our observations support the omentum as an alternative site for immature porcine islet cells, with beneficial effects on proliferation and implant revascularization.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Amyloid-containing (A+) islets are characteristic for type-2 diabetes (T2D), but their abundance seems variable among patients. It is unclear whether the distribution of A+ islets follows a certain pattern or occurs randomly throughout the pancreatic organ. We investigated the topography of A+ islets in eight pancreata of T2D patients and eight sex- and age-matched non-diabetic subjects. Transversal sections of head, body and tail segments were stained with synaptophysin combined with Congo red to map/quantify islet tissue and amyloid. In the eight T2D pancreata, the overall percentage of A+ islets varied from 4% to 85%. Further analysis in body and tail indicated that peripheral regions exhibited higher percentages of A+ islets than central regions (averages of, respectively, 30% and 17%, P<0.05). Non-diabetic control pancreata also exhibited A+ islets, albeit at a 25-fold lower frequency; a tendency towards higher percentage of A+ islets in peripheral versus central regions was also observed. The higher percentage A+ islets in peripheral regions was associated with a higher density and relative islet over exocrine surface area. These observations on heterogeneity in abundance and distribution of A+ islets need consideration when sampling tissue for studies on human islet amyloidosis. The present methodology allows us to further investigate the susceptibility to amyloidosis of islets in peripheral regions of the pancreas.

An infertile couple was referred for intracytoplasmic sperm injection (ICSI) because of primary infertility and oligoasthenoteratozoospermia (OAT) in the male. It was observed that although the sperm cells presented with an unusual head size and multiple tails they were able to fertilize the oocytes after ICSI. Subsequent molecular cytogenetic analysis demonstrated de-novo chromosome abnormalities in virtually all sperm cells with 40% diploidy and 24% triploidy in addition to aneuploidy for the sex chromosomes.

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.

We present a case of prenatal diagnosis of Fanconi anaemia (FA) in a pair of twins at 14 weeks of gestation. The parents had previously had two children: a healthy boy and a boy with FA belonging to complementation group C (FAC). The FA patient is a compound heterozygote, carrying a 322delG and a IVS4+4A-->T mutation in the FAC gene. Prenatal DNA analysis showed that both fetuses were heterozygous for different mutations in the FAC gene. Both fetuses had normal male karyotypes. At 36 weeks the twins were born. They did not show congenital anomalies.

We studied 201 pregnancies that were established by in vitro fertilization and embryo transfer (IVF-ET) and compared the frequency of cytogenetic abnormalities with that found in a large control population matched for indication group (advanced maternal age) and time of sampling. A total of 252 IVF-ET fetuses were cytogenetically analysed by either chorionic villus sampling (CVS; n = 80) or amniocentesis (n = 172). Eleven chromosome abnormalities were found in the CVS group (13.8 per cent); among them, a 45,X/46,X,dic(Y)(q11)/46,X,del(Y)(q11) mosaic that was found in an IVF pregnancy established by intracytoplasmic sperm injection (ICSI), four cases of trisomy 21, and three cases of trisomy 7 confined to the placenta. The results indicate a statistically significant three- to five-fold increase in both confined placental abnormalities (P < 0.008) and true fetal chromosome anomalies (P < 0.04). In the amniocentesis group, identical rates (1.7 per cent) of chromosome abnormalities were found in the IVF-ET and control groups. It is concluded that late first trimester, but not early second trimester, IVF-ET pregnancies are characterized by an increased frequency of cytogenetic abnormalities found at prenatal diagnosis.

A 5-day culture of adult rat islets with human recombinant IL-1 beta (3 U/ml) resulted in the death of most alpha-cells and 50% of beta-cells. The IL-1--exposed islet tissue contained--in addition to poorly granulated beta-cells--patches of outgrowing monolayers and dispersed activated macrophages. In purified alpha- and beta-cell preparations, no cytodestructive effects of IL-1 (as high as 30 U/ml) were noticed, indicating that the cytokine is in itself not a beta-cell--selective killer. Pure beta-cells were, on the other hand, more sensitive (from 0.3 U/ml on) than intact islets to an IL-1--induced suppression of hormone synthesis. This inhibitory action was reversible and affected predominantly the production of insulin, leading to degranulated cells with modified shape and attachment. Further studies with IL-1 should take into account that isolated islet preparations do not allow distinction between its irreversible, indirect, and aspecific beta-cell toxicity and its reversible, direct, and specific suppression of beta-cell functions. It is not yet known whether IL-1--suppressed beta-cells exhibit an altered sensitivity to beta-cell--toxic conditions.

