Poster Session 2 - E14
1Hima Gohil, 1Ashley Untereiner, 1Alpana Bhattacharjee, 1Feihan Dai, 1Ying Liu, 1.and Michael Wheeler
1,Department of Physiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building rm#3352, Toronto, Ontario, CANADA M5S 1A8
Introduction: Diabetes is a widespread disease impacting an estimated 8.8% of adults in the world in 20171. Inadequate insulin secretion resulting from progressive loss of functional β-cell mass is the characterizing feature of type 1 and type 2 diabetes. Thus, replenishment of the β-cell mass is a prevailing option for diabetes treatment and it is being explored through numerous avenues. One such option is γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter. We have shown that GABA administration in drinking water (6mg/ml) for 10 weeks can increase beta cell mass and improve glucose tolerance and insulin secretion in vivo. However, the mechanisms underlying GABA’s action and GABA’s application to a T2D mouse model have yet to be studied. Thus, we hypothesize that (i) GABA may function through the GABAA receptor/ shunt and (ii) its aforementioned effects may be extrapolated to a T2D model to improve beta cell mass and ultimately glucose tolerance.
Methods and Results:
i) GABA’s mechanism of action: In order to study the mechanism of action of GABA, we examined the GABAA receptor and GABA-shunt in primary mouse islets.GABAA receptor: CD1 mouse islets were treated with GABA for 4-5 days and Ki67+ staining was used as a marker for cell proliferation. In the GABA-treated group, a 4.5-fold increase was observed in Ki67+ cells in insulin+ cells. Co-treatment with GABA and bicuculline (a GABAA receptor antagonist) resulted in the complete ablation of beta-cell proliferation, suggesting that the coupling of GABA to the GABAA receptor is a crucial mechanism in GABA-stimulated beta-cell proliferation. Following, as the GABAA receptor is not well characterized in islets, we performed droplet digital PCR (high-throughput digital PCR that fractionates samples into 20,000 droplets in order to get precise and absolute quantification) to determine the subunit composition of GABAA receptor in islets. Through ddPCR and corresponding IF studies of the critical subunits, we show that α2/5, β1/3 and γ1 makeup the receptor in beta cells.
GABA-shunt: The GABA shunt pathway is a series of reactions intertwined with the Krebs cycle that metabolizes and synthesizes GABA. GABA shunt’s activity can be observed by studying islet metabolism changes in response to exogeneous GABA. Thus, islet metabolism changes were studied using Rhodamine123 (a mitochondrial membrane potential dye) and Seahorse XFe24 (measures mitochondrial function via oxygen consumption rate) in CD1 islets. Accordingly, acute 30 minute and overnight treatment with GABA (100μM and 1mM) revealed no significant changes in oxygen consumption rate, suggesting that the GABA shunt is not activated with increased extracellular GABA concentration in pancreatic islets
ii) Type 2 Diabetes mouse model: We employ a HFD-fed CD1 disease prevention and reversal model in order to study if GABA can prevent or reverse glucose intolerance, insulin resistance and increase β-cell mass in a T2D model. a) Disease prevention model: 9-week-old CD1 mice were administered GABA (6mg/ml; via drinking water) and high-fat diet (HFD; 60% kcal from fat) simultaneously for 20 weeks. Upon completion of the 20 weeks, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed. Thereafter, mice were sacrificed and the tissues were collected for histology. The OGTT and ITT revealed no significant difference between the HFD control and the GABA-treated HFD group, suggesting GABA does not improve glucose or insulin tolerance in HFD CD1 mice. b) Disease reversal mouse model: 9-week-old CD1 mice were placed on a HFD for 10 weeks prior to being segregated into control or GABA-treated group (10 weeks; via drinking water). Similar to prevention model (i), OGTT and ITT showed no difference between the control and GABA-treated group. Additional, there was no difference in β-cell mass.
Conclusion: Herein, we demonstrate that GABA may act through GABAA receptor to induce beta-cell proliferation, but not the GABA-shunt. We show that α2/5, β1/3 and γ1 subunits makeup the GABAA receptor in beta cells. Additionally, both of the metabolically challenged mice models showed no significant change in glucose tolerance, insulin tolerance, nor β-cell mass between the HFD-fed GABA-treated and HFD-fed control mice. The lack of improvement could be attributed to GABA’s inability to increase β-cell mass in our HFD-induced obese mice, which may have improved glucose tolerance.
References:
- Statista - The Statistics Portal for Market Data, Market Research and Market Studies. Statistacom. 2018. Available at: https://www.statista.com/. Accessed March 11, 2018.