UCL - SSS/IREC/EDIN - Pôle d'endocrinologie, diabète et nutrition, UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, Tariq, Mohammad, de Souza, Arnaldo H., Bensellam, Mohammed, Chae, Heeyoung, Jaffredo, Manon, Close, Anne-Françoise, Deglasse, Jean-Philippe, Santos, Laila R. B., Buemi, Antoine, Mourad, Nizar I., Wojtusciszyn, Anne, Raoux, Matthieu, Gilon, Patrick, Broca, Christophe, Jonas, Jean-Christophe, UCL - SSS/IREC/EDIN - Pôle d'endocrinologie, diabète et nutrition, UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, Tariq, Mohammad, de Souza, Arnaldo H., Bensellam, Mohammed, Chae, Heeyoung, Jaffredo, Manon, Close, Anne-Françoise, Deglasse, Jean-Philippe, Santos, Laila R. B., Buemi, Antoine, Mourad, Nizar I., Wojtusciszyn, Anne, Raoux, Matthieu, Gilon, Patrick, Broca, Christophe, and Jonas, Jean-Christophe
Aims/hypothesis: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Methods: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Results: Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were