Savita Dhanvantari

Glucagon secretion by pancreatic α cells is regulated by nutrient, paracrine, and neural inputs, and emerging evidence suggests that the islet endocannabinoid system (ECS) may also regulate α -cell function. While phytocannabinoids such as cannabidiol (CBD) have been shown to interact with the ECS, and negatively regulate insulin secretion from pancreatic β cells, whether CBD alters α cell function in ways that disrupt glucagon trafficking and secretion remains unclear. This study investigates how acute CBD exposure affects transcriptional and secretory networks governing α cell function. Using the α TC1-6 glucagon-secreting cell line, we exposed the cells to 0.3 and 3 µM CBD for RT-qPCR analysis, 3 µM CBD for bulk RNA sequencing, and 3 µM CBD or its inactive metabolite 7-COOH-CBD for imaging and secretion experiments. Transcriptomic analysis revealed that treatment with CBD did not alter the expression of ECS receptors and enzymes. However, pathways involved in cellular excitability, peptide hormone trafficking, and secretion were significantly downregulated. Using confocal microscopy, we found that CBD exposure increased the colocalization of glucagon with LAMP1 (a lysosomal marker), and that CBD rerouted peripheral glucagon, and glucagon-containing lysosomes towards the intracellular region of the cell. In conjunction with these findings, we observed reduced glucose-stimulated glucagon secretion under high glucose conditions. Overall, our data supports a model in which acute CBD exposure downregulates processes governing cellular excitability and peptide secretion and negatively regulates glucagon trafficking and secretion by rerouting peripheral glucagon into lysosome-associated intracellular compartments in α TC1-6 cells.

The secretion of glucagon from the pancreatic alpha (α) cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters and paracrine hormones. Insulin and somatostatin form an intra‐islet paracrine network to control glucagon secretion through direct inhibitory effects on α cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin‐2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined whether insulin and somatostatin direct glucagon to lysosomes in a Stmn2‐dependent manner as part of their paracrine mechanisms. Using the αTC1‐6 glucagon‐secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1‐RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA‐mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2‐dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in αTC1‐6 cells through the Arf‐like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2‐mediated lysosomal transport may be a potential new pathway through which insulin and somatostatin regulate glucagon secretion in the pancreatic α cell. image Key points: Glucagon is produced by the pancreatic islet alpha cell to maintain blood glucose levels, and its secretion is tightly regulated by the islet hormones, insulin and somatostatin. Insulin and somatostatin are known to inhibit glucagon secretory granule exocytosis. In this study, we report a novel pathway through which insulin and somatostatin may inhibit glucagon secretion by directing glucagon to lysosomes through the actions of stathmin‐2, a negative regulator of glucagon secretion. We show that insulin directs glucagon and lysosomes away from sites of exocytosis in a stathmin‐2‐dependent manner, while somatostatin retains lysosomes intracellularly in a stathmin‐2‐dependent manner. These results demonstrate a new pathway by which insulin and somatostatin regulate glucagon secretion, furthering our understanding of alpha cell physiology.

The secretion of glucagon from the pancreatic alpha (α) cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on α cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the αTC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in αTC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

Purpose : In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2- 18 F-fluoroethoxy)-L-tryptophan ( 18 F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18 F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18 F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18 F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18 F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18 F-L-FEHTP uptake with beta cell mass. Conclusions: 18 F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18 F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.

Glucagon is a 29-amino acid hormone synthesized and secreted by the pancreatic alpha cell in the islets of Langerhans. It is the primary glucose counter-regulatory hormone, secreted by the alpha cell to maintain euglycemia by stimulating hepatic gluconeogenesis and glycogenolysis. In addition to glucose, the alpha cell senses and responds to a number of inputs, such as paracrine factors, neurotransmitters, and other nutrients, including amino acids, to regulate the secretion of glucagon. Disruption of this fine regulation results in excessive glucagon secretion (hyperglucagonemia) and contributes to the pathogenesis of diabetes. In this mini-review, we summarize the current understanding of glucagon biosynthesis and intracellular trafficking, and we discuss emerging concepts in amino acid sensing and signaling that underpin the biology of the alpha cell and that may provide clues to the control of the hyperglucagonemia of diabetes.