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 beta-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 beta-cell suspensions of greater than 95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet beta-cells from islet endocrine non-beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 10(6) pure beta-cells with or without 8 x 10(5) pure endocrine non-beta-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure beta-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified beta-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Aberrant expression of MHC class II molecules on endocrine cells has been proposed to induce autoimmune reactions in thyroid and endocrine pancreas. The present study examines whether MHC class II positive insulin-containing islet cells occur at the onset of diabetes in rats, in analogy to the findings in man. At the onset of diabetes, both streptozotocin-treated and diabetes-prone BB rats exhibited numerous class II positive islet cells that presented ultrastructural features of monocytes and were surrounded by class II negative islet B cells. These class II positive cells were characterized by vacuoles that contained insulin immunoreactive granules and disrupted membranes. Similar cells also appeared positive for the monocyte marker OX-42. The presence of class II positive monocytes with insulin-containing vacuoles may indicate a removal of damage B cells by infiltrating leukocytes. A similar electron microscopical study in man will be necessary to distinguish the putative endocrine pancreatic B cells with aberrant class II expression from infiltrating nonendocrine class II positive cells with insulin-containing phagosomes.

A method is developed for the preparation of single, pure, and viable rat pancreatic A and B cells in numbers sufficient for in vitro analysis. Islet isolation and dissociation techniques have been modified to increase the yield in islet cells per pancreas and per experiment. Islet cells are separated on the basis of their light scatter activity and flavin adenine dinucleotide autofluorescence into single non-B cells, single B cells, and structurally coupled B cells. Islet non-B cells are further purified into single A cells by autofluorescence-activated sorting according to the cellular nicotinamide adenine dinucleotide phosphate content at 20 mM glucose. Apart from offering the advantage of separating cells according to their functional characteristics, this procedure succeeds in the simultaneous isolation of 95-100% pure A and B cells. More than 50% of the cells in the initial islet preparation are recovered as single purified cells which can be maintained in culture. The isolated pancreatic A and B cells have been defined in terms of their cell volume, DNA and hormone content, and ultrastructural characteristics. The availability of pure pancreatic A and B cells is expected to contribute to our understanding of the regulation of glucagon and insulin release.

In rat pancreatic islets, gap junctional subunits (GJS) occur under two different configurations, namely in linear single strands and in polygonal particle aggregates. The present freeze-fracture study demonstrates that GJS can rapidly (dis)assemble into one of these membrane specializations without changes in their total number. Isolation of the pancreatic gland and its perfusion at 2.8 mM glucose is accompanied by a decrease in polygonally packed GJS from 46 to 16%. A rise in medium glucose concentration is followed, within 10 min, by a dose-dependent increase in the percent polygonal particles. This glucose effect on gap junction configuration is calcium dependent and reversible upon glucose removal; it is still entirely detectable when protein synthesis is blocked by cycloheximide. These results indicate that islet gap junctions are dynamic structures that rapidly adjust their configuration to extracellular regulators of beta-cell function. In the light of previous observations, it is suggested that this rapid (dis)assembly of gap junctional structures be considered as a component in the ionic and metabolic coupling between insulin-containing beta-cells of the pancreas.

Immunogold-silver staining is a relatively new technique in light-microscopic immunocytochemistry. It is based on silver enhancement of 1 to 10 nm gold probes immunologically bound to antigenic determinants in tissue sections. The detection efficiency of this third-generation detection technique is compared to that of the peroxidase-based ABC technique in routinely processed, paraffin-embedded, human biopsy material.

