Laila Ahmed Eissa

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.
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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.
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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.
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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.
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