Dilated cardiomyopathy affects greater than 1 in 2500 patients worldwide, including those with the neuromuscular disorder Duchenne muscular dystrophy (DMD). While inflammation within skeletal muscle is strongly associated with DMD pathology, the key biomarkers for inflammation and possible targets for therapy within cardiac tissue in DMD-associated dilated cardiomyopathy remain to be identified. One such potential target is the myocardial ghrelin-growth hormone secretagogue receptor (GHSR) system, which is associated with cardiomyocyte survival and inhibition of inflammation. We sought to determine alterations in myocardial GHSR together with markers of cardiac inflammation using mdx:utrn^−/− mice as a model for DMD-associated dilated cardiomyopathy. With traditional histopathology, we determined that the pathology of DMD in mdx:utrn^−/− mice was characterized by disruption of myofiber organization, lymphocytic infiltration, and extensive cardiomyocyte vacuolization and necrosis surrounding areas of fibrosis in the left ventricular wall and apex. Using a fluorescent ghrelin analog, Cy5-ghrelin (1–19), to visualize GHSR with fluorescence confocal microscopy, we demonstrate that GHSR is elevated in mdx/utrn^−/− myocardial tissues and correlates strongly with both F4-80 (activated macrophages) and IL-6 (pro-inflammatory cytokine), and negatively with cardiac function. We also show that GHSR can be visualized in pro-inflammatory macrophages, suggesting a direct role for GHSR in the inflammatory progression of DMD.

The peptide hormone ghrelin is produced in cardiomyocytes and acts through the myocardial growth hormone secretagogue receptor (GHSR) to promote cardiomyocyte survival. Administration of ghrelin may have therapeutic effects on post–myocardial infarction (MI) outcomes. Therefore, there is a need to develop molecular imaging probes that can track the dynamics of GHSR in health and disease to better predict the effectiveness of ghrelin-based therapeutics. We designed a high-affinity GHSR ligand labeled with ¹⁸F for imaging by PET and characterized its in vivo properties in a canine model of MI. Methods: We rationally designed and radiolabeled with ¹⁸F a quinazolinone derivative ([¹⁸F]LCE470) with subnanomolar binding affinity to GHSR. We determined the sensitivity and in vivo and ex vivo specificity of [¹⁸F]LCE470 in a canine model of surgically induced MI using PET/MRI, which allowed for anatomic localization of tracer uptake and simultaneous determination of global cardiac function. Uptake of [¹⁸F]LCE470 was determined by time–activity curve and SUV analysis in 3 regions of the left ventricle—area of infarct, territory served by the left circumflex coronary artery, and remote myocardium—over a period of 1.5 y. Changes in cardiac perfusion were tracked by [¹³N]NH₃ PET. Results: The receptor binding affinity of LCE470 was measured at 0.33 nM, the highest known receptor binding affinity for a radiolabeled GHSR ligand. In vivo blocking studies in healthy hounds and ex vivo blocking studies in myocardial tissue showed the specificity of [¹⁸F]LCE470, and sensitivity was demonstrated by a positive correlation between tracer uptake and GHSR abundance. Post-MI changes in [¹⁸F]LCE470 uptake occurred independently of perfusion tracer distributions and changes in global cardiac function. We found that the regional distribution of [¹⁸F]LCE470 within the left ventricle diverged significantly within 1 d after MI and remained that way throughout the 1.5-y duration of the study. Conclusion: [¹⁸F]LCE470 is a high-affinity PET tracer that can detect changes in the regional distribution of myocardial GHSR after MI. In vivo PET molecular imaging of the global dynamics of GHSR may lead to improved GHSR-based therapeutics in the treatment of post-MI remodeling. Visual Abstract

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as an α-cell protein that regulates glucagon secretion by directing glucagon toward the endolysosomal system in αTC1-6 cells. We hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes in diabetes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ), glucagon secretion and cellular content were augmented, but cellular Stmn2 levels were reduced (p < .01), as measured by both ELISA and immunofluorescence intensity. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A⁺ lysosomes was dramatically reduced (p < .001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p < .01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for 2 weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K⁺-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A⁺ lysosome toward the secretory Lamp1⁺ lysosome.