Abstract
Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterised by hyperglycaemia and loss of peripheral tissue insulin sensitivity. Prolonged hypersecretion of insulin by the pancreatic beta cells to compensate this insulin resistance causes reactive oxygen species (ROS) accumulation. Due to low expression of detoxifying enzymes in the (3-cells, ROS accumulation leads to inflammation, mitochondrial and/or ER stress and eventually loss of functional 3- cells. Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by intestinal endocrine L-cells that potentiates postprandial glucose-stimulated insulin secretion and promotes 3-cell proliferation and survival. It has been reported previously that incretin effects are diminished in T2DM. Thus, any therapeutic agents that can stimulate endogenous GLP-1 secretion, enhance insulin secretion and protect the pancreatic 3-cells from detrimental effects of ROS and inflammation might have an anti-diabetic potential. Gypenosides (GYP) are triterpene glycosides that are known for their anti-diabetic properties in rat and mouse diabetic models, although their mechanisms of action remain unclear. Costus pictus D. Don leaf extract (CPE) is known for its stimulatory effect on insulin secretion from islets although its effect on GLP-1 secretion and 3-cell protection are unknown. Thus, this study aimed to elucidate the mechanisms of GYP on insulin secretion and the potential of GYP and CPE to protect 3-cells against oxidative stress using the rat BRIN-BD11 3-cell model. This study also investigated effects of GYP and CPE on GLP-1 secretion from L-cells using the well characterised murine GLUTag L-cell model.Cell viability was assessed by MTT assay and mRNA and protein levels were quantified by real time PCR and western-blot analysis. Intracellular calcium ([Ca2+]i) changes were measured in FURA-2AM loaded cells. Insulin and GLP-1 release XVI studies using BRIN-BD11 and GLUTag cells were performed and hormone levels measured using insulin and total GLP-1 ELISA, respectively.
In pancreatic BRIN-BD11 cells, both GYP and CPE showed a significant protective effect against palmitate and cytokine cocktail induced toxicity and enhanced cell viability over long term treatment of 48-72h. Over 24h treatment, both GYP and CPE significantly enhanced detoxifying enzymes including Sod1, Cat and Gpx1 and downregulated proinflammatory NFkB expression. GYP enhanced [Ca2+]i acutely in both BRIN-BD11 and GLUTag cells and this effect was completely blocked by the sodium/calcium exchanger (Ncx) reverse mode inhibitor SN-6. Non-specific beta-adrenergic receptor blockers (propranolol and alprenolol) and the specific 5HT-7 receptor blocker, SB269970, also completely abolished GYP ability to increase [Ca2+]i. GYP concentration-dependently enhanced insulin secretion from BRIN-BD11 cells irrespective of glucose concentration. Both GYP and CPE enhanced GLP-1 secretion from GLUTag cells irrespective of glucose concentration although long term treatment downregulated proglucagon gene expression as measured by real time PCR.
These results indicate that GYP induced [Ca2+]i influx into p-cells are mediated by the activation of Ncx reverse mode and possibly through activation of the 5HT-7 receptor. Both GYP and CPE stimulate GLP-1 secretion and protect p-cells against detrimental effects of free fatty acids (FFA) and cytokines. Thus, both GYP and CPE might be beneficial to enhance p-cell function and enhance endogenous GLP-1 levels, which may have therapeutic potential in T2DM. Further studies are necessary confirm these results in primary cells and in vivo model systems.
| Date of Award | 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Steven Patterson (Supervisor), Andrew Collier (Supervisor) & Sharron Dolan (Supervisor) |