>Magister Scientiae - MSc / The plant Hypoxis hemerocallidea, also known as the African potato, is commonly used as a traditional medicine to treat diabetes in South Africa. The mechanism by which it lowers blood glucose levels is not known. The main aim of this research was to study the possible hypoglycemic effect of HH using RIN-5 F pancreatic tumor cells. To accomplish this, the study was divided into three parts: (1) to test whether exposure of RIN-5F cells to glucose and HH extract affect the cell proliferation and cell viability, (2) to test whether the HH extract have an effect on insulin secretion, and (3) to test whether the HH extract has an effect on alpha amylase and alpha glucosiadase enzyme activity. The RIN-5F cells were exposed to different concentrations of glucose (5, 10, 20, 37.5, 50, 55, 74, and 92.3 mM) for different times (1, 3, 6 and 24 hours). The RIN-5F cells were also exposed to different concentrations of HH (50, 100, 150, 200 and 500 μ/ml) for different times (1, 3, 6 and 24 hours). Cell proliferation was evaluated using crystal violet staining and cell viability was evaluated using the XTT assay. To evaluate the effect of glucose and HH on RIN-5 F cell insulin secretion the cells were exposed to HH (100 μg/ml or 500 μg/ml) and / or glucose (2 mM or 50 mM) for 30 or 90 minutes. Insulin, α-amylase activity and α-glucosidase activity were evaluated by using commercially available colorimetric assays. Enzymatic activity in the presence of HH was compared with positive controls for α-amylase activity or α-glucosidase activity. Results are expressed as means ± SEM or median. Statistical differences among groups were analyzed by analyses of variance. P < 0.05 was considered as significant. An increase in the cell viability and cell proliferation was found when RIN-5 F cells were exposed to high glucose concentrations and a high dose of HH extract for a short time period (1, 3 and 6 hours). When the cells were exposed to the HH extract over 24 hours, HH did not affect cell viability significantly. Insulin secretion from RIN-5 F cells was increased when exposed to low glucose (2 mM) or high glucose (50 mM) for 30 minutes. Insulin secretion was increased from RIN5F cells after exposure to low HH (100 μg/ml) or high HH (500 μg/ml) for 30 minutes. Exposure of RIN5-F cells to HH for 90 minutes caused a further increase in insulin secretion from (4.3±0.17 mIU/mg protein; P ≤ 0.01) in 100 μg/ml, to (7.87±0.17 mIU/mg protein; P ≤ 0.001) in 500 μg/ml. At both 30 minutes and 90 minutes, insulin secretion was significantly higher when cells where exposed to 500 μg/ml HH compared to 100 μg/ml HH. Insulin secretion by cells exposed to 2 mM glucose + 100 μg/ml HH (4.69±0.16 mIU/mg protein; P ≤ 0.001) was significantly higher than when exposed to 2 mM glucose only (2.27±0.17 mIU/mg protein), while the insulin secretion in 2 mM glucose + 500 μg/ml HH (2.56±0.17 mIU/mg protein; P > 0.05) was not significantly different from that in 2 mM glucose treated cells (2.27±0.17 mIU/mg protein). Similar results are obtained after 90 minutes. In the presence of high-glucose (50 mM), at both 30 minutes and 90 minutes, insulin secretion was significantly decreased when cells where exposed to low concentration of HH (100 μg/ml) and high concentration of HH (500 μg/ml). The HH extract produced α-amylase enzyme inhibition. The maximum inhibition was at a concentration of 10μg/ml (922±117U/ml; P ≤ 0.01). The 5 μg/ml concentrations failed to produce significant inhibition. The HH extract had significant α- glucosidase inhibitory activity at a concentration of 5μg/ml (0.12±0.3U/ml; P ≤ 0.001) or 10μg/ml (0.13±0.3U/ml; P ≤ 0.001). In conclusion, based on its ability to inhibit α-amylase and α- glucosidase activity HH has the potential to be used in control of blood glucose levels. The HH aqueous extract increased insulin secretion under our basic experimental conditions and in the presence of low glucose levels, but not at high (50 mM) glucose concentrations. Insulin secretion in the presence of different glucose concentrations, in the presence of HH, needs further investigation. It is recommended that the ability of HH to stimulate insulin secretion be evaluated at 15-20 mM glucose.

