1*Ashley Untereiner, 1*Shaaban Abdo, 2Alpana Bhattacharjee, 1Himaben Gohil, 2Farzaneh Pourasgari, 4Neke Ibeh, 2Mi Lai, 1Anthony Wong, 3Nicholas Khuu, 2Ying Liu, 3Neil Winegarden, 4Carl Virtanen, 1,2Feihan F. Dai, 1,2Michael B. Wheeler
1 Department of Physiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada; 2 Toronto General Hospital Research Institute, University Health Network, 101 College Street, Toronto, ON, M5G 1L7 Canada; 3 Princess Margaret Genomics Centre, 101 College Street, Toronto, ON, M5G 1L7 Canada; 4 Princess Margaret Cancer Centre, University Health Network, 101 College Street, PMCRT 11-313, Toronto, ON, M5G 1L7 Canada; *Authors contribute equally
Introduction: GABA is an inhibitory neurotransmitter that is also synthesized in the pancreatic beta-cell. Recently, groundbreaking studies showed that GABA administration in mice increased beta-cell mass by stimulating the conversion of glucagon-producing alpha-cells into beta-like cells, leading to a reversal of T1D. This has prompted us to determine if GABA has any impact on beta-cells of normoglycemic mice and those in a model of prediabetes/T2D. Results: i) Effects of GABA on beta-cell mass, islet function and glucose tolerance in normoglycemic mice: In the present study, we show that oral GABA administration (ad libitum; 10 weeks) to normoglycemic mice increased pancreatic beta-cell mass; leading to a significant enhancement of glucose tolerance and insulin secretion. To prove a direct effect of GABA on the islet, dispersed mouse and human islets were treated with GABA for 4-5 days. GABA treatment led to a 2.6-fold increase in Ki67+ glucagon+ cells and a 3.5-fold increase in Ki67+ insulin+ cells in dispersed mouse islets; an effect blocked with bicuculline (a GABAA receptor antagonist), indicating that GABA promotes alpha- and beta-cell proliferation in islets via coupling to GABAA receptor. Importantly, GABA treatment also induced alpha- and beta-cell proliferation in isolated human islets, however, this was not universal as some healthy donor islets lacked a response to GABA. ii) GABA mechanism of action: To link GABA actions directly to the beta-cell, cytospin was used to define both alpha- and beta-cell GABAA receptor subunit expression. In mouse and human beta-cells, alpha2/5, beta1/3 and gamma1/2 were detected, while alpha-cells surprisingly appeared to lack a full repertoire of subunits to constitute a functional GABAA receptor. In functional assays, GABA did not alter islet glucose-stimulated insulin/glucagon secretion, nor did it affect glucose metabolism and mitochondrial function; strongly suggesting improvements in glucose tolerance was primarily due to increased beta-cell number. To define the direct impact more precisely, mouse islets were treated overnight with GABA and subsequently analyzed by single cell RNA-seq. In beta-cells, the most significantly enriched pathways included cell cycle, TGF-beta, MAPK and Wnt signaling, all of which contribute to cell proliferation. Single cell analysis also revealed four distinct subpopulations of beta-cells in normal islets and six in the GABA-treated group. Intriguingly, cluster 3 in the GABA-treated group represented a newly emerged beta-cell subpopulation. iii) Effects of GABA in model of prediabetes/T2D: Mice were placed on high-fat high-caloric diet for 16 weeks and treated with/out GABA for the last 10 weeks. Under these conditions, GABA treatment did not further increase beta-cell mass and was unable to prevent glucose intolerance and insulin resistance associated with diet-induced obesity and diabetes. Conclusions: We demonstrate that GABA directly increases beta-cell mass via proliferation, not trans-differentiation of alpha-cells. GABA gives rise to a new subpopulation of beta-cells and enhances pathways preferentially involved in proliferation. Under the conditions studied, GABA does not improve islet capacity nor function associated with diet-induced prediabetes. We speculate this may be due to a common pathway for proliferation used by GABA and the proliferative factors associated with insulin resistance.