Search

A correct biosynthetic activity is thought to be essential for the long-term function and survival of islet cells in culture and possibly also after islet transplantation. Compared to the secretory activity, biosynthetic activity has been poorly studied in pancreatic islet cells. Here we aimed to assess biosynthetic activity at the single cell level to investigate if protein synthesis is dependent on secretagogues and increased as a consequence of hormonal secretion. Biosynthetic activity in rat islet cells was studied at the single cell level using O-propargyl-puromycin (OPP) that incorporates into newly translated proteins and chemically ligates to a fluorescent dye by "click" reaction. Heterogeneous biosynthetic activity was observed between the four islet cell types, with delta cells showing the higher relative protein biosynthesis. Beta cells protein biosynthesis was increased in response to glucose while 3-isobutyl-1-methylxanthine and phorbol-12-myristate-13-acetate, 2 drugs known to stimulate insulin secretion, had no similar effect on protein biosynthesis. However, after several hours of secretion, protein biosynthesis remained high even when cells were challenged to basal conditions. These results suggest that mechanisms regulating secretion and biosynthesis in islet cells are different, with glucose directly triggering beta cells protein biosynthesis, independently of insulin secretion. Furthermore, this OPP labeling approach is a promising method to identify newly synthesized proteins under various physiological and pathological conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Due to the shortage of multi-organ donors, human pancreatic islet transplantation has now been extended to islets originating from obese subjects. In this study, our aim is to compare the respective sensitivity of human islets from lean vs obese donors to chronic high glucose or high palmitate., Methods: Human islets were isolated from pancreases harvested from brain-dead multi-organ donors. Islets were cultured during 72 hours in the presence of moderate (16.7 mmol/L) or high (28 mmoL/L) glucose concentrations, or glucose (5.6 mmoL/L) and palmitate (0.4 mmoL/L), before measurement of their response to glucose., Results: We first observed a greater insulin response in islets from obese donors under both basal and high-glucose conditions, confirming their hyperresponsiveness to glucose. When islets from obese donors were cultured in the presence of moderate or high glucose concentrations, insulin response to glucose remained unchanged or was slightly reduced, as opposed to that observed in lean subjects. Moreover, culturing islets from obese donors with high palmitate also induced less reduction in insulin response to glucose than in lean subjects. This partial protection of obese islets is associated with less induction of inducible nitric oxide synthase in islets, together with a greater expression of the transcription factor forkhead box O1 (FOXO1)., Conclusions: Our data suggest that in addition to an increased sensitivity to glucose, islets from obese subjects can be considered as more resistant to glucose and fatty acid excursions and are thus valuable candidates for transplantation., (© 2020 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated. [ABSTRACT FROM AUTHOR]

Obesity and lipid metabolism dysregulation are often associated with insulin resistance, and can lead to type 2 diabetes. However, mechanisms linking insulin resistance, high levels of plasma free fatty acids (FFA), and β cell failure remain unclear. The aim of this work was to search for proteins whose synthesis was modified by a short exposure to FFA. This could help in the future to identify molecular mechanisms underlying islet dysfunction in the presence of FFA. Therefore, we assessed by mass spectrometry de novo protein synthesis of freshly isolated rat islets after palmitate short exposure. Quantitative proteome and secretome analyses were performed by combining metabolic incorporation of azidohomoalanine (AHA) and pulse labeling with stable isotope labeling by amino acids in cell culture (SILAC). We showed that pancreatic islets, in response to 4-h exposure to palmitate, increased the synthesis of ribosomal proteins and proteins of the cytoskeleton, and increased their secretion of proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. First, these results show that de novo protein quantification analysis by LC-MS/MS is a useful method to investigate cellular modifications induced by FFA on pancreatic islets. Also, these results show that short exposure to palmitate increases the expression of ribosomal proteins and proteins involved in insulin secretion, and it remains to be determined if these effects are responsible or linked to the harmful effect of palmitate on β cells. [ABSTRACT FROM AUTHOR]