Faculty of Pharmacy Mansoura University Department of Biochemistry Effect of omega -3 fatty acids on toll -like receptor 4 and fibroblast growth factor in experimentally induced diabetes in rats Presented by Salma Mossad Eraky Nasr B. Pharm. Sci., (2012) Supervisors Prof. Dr. Laila Ahmed Eissa Professor and Head of Biochemistry Department Faculty of Pharmacy Mansoura University Dr. Noha Abdel-Rahman Lecturer of Biochemistry Faculty of Pharmacy Mansoura University Thesis submitted as a Partial Fulfillment for Master Degree in Pharmaceutical Sciences (Biochemistry) 2015 Summary and conclusion Summary and conclusion Type 2 diabetes mellitus (T2DM), which accounts for about 90–95% of those with diabetes is mainly characterized by insulin resistance (IR) (ADA, 2014). Management of T2DM cannot depend only on medications, but lifestyle modifications should be considered. Omega-3 fatty acids are known to have anti-inflammatory and lipid-lowering effects, suggesting that they may be beneficial in T2DM management (Flachs et al., 2014). Pioglitazone, a drug belongs to thiazolidinedione group of insulin sensitizers, which act by activating peroxisome-proliferator activated receptor gamma (PPAR- γ) (Konda et al., 2014). The present study aimed to evaluate the anti-diabetic activity of omega-3 fatty acids, pioglitazone and their combination, and to understand their mechanism through measuring the gene expression and protein concentration of toll-like receptor 4 (TLR-4) and fibroblast growth factor 21 (FGF21) in a rat model of T2DM. Protocol of type 2 diabetes mellitus induction: Rats were fed high fat diet (HFD) for 4 weeks, then injected 35 mg/kg streptozotocin (STZ) after fasting for 12 hours (Srinivasan et al. 2005), and continued on HFD until the end of study. STZ was freshly dissolved in (0.1 M) citrate buffer (pH 4.5) and immediately injected into rats. To overcome hypoglycemia which follows STZ during the first 24 hours after their injection, diabetic rats were given 5% glucose solution to drink instead of tap water. Diabetes was approved 3 days after STZ injection by measuring blood glucose from tail vein using (ACCU-CHECK GO, Roche Diagnostics, Mannheim, Germany) glucometer. Rats having blood glucose > 250 mg/dl were considered diabetic. 124 Summary and conclusion The animals were randomly divided into 5 groups: (1) Control group (6 rats): maintained on normal pellet diet (3.15 kcal/g), and received single dose of citrate buffer (0.1 M, pH 4.5) intraperitoneally (i.p). (2) Diabetic group (8 rats): in which diabetes was induced by HFD and low-dose STZ. (3) ω-3 group: diabetic rats received omega-3 fatty acids (EPA and DHA) (10% of diet) (Devarshi et al., 2013) for 4 weeks. (4) PIO group (8 rats): diabetic rats treated with pioglitazone (20 mg/kg/day, orally) (Ding et al., 2005) for 4 weeks. (5) ω-3+ PIO group (8 rats): diabetic group treated with a combination of both omega-3 fatty acids (10% of diet) and pioglitazone (20 mg/kg/ day, orally) for 4 weeks. At the end of the study period, rats were denied from food for 12 hours. Blood glucose was measured and then rats in each group were weighed. Blood (for sera preparation) and liver samples were collected. Sera were used for analysis of: 1. Total cholesterol, triglycerides, HDL-cholesterol, and Total lipids by spectrophotometry method. 2. Insulin and FGF21 by ELISA method. Liver tissues were divided into three sections: The first section: was immediately immersed in liquid nitrogen and stored at -80°C for quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of TLR-4 and FGF21. The second section: homogenized in ice-cold phosphate buffered saline (PBS) (0.02 M, pH 7.4) (10% w/v), centrifuged, and kept at -80˚C until further analysis of 1. TLR-4 concentration by ELISA assay. 2. Malondialdehyde (MDA) concentration by spectrophotometry. 125 Summary and conclusion The third section: homogenized in ice-cold PBS (0.02 M, pH 7.4) (10% w/v) with a glass homogenizer. The resulting suspension was subjected to ultra-sonication using Ultrasonic homogenizer, centrifuged, and stored at -80°C for further analysis of FGF21 by ELISA assay. The most important results of the present study are: 1. Omega-3 fatty acids, pioglitazone, and their combination significantly reduced fasting blood glucose concentrations. 2. Pioglitazone, and the combination significantly reduced HOMA-IR (Homeostasis model of insulin resistance). 3. Omega-3 fatty acids, pioglitazone, and their combination significantly reduced total lipids, total cholesterol, and triglycerides. 4. Combination therapy showed synergistic triglycerides-lowering effect than either omega-3 fatty acids or pioglitazone alone. 5. Omega-3 fatty acids, pioglitazone, and their combination significantly reduced hepatic MDA concentration. 6. Combination therapy showed synergistic MDA-lowering effect than either omega-3 fatty acids or pioglitazone alone. 7. Omega-3 fatty acids, pioglitazone, and their combination significantly reduced serum FGF21, alleviating the state of FGF21 resistance. 8. Pioglitazone significantly increased hepatic FGF21 gene expression, while omega-3 fatty acids and the combination treatment caused non-significant decrease . 9. Omega-3 fatty acids and the combination significantly increased inactive TLR-4 concentration, leading to reduction of its protein activation, and inhibiting the inflammatory pathway. 10. Omega-3 fatty acids, pioglitazone, and the combination significantly reduced gene expression of TLR-4. 126 Summary and conclusion 11. A significant positive correlation was found between serum FGF21 and fasting blood glucose, HOMA-IR, serum total cholesterol, serum triglycerides, serum total lipids and hepatic MDA. 12. A significant negative correlation was found between hepatic inactive TLR-4 and fasting blood glucose, HOMA-IR, serum total cholesterol, serum triglycerides, serum total lipids and hepatic MDA. 13. A significant negative correlation was found between hepatic inactive TLR-4 and serum FGF21. Conclusion: Combining PPAR-α agonist, as omega-3 fatty acids (natural product), with PPAR-γ agonists, as pioglitazone (commercially available drug), showed potential effects in lowering blood glucose concentrations and improving lipid profile and IR. Such effects are mediated through modulation of TLR-4 and FGF21 as well as inhibition of lipid peroxidation. Clinical studies should also be conducted to evaluate the potentiatial anti-diabetic effect of omega-3 fatty acids alone or in combination with pioglitazone in patients with T2DM. 127
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