(175l) Investigating the Cross-Talk between Insulin and Glucagon Secretion By Pancreatic Alpha- and Beta-Cells

Glucose homeostasis relies on the balanced action of insulin produced by pancreatic beta-cells and glucagon released mainly by alpha-cells. Insulin acts to reduce blood sugar, for example, postprandially, whereas glucagon stimulates the liver to release glucose upon hypoglycemia. A hallmark of diabetes is the loss of insulin secretion associated with damage of beta-cells due to their autoimmune ablation (type 1 diabetes) or insulin resistance (type 2 diabetes). This also results in aberrant levels of glucagon pointing to a cross-talk between insulin- and glucagon-releasing cells in their hormonal response [1]. Previously, we showed that insulin production from rodent [2, 3] and human [4] pancreatic beta-cells can be enhanced simply by cell aggregation (forming clusters termed pseudoislets) or optogenetic modulation of cyclic adenosine monophosphate (cAMP) via the action of a photoactivatable adenylyl cyclase. Besides communication among beta-cells, mixed cultures of beta- and non-beta-cells may recapitulate more closely the glucose-stimulated insulin secretion (GSIS) of native pancreatic islets compared to those from beta-cells only. Reportedly however, this enhancement of insulin release was not observed for the beta-cells in co-culture with non-beta-cells [5]. Yet, in other reports glucagon was shown to enhance the insulin response of primary beta-cells and isolated islets [6, 7], so theoretically beta-cells secretion could be enhanced by co-culturing with alpha-cells. With these studies, we wish to investigate the possibility of tuning GSIS by cultivating beta-cells together with glucagon secreting alpha-cells.

Murine beta-cells (betaTC-6) were cultured with alpha-cells (alphaTC1-6) to evaluate changes in baseline insulin and glucagon secretion. Both cell types retain many characteristics of the differentiated beta- and alpha-cells. Compared to monocultures of beta-cells, alpha-/beta-cell mixed cultures over the course of one hour secrete 3.8-fold less insulin at 1 mM glucose (p=0.046, n=3) to 2.1-fold less at 25 mM glucose (p=0.010, n=3) and maintaining the downward trend in intermediate glucose concentrations. The overall trend in increasing insulin secretion with increasing glucose holds true for both alpha-/beta- and only beta-cell cultures.

Conversely, glucagon release from the alpha-cells changed in both magnitude and trend. When cultured alone, alpha-cells maximize glucagon secretion at 5 mM glucose and display a U-shaped secretion trend when varying glucose from 1 to 25 mM glucose. Cocultured alpha-cells elevate their glucagon secretion with increasing glucose concentration exhibiting the same pattern as that of beta-cell insulin release. At 1 mM glucose, no significant differences in glucagon secretion were observed between cultures of alpha-cells alone vs. those of cocultures (p=0.860, n=3). Yet, at 5 mM glucose, alpha-cells alone secrete 4.4-fold more glucagon than cocultured cells (p=0.011, n=3). Similarly, significant changes are measured at 12.5 and 25 mM glucose, with cocultured cells secreting 2-fold and 4-fold higher glucagon, respectively, than independent alpha-cells.

The findings motivate the question of whether the difference in insulin secretion can be attributed to paracrine signaling or potentially to cell-cell contact. To pursue this question, beta-cells will be exposed to exogenous glucagon. Similarly, alpha-cells will be exposed to added insulin. Furthermore, optogenetics will be employed to better address the interplay between alpha- and beta-cells with respect to their hormonal responses. Beta-cells engineered to express a photoactivatable adenylyl cyclase will be co-cultured with alpha-cells to confirm if the reduction in insulin secretion holds true when engineered cells are activated.

Results so far are contrasting those of primary isolated islets where co-culturing reduces insulin secretion rather than enhancing it, but the findings corroborate research conducted with the exact same cell lines [8]. These conflicting trends may be due to factors such as cell line vs. primary cell differences and cell-cell contact signaling, requiring future studies to assess these factors. This work shines light on aspects of regulation of insulin and glucagon production centered on the interplay of the two hormones, and expands our understanding of the pancreas function under normal and pathological conditions.

References:

1. Brown, E. S. Tzanakakis, Mathematical modeling clarifies the paracrine roles of insulin and glucagon on the glucose-stimulated hormonal secretion of pancreatic alpha- and beta-cells, Front. Endocrinol., 14, 2023.
2. Zhang, E. S. Tzanakakis, Amelioration of diabetes in a murine model upon transplantation of pancreatic β-cells with optogenetic control of cyclic adenosine monophosphate, ACS Synth. Biol., 8: 2248-55, 2019.
3. Zhang, E. S. Tzanakakis, Optogenetic regulation of insulin secretion in pancreatic beta-cells, Sci. Rep., 7: 1-10, 2017.
4. Chen, D. Stoukides, E. S. Tzanakakis, Light-mediated enhancement of glucose-stimulated insulin release of optogenetically engineered human pancreatic beta-cells, ACS Synth. Biol., 13: 825-836, 2024.
5. Bosco, D., Orci, L., Meda, P. Homologous but not heterologous contact increases the insulin secretion of individual pancreatic beta-cells, Cell Res., 131:1561-72, 1995.
6. Huypens, P., et al. Glucagon receptors on human islet cells contribute to glucose competence of insulin release, Diabetologia, 43:1012-19, 2000.
7. Moede, Tilo, et al., Alpha-cell regulation of beta cell function, Diabetologia, 63: 2064–75, 2020.
8. Hamaguchi, Kazuyuki, et al. Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: Possible contact-dependent inhibition of insulin secretion, Biol. Med., 228:1227–33, 2003.