Division of Diabetes and Nutrient Science
King's College London
Glutamine plays an essential role in the functional and structural integrity of isolated human islets
Guo Huang1,2, Min Zhao PhD2, Pratik Choudhary Dr1, Attilio Pingitore PhD1, Bo Liu PhD1, Helen Tang Ms2, Nigel Heaton Prof.2, Meimei Fung Ms2, Andreas Barthel Prof.3, Shanta Persaud Prof.1, Stefan Bornstein Prof.1,3, Stephanie Amiel Prof.1.
1Division of Diabetes and Nutrient Science, King's College London, London, United Kingdom; 2Liver Caregroup, King's College Hospital, London, United Kingdom; 3Centre for Diabetes Research, , Paul Langerhans Institute , Dresden, Germany
Diabetes Study Group.
Background: Successful human islet transplantation depends on an adequate quantity of high quality human islets. After isolation, islets are cultured before transplantation, but optimal culture systems are debated. We evaluated the role of glutamine in optimising this culture period and in maintaining islet structure and function in the post-isolation period.
Methods and materials: Human islets not suitable for clinical transplantation due to inadequate yield post-isolation, and with consent for academic research, were cultured at different concentrations of L-glutamine (0-4mM) and analysed at different time points for physical intactness, viability and glucose-induced insulin secretion. The capacity of the human islets to correct hyperglycaemia following transplantation into diabetic SCID mice was assessed by measuring changes in blood glucose in mice and by intraperitoneal glucose tolerance tests. The graft-bearing kidneys were examined for the presence of insulin expressing cells and endothelial cell markers by immunohistochemical staining.
Results: Human islets maintained in culture in the absence of glutamine for 24-36 hours did not show glucose-induced insulin secretion. In fact, exposure of these islets to 20mM glucose in dynamic perifusion resulted in a paradoxical reduction in insulin secretion (2mM glucose: 11.4±1.1 pg/islet/min; 20mM glucose: 4.80±0.7) that was coupled to decreased ATP production upon substrate availability (20mM glucose 71.7±6.6% ATP generation vs 2mM glucose; p<0.05). Islets cultured for 48 hours in the presence of 1mM glutamine showed a poor ability to correct hyperglycaemia following transplantation into diabetic SCID mice (15.1±2.1 mM blood glucose pre-transplant vs. 14±1.9 mM blood glucose 7 days post-transplant). Immunohistochemical analysis on day 7 post-transplant showed that the islets had not engrafted well. In contrast, islets that had been maintained with 4mM glutamine showed superior ability to correct hyperglycaemia (15.3±2.8 mM blood glucose pre-transplant vs. 5.4±0.3 mM blood glucose 3 days post-transplant). Immunohistochemical analysis 7 days post-transplant showed that islet grafted well with appropriate insulin expression, well engrafment and showed appropriate endothelial blood vessel formation. The improved in vivo function of islets after exposure to 4mM glutamine was associated with robust glucose-induced secretion in vitro (2mM glucose: 8.5±1.2 pg/islet/min; 20mM glucose: 95.2±10.1).
Conclusion: Our data indicate that the presence of glutamine in human islet culture medium is essential for appropriate function in vitro and in vivo, and that the optimal concentration of glutamine for isolated human islets is 4mM rather than 2mM, which is routinely used in cell culture. We recommend that human islets should be maintained in medium supplemented with 4mM glutamine to improve their functional and structural integrity.
07:00 - 08:00
|Mini-Orals: Experimental islet transplantation||Glutamine plays an essential role in the functional and structural integrity of isolated human islets||Room 110|