Submitted in fulfillment of the requirement for the degree of Doctor of Philosophy in the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg / Renewed interest in cannabinoid compounds arose since the discovery of the endocannabinoid system in the early 1990’s and its role in mediating the body’s energy balance. The aim of this study was to investigate the effect of an organic Cannabis sativa (hereafter referred to as C. sativa) extract on β-cell secretory function using an in vivo diet-induced obese rat model and an in vitro isolated rat pancreatic islet model and to determine the associated molecular changes within the pancreatic tissue. Materials and methods: Diet-induced obese Wistar rats and rats fed on standard pellets were subcutaneously injected, over a 28 day period, with an organic C. sativa extract or the vehicle (1% Tween 80® in saline). The effect of diet and treatment was evaluated using the intraperitoneal glucose tolerance tests (IPGTTs) and quantitative polymerase chain reaction (qPCR) analysis on rat pancreata. In vitro studies were conducted using isolated rat islets exposed to 11.1 (representative of normoglycemic conditions) and 33.3 mM glucose levels (representative of hyperglycemic conditions) over a 24-(D1; acute) and 96-hour (D4; chronic) period, and treated with C. sativa extract containing an equivalent of 2.5 (T1) and 5 ng/mL (T2) tetrahydrocannabinol (THC). Glucose-stimulated insulin secretion (GSIS), immunohistocytochemistry for apoptosis and proliferation detection and western blotting for detection of cannabinoid receptor type 1 (CB1), CB2 receptors and specific transduction factors were undertaken. Antagonist studies were conducted using AM251 (A1) and AM630 (A2) to block CB1 and CB2, respectively, to determine the role of cannabinoid receptors in insulin secretion. Results: The overall increase in body weight in the experimental groups occurred at a significantly slower rate than the control groups (P < 0.01), irrespective of diet. In the lean group, the area under the curve for glucose (AUCg) was significantly higher compared to the diet-induced obese group (P < 0.001), while C. sativa treatment significantly improved the AUCg in the lean rats (P < 0.05). The cafeteria diet did not induce hyperglycemia and insulin resistance in the obese rats and C. sativa treatment maintained a plasma glycemic profile similar to the obese control rats. The lower AUCg values in the obese group may, in part, be due to the inclusion of milk products (shown to be beneficial in reducing diabetes) in the cafeteria diet. qPCR analysis showed that the cafeteria diet induced down-regulation of the following genes in the obese control group, relative to lean controls: UCP2 (P < 0.01), c-MYC (P < 0.05) and FLIP (P < 0.05), and upregulation of CB1 (P < 0.01), GLUT2 (P < 0.001), UCP2 (P < 0.001) and PKB (P < 0.05), relative to the obese control group, while c-MYC levels were down-regulated (P < 0.05), relative to the lean control group. In the in vitro study, results showed C. sativa treatment decreased chronic insulin secretion in islets cultured under normoglycemic condition for D1 (P < 0.05), but not for D4. In islets cultured under hyperglycemic conditions, C. sativa treatment for the D4 period showed a significant increase in their chronic insulin secretion (HD4T1, P = 0.07; HD4T2, P < 0.001), increase in basal insulin secretion (HD4T1, P < 0.001; HD4T2, P < 0.001), increase in GSIS (HD4T1, P < 0.05; HD4T2, P < 0.001), reduction in glucose-stimulated:basal insulin production (HD4T1, P < 0.05; HD4T2, P < 0.05), reduction in insulin content (HD4T1, P < 0.001), increase in the percentage basal : content ratio (HD4T1, P < 0.001; HD4T2, P < 0.01) and increase in the percentage GSIS : content ratio (HD4T1, P < 0.001; HD4T2, P < 0.05), relative to ND4C islets. In antagonist studies, A2 preconditioning did not affect suppress the stimulatory effect of C. sativa treatment on chronic insulin secretion under normo- and hyperglycemic conditions, relative to the NC and HC islets, respectively. qPCR studies showed that C. sativa exposure induced a 2.2-fold increase in CB1 gene expression, relative to normoglycemic control islets (P < 0.05), while c-MYC and FLIP expression was significantly reduced by 12% (ND4T1, P < 0.05) and 37% (HD4T1, P < 0.05), respectively. C. sativa treatment also induced increased secretion of anti-inflammatory cytokines/chemokines under hyperglycemic conditions. Conclusion: These results suggest that C. sativa protects pancreatic islets against the negative effects of obesity (in vivo studies) and hyperglycemia (in vitro studies). In light of these findings, further investigation into the potential of C. sativa as a complementary therapeutic agent in the treatment of the deleterious effects of hyperglycemia in diabetic patients is warranted. In addition, the significant effect of C. sativa treatment on adipose tissue in experimental rats needs further investigation to determine how the cannabinoids affect the mechanisms of adipogenesis and lipolysis in diet-induced obesity. Keywords: Diet-Induced Obesity, Cannabinoids, C. sativa, THC, β-cell, AM251, AM630.

Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of cannabinoids and also their potentials for cartilage repair and regeneration. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602 and HU-308 with and without their antagonists on the wound healing of chondrocyte monolayers was investigated using a simple scratch assay model. The three cannabinoids were found to increase wound healing of chondrocyte monolayers, but at different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of increasing both migration and proliferation of chondrocytes cultured in a chondrogenic media, which increased the rate of wound closure. It was also found that treating the cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the rate of wound closure. These findings were further investigated, by studying the effect of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen type-II, fibronectin and S100 proteins were detected using immunofluorescence and verified quantitatively using ELISA based techniques, following treatment with 1μM and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with 1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and significantly reduced collagen type-I compared to control groups in monolayers and chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO, and MMP-2, in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory agent, which could be used to enhance tissue engineering protocols aimed at cartilage repair.

Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of cannabinoids and also their potentials for cartilage repair and regeneration. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602 and HU-308 with and without their antagonists on the wound healing of chondrocyte monolayers was investigated using a simple scratch assay model. The three cannabinoids were found to increase wound healing of chondrocyte monolayers, but at different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of increasing both migration and proliferation of chondrocytes cultured in a chondrogenic media, which increased the rate of wound closure. It was also found that treating the cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the rate of wound closure. These findings were further investigated, by studying the effect of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen type-II, fibronectin and S100 proteins were detected using immunofluorescence and verified quantitatively using ELISA based techniques, following treatment with 1μM and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with 1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and significantly reduced collagen type-I compared to control groups in monolayers and chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO, and MMP-2, in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory agent, which could be used to enhance tissue engineering protocols aimed at cartilage repair. / Egyptian Government