Stathmin-2 Mediates Glucagon Secretion from Pancreatic α-Cells

Abstract

21Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of 22 diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules 23 will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon 24 secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules 25 of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon 26 secretion. In the present study, we show that one of these interactome proteins, the neuronal protein 27 stathmin-2, is expressed in aTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of 28 glucagon secretion. Stathmin-2 was co-secreted with glucagon in response to 55 mM K+, and 29 immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the 30 secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, 31Stathmin-2 co-localized with glucagon, but not with insulin, indicating that it is an alpha cell protein.To 32 show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA -mediated 33 depletion of stathmin-2 in aTC1-6 cells caused glucagon secretion to become constitutive without any effect 34 on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and 35 K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into 36 the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization 37 of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule 38 protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These 39 findings describe a potential new pathway for the regulation of glucagon secretion, and may have 40 implications for controlling glucagon hypersecretion in diabetes. 41 42 43 44 Glucagon secretion and Stathmin-2 3 Introduction 45 Hyperglucagonemia is a characteristic sign of diabetes, causing fasting hyperglycemia and 46 glycemic volatility. Clinically, glycemic variability contributes to the development of diabetes 47 complications (1). Persistent hyperglucagonemia may exacerbate abnormal glucose metabolism 48 in patients with type 2 diabetes and lead to metabolic disturbances in obese and prediabetic 49 individuals (2). Accordingly, controlling this excess glucagon secretion may be a potential 50 therapeutic strategy for diabetes (3) so that glycemia and glucose metabolism may be better 51 regulated. Such an approach has been suggested as a priority for the treatment of diabetes (1). 52Combating hyperglucagonemia could be theoretically achieved by i) inhibition of glucagon action 53 at target organs by blocking the glucagon receptor, or ii) inhibition of glucagon secretion from the 54 pancreatic α cells. While in the short-term the former...

Misrouting of Glucagon and Stathmin-2 Towards Lysosomal System of α-Cells in Glucagon Hypersecretion of Diabetes

“…In particular, treatment of α TC1-6 cells with insulin, which can override glucose-mediated regulation of glucagon secretion (Kawamori et al, 2009), appeared to recruit stathmin-2 (Stmn2 or SCG10) to the interactome, thus potentially identifying another inhibitor of glucagon secretion. In a subsequent study, we showed that Stmn2 is co-secreted in a regulated manner with glucagon, and when over-expressed, suppressed glucagon secretion by increasing its trafficking through the endolysosomal pathway in α TC1-6-cells (Asadi and Dhanvantari, 2020).…”

“…NA plan-Apochromat oil differential interference contrast objective and NIS-Elements software (Nikon, Mississauga, Canada). Acquisition of high-resolution images was done by selecting Nyquist XY scan area, 1024×1024-pixel size scanning of the selected area and 2D-Deconvolution of the captured images, as we have done previously (Asadi and Dhanvantari, 2020).…”

“…Bruce Verchere, University of British Columbia, Vancouver, BC) were cultured in DMEM containing 16.7 mM glucose, L-glutamine, 15% horse serum and 2.5% fetal bovine serum, as described previously (Asadi and Dhanvantari, 2019;Asadi and Dhanvantari, 2020). As we have shown previously, after long-term culturing in high glucose conditions, cells demonstrated the phenotype of glucagon hypersecretion of diabetes (Asadi and Dhanvantari, 2019;McGirr et al, 2005).…”

“…For lysosomal inhibition, cells were treated with 10 nM Bafilomycin A1 (Cat# B1793, Sigma) for 2 h. Cells were then rinsed and incubated in HBSS containing 16.7 mM glucose and 0.5% BSA for 2h. Secretion studies were done by addition of 55 mM KCl for 20 min as we have done previously (Asadi and Dhanvantari, 2020). Media were then collected into microfuge tubes containing PMSF (45 mM) and Aprotinin (5µg/mL), centrifuged at 14000×g for 15 minutes at 4°C, and then stored at -80°C until analysis.…”

“…show that the endolysosomal system may figure prominently in the intracellular trafficking of glucagon as it is directed through the regulated secretory pathway (Asadi and Dhanvantari, 2020).…”