Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Aims/hypothesis: We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. Methods: Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1h of stimulation with different secretagogues. Results: Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2mmol/l glucose were observed in murine but not in human islets. Conclusions/interpretation: Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islet

Klf6 protects β-cells against insulin resistance-induced dedifferentiation.

In the pathogenesis of type 2 diabetes, development of insulin resistance triggers an increase in pancreatic β-cell insulin secretion capacity and β-cell number. Failure of this compensatory mechanism is caused by a dedifferentiation of β-cells, which leads to insufficient insulin secretion and diabetic hyperglycemia. The β-cell factors that normally protect against dedifferentiation remain poorly defined. Here, through a systems biology approach, we identify the transcription factor Klf6 as a regulator of β-cell adaptation to metabolic stress. We used a β-cell specific Klf6 knockout mouse model to investigate whether Klf6 may be a potential regulator of β-cell adaptation to a metabolic stress. We show that inactivation of Klf6 in β-cells blunts their proliferation induced by the insulin resistance of pregnancy, high-fat high-sucrose feeding, and insulin receptor antagonism. Transcriptomic analysis showed that Klf6 controls the expression of β-cell proliferation genes and, in the presence of insulin resistance, it prevents the down-expression of genes controlling mature β-cell identity and the induction of disallowed genes that impair insulin secretion. Its expression also limits the transdifferentiation of β-cells into α-cells. Our study identifies a new transcription factor that protects β-cells against dedifferentiation, and which may be targeted to prevent diabetes development

Detection of TMPRSS2 : ERG fusion gene in circulating prostate cancer cells

Creative Commons Attribution-NonCommercial-Share Alike 3.0 license (CC BY-NC SA)Aim: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. Methods: We analyzed the frequency of TMPRSS2: ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. Results: TMPRSS2: ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However, TMPRSS2:ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. Conclusion: Although further study is required to address the association between TMPRSS2:ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis.Peer reviewedFinal Published versio

A Genetic Screen Identifies Hypothalamic Fgf15 as a Regulator of Glucagon Secretion.

The counterregulatory response to hypoglycemia, which restores normal blood glucose levels to ensure sufficient provision of glucose to the brain, is critical for survival. To discover underlying brain regulatory systems, we performed a genetic screen in recombinant inbred mice for quantitative trait loci (QTL) controlling glucagon secretion in response to neuroglucopenia. We identified a QTL on the distal part of chromosome 7 and combined this genetic information with transcriptomic analysis of hypothalami. This revealed Fgf15 as the strongest candidate to control the glucagon response. Fgf15 was expressed by neurons of the dorsomedial hypothalamus and the perifornical area. Intracerebroventricular injection of FGF19, the human ortholog of Fgf15, reduced activation by neuroglucopenia of dorsal vagal complex neurons, of the parasympathetic nerve, and lowered glucagon secretion. In contrast, silencing Fgf15 in the dorsomedial hypothalamus increased neuroglucopenia-induced glucagon secretion. These data identify hypothalamic Fgf15 as a regulator of glucagon secretion

Proliferation of sorted human and rat beta cells

Aims/hypothesis: The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors. Methods: Human beta cells were purified by FACS by virtue of their high zinc content using Newport Green, and excluding ductal and dead cells. Rat beta cells were sorted by autofluorescence or using the same method developed for human cells. Cells were plated on poly-l-lysine or ECMs from rat or human bladder carcinoma cells or bovine corneal ECM and incubated in the presence of BrdU with or without growth factors. Results: The newly developed method for sorting human beta cells yields a population containing 91.4 ± 2.8% insulin-positive cells with a low level of spontaneous apoptosis and a robust secretory response to glucose. Beta cells from 8-week-old rats proliferated in culture and this was increased by ECM. Among growth factors, only human growth hormone (hGH) and the glucagon-like peptide-1 analogue liraglutide enhanced proliferation of rat beta cells, with a significant increase on both poly-l-lysine and ECM. By contrast, sorted adult human beta cells from 16 donors aged 48.9 ± 14.3years (range 16-64years) failed to replicate demonstrably in vitro regardless of the substratum or growth factors used. Conclusions/interpretation: These findings indicate that, in our conditions, the fully differentiated human adult insulin-producing beta cell was unable to proliferate in vitro. This has important implications for any attempt to expand cells from pancreases of donors of this age group. By contrast, the rat beta cells used here were able to divide in vitro, and this was enhanced by ECM, hGH and liraglutid

Sympathetic activity and early mobilization in patients in intensive and intermediate care with severe brain injuries: a preliminary prospective randomized study.

Patients who experience severe brain injuries are at risk of secondary brain damage, because of delayed vasospasm and edema. Traditionally, many of these patients are kept on prolonged bed rest in order to maintain adequate cerebral blood flow, especially in the case of subarachnoid hemorrhage. On the other hand, prolonged bed rest carries important morbidity. There may be a clinical benefit in early mobilization and our hypothesis is that early gradual mobilization is safe in these patients. The aim of this study was to observe and quantify the changes in sympathetic activity, mainly related to stress, and blood pressure in gradual postural changes by the verticalization robot (Erigo®) and after training by a lower body ergometer (MOTOmed-letto®), after prolonged bed rest of minimum 7 days. Thirty patients with severe neurological injuries were randomized into 3 groups with different protocols of mobilization: Standard, MOTOmed-letto® or Erigo® protocol. We measured plasma catecholamines, metanephrines and blood pressure before, during and after mobilization. Blood pressure does not show any significant difference between the 3 groups. The analysis of the catecholamines suggests a significant increase in catecholamine production during Standard mobilization with physiotherapists and with MOTOmed-letto® and no changes with Erigo®. This preliminary prospective randomized study shows that the mobilization of patients with severe brain injuries by means of Erigo® does not increase the production of catecholamines. It means that Erigo® is a well-tolerated method of mobilization and can be considered a safe system of early mobilization of these patients. Further studies are required to validate our conclusions. The study was registered in the ISRCTN registry with the trial registration number ISRCTN56402432 . Date of registration: 08.03.2016. Retrospectively registered

Tumor microenvironment defines the invasive phenotype of AIP-mutation-positive pituitary tumors

The molecular mechanisms leading to aryl hydrocarbon receptor interacting protein (AIP) mutation-induced aggressive, young-onset growth hormone-secreting pituitary tumors are not fully understood. In this study, we have identified that AIP-mutation-positive tumors are infiltrated by a large number of macrophages compared to sporadic tumors. Tissue from pituitary-specific Aip-knockout (AipFlox/Flox;Hesx1Cre/+) mice recapitulated this phenotype. Our human pituitary tumor transcriptome data revealed the "epithelial-to-mesenchymal transition (EMT) pathway" as one of the most significantly altered pathways in AIPpos tumors. Our in vitro data suggest that bone marrow-derived macrophage-conditioned media induces more prominent EMT-like phenotype and enhanced migratory and invasive properties in Aip-knockdown somatomammotroph cells compared to non-targeting controls. We identified that tumor-derived cytokine CCL5 is upregulated in AIP-mutation-positive human adenomas. Aip-knockdown GH3 cell-conditioned media increases macrophage migration, which is inhibited by the CCL5/CCR5 antagonist maraviroc. Our results suggest that a crosstalk between the tumor and its microenvironment plays a key role in the invasive nature of AIP-mutation-positive tumors and the CCL5/CCR5 pathway is a novel potential therapeutic target