An in situ approach to study alpha cell physiology in human diabetes pathogenesis

PhD student: Denise Minerva Drotar
Supervisor at TUD: Stephan Speier
Supervisor at KCL: Shanta Persaud
Start date: 14.08.2017  Date of defense: 16.12.2020  PhD

Glucose homeostasis is tightly regulated by hormones secreted within the pancreatic islets of Langerhans. The most important are insulin and glucagon produced by beta and alpha cells respectively. Changes in beta cell mass and/or their functional deficit can lead to hyperglycemia, a major hallmark of both type 1 (T1D) and type 2 (T2D) diabetes. Moreover, a dysregulation in glucagon secretion is thought to also play a major role in patients with diabetes, suggesting a failure in the counterregulatory mechanisms of glucose homeostasis in disease pathogenesis. Dysfunction at the alpha cell level in T1D manifests as blunt glucagon response to low glucose levels, which can cause severe hypoglycemic events in patients with T1D. Furthermore, exaggerated glucagon responses to glucose or amino acid intake significantly contributes to dysglycemia in both T1D and T2D patients.

Most of our knowledge about glucagon and alpha cell physiology in the human setting was generated using in vivo systemic assessments or in vitro investigations of isolated human islets or dispersed single cells. Despite the increasing knowledge regarding alpha cells and glucagon biology, the underlying mechanisms of alpha cell dysfunction are still uncertain. Studies on alpha cell physiology were hindered by limited human tissue accessibility, technical methodologies and translational value of findings from rodents to humans. To fill the gap between the currently available in vivo and in vitro approaches and a more precise understanding of mechanisms of diabetes pathogenesis, detailed investigation of islet cells within their native environment is needed.

The overall objective of this thesis was to characterize alpha cell function in diabetes pathogenesis. To this end, the human pancreas slice preparation would to be adapted and advanced for the study of alpha cell physiology. These adjustments would be then used to investigate changes in alpha cell mass and function in T1D and T2D.

Pancreas tissue slices were prepared from donor organs with and without T1D and from tissue donors after pancreatectomy at different stages of T2D. Immunofluorescent staining with subsequent 3D morphometry was used to quantify alpha cell volumes from 120μm thick tissue slices. Furthermore, human tissue slices were subjected to dynamic slice perifusion for the assessment of glucagon and insulin secretion kinetics in response to specific stimuli. Finally, functional and morphometrical analysis was performed on the same tissue slices to enable direct correlation of glucagon secretion and alpha cell volume in a subset of cases in the context of T1D.

Here we developed a semi-automatic 3D approach to quantify total endocrine cell volumes within a given volume of pancreas tissue. In addition, we established an in situ method for dynamic insulin release measurements from islets preserved in their native environment. We successfully modified this protocol to allow the measurement of glucagon release in slices from organ donors. After further optimizations, we were additionally able to also measure alpha cell function from surgical specimens after pancreatectomy.

To gain insight into alpha cell pathophysiology in T1D we investigated alpha cell volume in donor organs with different disease duration and age at onset. Alpha cell volumes in slices of individuals with T1D did not show a dramatic change (neither increase nor decrease) in comparison to slices generated from non-diabetic (ND) pancreata. Furthermore, functional assessment of glucagon release using a specific stimulation protocol for alpha cells suggests preserved stimulatory capacity of these cells in slices from autoantibody positive donors. Interestingly, this is also the case in the so far studied slices from donors with different durations of diabetes. Nevertheless, normalization of secreted glucagon to the total alpha cell mass within the slice indicated reduced glucagon release in the here investigated two cases of T1D.

In the context of T2D, 3D morphometrical analysis revealed that overall endocrine cell volume, including alpha cell volume, is maintained in our cohort of IGT and T2D individuals. Glucagon release can also be measured in tissue procured from patients undergoing pancreatectomy, given the presence of amino acids in the perifusion media and increased trypsin inhibitors. While we provided proof of concept using tissue from ND individuals, we are confident that the approach will give valuable insight in different states of diabetes.

These results demonstrate that human pancreas tissue slices represent a complementary platform to study alpha cell pathophysiology in both major types of diabetes. We provide evidence that this approach can be used to study alpha cell pathophysiology in T1D and T2D. Our preliminary data indicates no defect in the stimulatory capacity in slices from Aab+ and T1D donors, however more cases need to be investigated given the heterogeneous nature of the disease. We anticipate that the here proposed protocol for measurement of glucagon release from tissue slices will help us to gain insight in the role of alpha cells in diabetes pathophysiology.

Publications:

Liposomal prednisolone phosphate potentiates the antitumor activity of liposomal 5-fluorouracil in C26 murine colon carcinoma in vivo. L. Patras, B. Sylvester, L. Luput, A. Sesarman, E. Licarete, A. Porfire, D. Muntean, D.M. Drotar, A.D. Rusu, A.L. Nagy, C. Catoi, I. Tomuta, L. Vlase, M. Banciu, M. Achim. Cancer Biol Ther. 2017;18:616-626. 

In vivo Double Targeting of C26 Colon Carcinoma Cells and Microenvironmental Protumor Processes Using Liposomal Simvastatin. L. Luput, E. Licarete, D.M. Drotar, A.L. Nagy, A. Sesarman, L. Patras, V.F. Rauca, A. Porfire, D. Muntean, M. Achim, I. Tomuta, L. Vlase, C. Catoi, N. Dragos, M. Banciu. J Cancer. 2018;9:440-449. 

Dysfunction of Persisting beta Cells Is a Key Feature of Early Type 2 Diabetes Pathogenesis. C.M. Cohrs, J.K. Panzer, D.M. Drotar, S.J. Enos, N. Kipke, C. Chen, R. Bozsak, E. Schoniger, F. Ehehalt, M. Distler, A. Brennand, S.R. Bornstein, J. Weitz, M. Solimena, S. Speier. Cell Rep. 2020;31:107469. 

Overcoming Intrinsic Doxorubicin Resistance in Melanoma by Anti-Angiogenic and Anti-Metastatic Effects of Liposomal Prednisolone Phosphate on Tumor Microenvironment. E. Licarete, V.F. Rauca, L. Luput, D. Drotar, I. Stejerean, L. Patras, B. Dume, V.A. Toma, A. Porfire, C. Gherman, A. Sesarman, M. Banciu. Int J Mol Sci. 2020;21:2968. 

Liposomal simvastatin sensitizes C26 murine colon carcinoma to the antitumor effects of liposomal 5-fluorouracil in vivo.  L. Luput, A. Sesarman, A. Porfire, M. Achim, D. Muntean, T. Casian, L. Patras, V.F. Rauca, D.M. Drotar, I. Stejerean, I. Tomuta, L. Vlase, N. Dragos, V.A. Toma, E. Licarete, M. Banciu.  Cancer Sci. 2020;111:1344-1356. 

Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis. J.K. Panzer, H. Hiller, C.M. Cohrs, J. Almaca, S.J. Enos, M. Beery, S. Cechin, D.M. Drotar, J.R. Weitz, J. Santini, M.K. Huber, M. Muhammad Fahd Qadir, R.L. Pastori, J. Dominguez-Bendala, E.A. Phelps, M.A. Atkinson, A. Pugliese, A. Caicedo, I. Kusmartseva, S. Speier. JCI Insight. 2020;5:e134525. 

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices. M. K. Huber, D. M. Drotar, H. Hiller, M. L. Beery, P. Joseph, I. Kusmartseva, S. Speier, M. A. Atkinson, C. E. Mathews, E. A. Phelps. Accepted for publication in J Vis Exp. 2021.