Biology subjects

Dhanvantari, S.

Publications and source records attributed to Dhanvantari, S..

7 recordsLinked to original sources

bioRxiv2026

Cannabidiol Impairs Glucagon Trafficking and Secretion Through the Lysosomal System in Murine αTC1-6 cells

Glucagon secretion by pancreatic cells is regulated by nutrient, paracrine, and neural inputs, and emerging evidence suggests that the islet endocannabinoid system (ECS) may also regulate -cell function. While phytocannabinoids such as cannabidiol (CBD) have been shown to interact with the ECS, and negatively regulate insulin secretion from pancreatic {beta} cells, whether CBD alters 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 cell function. Using the TC1-6 glucagon-secreting cell line, we exposed the cells to 0.3 and 3 M CBD for RT-qPCR analysis, 3 M CBD for bulk RNA sequencing, and 3 M 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 TC1-6 cells.

cell biology↗

bioRxiv2026

Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

The secretion of glucagon from the pancreatic alpha () 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 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 TC1-6 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 TC1-6 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.

cell biology↗

bioRxiv2021

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

Glucagon hypersecretion from the pancreatic -cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from -cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as a protein that resides in -cell secretory granules, and showed that it regulates glucagon secretion by directing glucagon towards the endolysosomal system in TC1-6 cells. Here, we hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ) to induce diabetes, Arg-stimulated secretion of glucagon and Stmn2 was augmented. However, cell glucagon content was significantly increased (p<0.001), but Stmn2 levels were reduced (p<0.01) in STZ-treated mice, as measured by both ELISA and immunofluorescence intensity. Expression of Gcg mRNA increased ~4.5 times, while Stmn2 mRNA levels did not change. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p<0.001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p<0.01) increased. Further studies were conducted in TC1-6 cells cultured in media containing high glucose (16.7 mM) for two weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of TC1-6 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 towards the secretory Lamp1+lysosome.

cell biology↗

bioRxiv2020

Regional Differences in the Ghrelin-Growth Hormone Secretagogue Receptor Signalling System in Human Heart Disease

The hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR) are expressed in myocardium. GHSR binding activates signalling pathways coupled to cardiomyocyte survival and contractility. These properties have made the ghrelin-GHSR axis a candidate for a biomarker of cardiac function. The dynamics of ghrelin-GHSR are altered significantly in late stages of heart failure and cardiomyopathy, when left ventricular (LV) function is failing. We examined the relationship of GHSR with ghrelin in cardiac tissue from patients with valvular disease with no detectable changes in LV function. Biopsy samples from the LV and left atrium (LA) were obtained from 25 patients with valvular disease (of whom 13 also had coronary artery disease) and preserved LV ejection fraction, and compared control samples obtained via autopsy. Using quantitative confocal fluorescence microscopy, levels of GHSR were determined using a fluorescent peptide analog of ghrelin, Cy5-ghrelin(1-19); ghrelin, the heart failure marker natriuretic peptide type-b (BNP), and contractility marker sarcoplasmic reticulum ATPase pump (SERCA2a) were measured by immunofluorescence. A positive correlation between GHSR and ghrelin was apparent in only diseased tissue. Ghrelin and BNP significantly correlated in the LV and strongly co-localized to the same intracellular compartment in both diseased and control tissue. GHSR, ghrelin and BNP all strongly and significantly correlated with SERCA2a in the LV of diseased tissue only. Our results suggest that the dynamics of the myocardial ghrelin/GHSR axis is altered in cardiovascular disease in the absence of measurable changes in heart function, and may accompany a regional shift in endocrine programming.

molecular biology↗

bioRxiv2019

Stathmin-2 Mediates Glucagon Secretion from Pancreatic α-Cells

Inhibition of glucagon hypersecretion from pancreatic -cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the cell line, TC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in aTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. Stathmin-2 was co-secreted with glucagon in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in TC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, indicating that it is an alpha cell protein. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA - mediated depletion of stathmin-2 in aTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

cell biology↗

bioRxiv2018

Plasticity in the glucagon interactome reveals novel proteins that regulate glucagon secretion in alpha TC1-6 cells

Glucagon is stored within secretory granules of pancreatic alpha cells until a stimulus, such as a change in microenvironmental conditions, triggers its release. We hypothesized that the secretory response of the alpha cell to various stimuli could be determined by plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. To answer this question, we isolated secretory granules from alpha TC1-6 cells and identified glucagon-interacting proteins by affinity purification coupled with liquid chromatography/tandem mass spectrometry. Proteomic analyses revealed a network of cytoplasmic and histone proteins. Specifically, the interaction between glucagon and histone H4 and the ER stress protein GRP78 was confirmed through co-immunoprecipitation of secretory granule lysates, and co-localization within secretory granules using high-resolution confocal microscopy. The composition of these networks was altered at different glucose levels (25 mM vs 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. Finally, siRNA-mediated silencing of a subset of nonhistone proteins revealed novel proteins that may regulate glucagon secretion. We have therefore described a novel and dynamic glucagon interactome within alpha cell secretory granules, and suggest that plasticity in the interactome governs the alpha cell secretory response to paracrine and nutritional stimuli.

cell biology↗

bioRxiv2018

The Growth Hormone Secretagogue Receptor, Ghrelin and Biochemical Signaling Molecules in Human Heart Failure

BackgroundCurrently, the early pre-clinical detection of left ventricular (LV) dysfunction is difficult as biomarkers are not specific for the cardiomyopathic process. The underlying molecular mechanisms leading to heart failure remain elusive, highlighting the need for identification of cardiac-specific markers. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin are present in cardiac tissue and are known to contribute to myocardial energetics. Here, we examined tissue ghrelin-GHSR levels as specific markers of cardiac dysfunction in patients who underwent cardiac transplantation.\n\nMethods and ResultsSamples of cardiac tissue were obtained from 10 cardiac transplant patients at the time of organ harvesting, and during serial post-transplant biopsies. Quantitative fluorescence microscopy using a novel fluorescent ghrelin analog was used to measure levels of GHSR, and immunofluorescence was used to measure levels of ghrelin, b-type natriuretic peptide (BNP) and tissue markers of cardiomyocyte contractility and growth. GHSR and ghrelin expression levels were highly variable in the explanted heart, less in the grafted heart biopsies. GHSR and ghrelin were strongly positively correlated, and both markers were negatively correlated with LV ejection fraction. Ghrelin had stronger positive correlations than BNP with the signaling markers for contractility and growth.\n\nConclusionsThese data suggest that GHSR-ghrelin have potential use as an integrated marker of cardiac dysfunction. Interestingly, tissue ghrelin appeared to be a more sensitive indicator than BNP to the biochemical processes that are characteristic of heart failure. This work allows for further use of ghrelin-GHSR to interrogate cardiac-specific biochemical mechanisms in pre-clinical stages of HF.\n\nPrecisThis study shows the relationships between GHSR, ghrelin, and signaling molecules with relation to heart function in human heart failure with tissue from diseased heart and healthy heart biopsies.

cell biology↗