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Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Doutor em Genética e Bioquímica / CHAPTER II: Purpose: We performed a meta-analysis of randomized trials to assess the effect of metformin on inflammatory markers and metabolic parameters in subjects with diabetes. Methods: We performed comprehensive searches on NCBI, Cochrane, Science Direct databases from 1966 to Jun of 2015. We included randomized trials of at least 4 weeks duration that compared groups with diabetes before and after the treatment with metformin or metformin plus other drugs, and evaluated body mass index, blood glucose, HbA1c and inflammatory parameters such as C-reactive protein, tumor necrosis factor and adiponectin. Results: Pooled results of the 26 trials, with 1760 participants at the end of treatment reduce BMI in 0.9% p=0,0043, as well as, decrease of blood glucose level [SMD -0,411 mg/dL, 95%CI -0,463 to -0,369, I2= 56.62%], HbA1c [SMD -0.479%, 95%CI -0,568 to -0,390, I2= 55.02%], CRP levels [SMD -0,274mg/dL, 95%CI -0,419 to -0,129, I2= 72.78%], TNFα concentration [SMD -0,103pg/ml, 95%CI -0,514 to 0,309, I2= 87.67%] and increase of adiponectin [SMD 0,171μg/ml, 95%CI 0,098 to 0,440, I2= 81.09%] compared with pretreatment. Conclusion: The long-treatment with metformin monotherapy or metformin plus other drugs improves metabolic parameters and induced changes in inflammatory markers in diabetic subject. CHAPTER III: Background: Metformin increases insulin sensitivity by decreasing hepatic glucose production and increasing glucose disposal in skeletal muscle. However, modulation of inflammatory response and CaMKKβ/AMPK/Myosin V activation in gastrocnemius muscle by metformin treatment has not been demonstrated in hypoinsulinemic diabetic rats. Objective: The present study investigated how the metformin improve insulin sensitivity in skeletal muscle of hypoinsulinemic diabetic rats. Methods: Diabetes was induced by streptozotocin (45 mg/kg, intraperitoneally) 10 days prior treatments. On 11th day, diabetic rats were treated with metformin (500 mg/kg, oral gavage), insulin (2U at 08:00 h and 4U at 17:00 h, subcutaneously) or untreated. After 20 days, glycemia was measured and insulin sensitivity was determined by KITT. Serum Insulin, GLUT4, IRSthr, inflammatory markers (NF-κB, IκB, TNF-α and p-JNK) and CAMKK, AMPK and Myosin V in gastrocnemius muscle were determined by ELISA. Results: As expected, insulin and metformin improved the insulin sensitivity. Besides, metformin treatment promoted reduction in inflammatory response mediated by NF-κB, IκB, TNF-α and p-JNK, and that was accompanied by increased CaMKKβ/AMPK/Myosin V/GLUT4 pathway activity in gastrocnemius muscle of diabetic rats. Conclusion: Our findings suggest that metformin induces significant reductions in several inflammatory markers in skeletal muscle of diabetic rats. Metformin-induced increase in CaMKKβ/AMPK/Myosin V/GLUT4 pathway activity was associated with higher insulin sensitivity. CHAPTER IV: Diabetes is characterized by a proinflammatory state which can activate TLR2 and TLR4, and these receptors could induce NF-κB and JNK activation in skeletal muscle. In this study, we investigated the inflammatory and apoptotic signaling pathways triggered by TLRs/NF-κB and JNK activation in skeletal muscle of diabetic rats treated with metformin before and after an insulin tolerance test. Metformin treatment decreased p-JNK and NF-κB, and increased IκB concentrations. This attenuation leads to a decrease of TNFα and CXCL1/KC, and an increase of p-AMPK, BAX and Bcl2 concentration. Furthermore, KITT revealed an improvement of the insulin sensitivity in the diabetic rats treated with metformin. In addition, metformin was not capable of attenuating the changes in the inflammatory pathway triggered by insulin injection as the increase of TNFα and TLR4 in metformin treated rats, and IκB, CXCL1/KC, TNFα and p-AMPK increase in the untreated group. Taken together, these results point out that metformin may attenuate the activation of the inflammatory pathway TLRs/NF-κB/TNFα/CXCL1/KC and the apoptotic signaling BAX/Bcl2/p-JNK, which could be accompanied by a reduction of the inflammatory damage caused by hyperglycemia and an improvement of insulin sensitivity in diabetic rats.