Delineating the role of FANCA in glucose-stimulated insulin secretion in β cells through its protein interactome

Abstract

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas β cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas β cell line EndoC-βH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-βH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-βH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of β cell-specific endocrinopathies in FA patients.

An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry

“…This protocol was used in a recent publication by Lagundžin and co-authors ( Lagundzin et al., 2019 ), in which a proteomics-based approach was employed to study the role of Fanconi Anemia Complementation Group A (FANCA) in pancreas β islet cells. We initially tested the protocol using human embryonal kidney 293 FT, human pancreatic islet EndoC-βH3 and human pancreatic cancer MiaPaCa-2 cell lines.…”

“…Our conclusion is that the protocol is applicable to different cell lines and species. For complete details on the use and execution of this protocol, please refer to Lagundžin et al. (2019) .…”

“…For complete details on the use and execution of this protocol, please refer to Lagundžin et al. (2019) .…”

Impact of Epigenetics on Complications of Fanconi Anemia: The Role of Vitamin D-Modulated Immunity

“…In addition to these life-threatening and limiting complications, an FA individual is facing numerous other clinical dysfunctions. FA patients have a wide range of metabolic and endocrine impairments [63,64] affecting lipid metabolism [65], glucose and/or insulin homeostasis [66], the thyroid axis [67] and most important fertility [68]. These non-cancerous aspects of FA often dramatically reduce the quality of life of the individuals.…”

“…Moreover, altered mitochondrial functions [78][79][80] and increased oxidative stress [81,82] are linked to FA. This also implies direct interaction of FA proteins with altered insulin secretion [66] and lipid metabolism [83]. Furthermore, non-canonical functions of FA proteins in the control of cytokines, such as tumor necrosis factor [84] and transforming growth factor beta [85] have been described.…”