Single pancreatic B cells are purified by autofluorescence-activated cell sorting, and their secretory activity is measured after overnight culture. Compared to intact islets, the isolated cells release 2-fold more insulin under basal conditions and 5-fold less during nutrient stimulation. Their secretory activity can be induced by glucose, leucine, or arginine, but only 0.3-1.7% of their hormone content is liberated at 20 mM nutrient concentrations. This poor nutrient-induced insulin release from purified B cells is attributed to their low cAMP levels and is markedly increased after addition of (Bu)2cAMP, of glucagon, or of pancreatic A cells. These results strongly support the concept that the potent in vivo insulin-releasing action of glucose and leucine is not only dependent on their fuel capacity in pancreatic B cells but also on the concurrent cAMP levels in these cells. In isolated islets, endogenously released glucagon apparently determines the cAMP production in B cells and thus participates in the nutrient-induced secretory process. Somatostatin and epinephrine were shown to exert their suppressive effects via the glucagon-dependent messenger system. It is concluded that nutrients and hormones interact with two different messenger systems which amplify each others' stimulatory effect upon insulin release. cAMP might represent the hormone-induced messenger which sets the B cell's sensitivity and secretory capacity for nutrient stimuli such as glucose. The higher insulin secretory response observed after reaggregation of single B cells could not be attributed to an altered activity in the nutrient or hormonal regulatory units, raising the possibility that the aggregated state of the cells is rather responsible for a better organization or cooperation of the secretory effector unit.

Tight junctional fibrils were absent in freeze-fracture replicas of rat and human islets in situ, but were easily discerned in collagenase-isolated islets. Disruption of the pancreatic gland and its exposure to trypsin were each found to induce tight junction formation in rat islets. The amount of tight junctions between islet cells declined progressively during culture, but tight junctional structures remained detectable after 1 day of culture. It is suggested that rather than being involved in normal islet cell function tight junctions provide an adaptive mechanism intended to seal and hence to protect islet microdomains against sudden perturbations in local interstitial fluid.

In this study on purified rat pancreatic beta-cells, we show that the second-generation sulfonylurea glyburide stimulates insulin release through a direct interaction with the beta-cells. During static incubations, 2 microM glyburide releases 0.16 pg insulin per beta-cell, which corresponds to a half-maximal glucose stimulation. This effect occurs independently from the glucose-recognition unit, being detectable at both nonstimulatory and stimulatory glucose concentrations and proceeding without alterations in the rate of glucose oxidation. The secretagogue action of glyburide appears not to be mediated through cAMP but is potentiated by cAMP-generating substances such as glucagon (10(-8) M; 0.31 pg insulin released per beta-cell). Its 10-fold higher potency in isolated islets is attributed to the markedly higher cAMP levels that are maintained in islet beta-cells under the influence of locally released glucagon. Perifused pancreatic beta-cells respond to glyburide with a biphasic insulin release. After removal of the drug, the cells continue to secrete insulin at the same rate for greater than or equal to 30 min. This prolonged secretory activity coincides with a cellular accumulation of the drug, primarily in association with membranes of secretory vesicles and mitochondria. Tolbutamide also stimulates insulin release from pure beta-cells, but it is less powerful on a molar basis and does not lead to a sustained hormone release after its removal from the extracellular medium. We conclude that the hypoglycemic action of glyburide is at least partly the result of a direct interaction with pancreatic beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Several polycations added to the luminal solution were found to inhibit the vasopressin (ADH)-induced water flow in toad urinary bladder but not the ADH-induced increase in sodium transport or in urea permeability. Ultrastructural studies were conducted to evaluate the uptake of cationized ferritin. It was found that endocytosis of cationized ferritin by luminal cells was strikingly enhanced on exposure to ADH; this increased endocytosis was concomitant with inhibition of transepithelial ADH-induced water flow. Various maneuvers preventing endocytosis were also found to counteract the polycation-induced inhibition of the ADH effect. It is suggested that polycations are endocytosed in vesicles whose walls contain the water channels but not the urea or sodium channels.

Glucose is a well-known stimulus of proinsulin biosynthesis. In purified beta cells, the sugar induces a 25-fold increase in the synthesis of insulin immunoreactive material over 60-min incubation. Autoradiographic analysis of the individual cells shows that this effect is achieved via dose-dependent recruitment of pancreatic beta cells to biosynthetic activity. Recruitment of beta cells is also seen in isolated islets exposed to glucose. The sigmoidal dose-response curve for glucose-induced proinsulin biosynthesis thus reflects a heterogeneous responsiveness of pancreatic beta cells rather than a progressively increasing activity of functionally homogeneous cells. Dose-dependent recruitment of functionally diverse cells may be a ubiquitous mechanism in tissue function.