BIOCHEMICAL STUDIES ON AMELIORATIVE EFFECT OF PROBIOTIC ON INDUCED OBESITY IN RAT.

Mohamed Ahmed Kandeil 1 , Ghada Mohamed Safwat 1 , Mahmoud Mohamed Arafa 2 and Doaa Ahmed Mansour 2 . 1. Biochemistry Department, Faculty of Veterinary Medicine, Beni-Suef University,Beni-Suef, Egypt. 2. Biochemistry Department, Animal Health Research Institute, Dokki, Giza, Egypyt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Obesity is a significant cause of morbidity and mortality worldwide. Obesity was induced in our experiment by consumption of diet containing 15% palm oil and by adding of sucrose (20%) to the drinking water for a period of 16 weeks. Probiotics have several health benefits so we used PROBAC Plus (20 million mo s /g.) to evaluate its ameliorative effect on biochemical changes associated with obesity. It composed of Lactobacillus Acidophilus, Lactobacillus Planterum, Lactobacillus Bervis, Bifidobacteriais and Saccharomyces Cerevisiae. 80 male albino rats were divided into control group (Cr), High fat diet &sucrose group (HFDS), probiotic group (Pro.), High fat diet & sucrose + probiotic group (HFDS+ Pro.) and High fat diet& sucrose then probiotic group (HFDS then Pro.). Consumption of palm oil and sucrose for 16 weeks leads to a significant increase in body weight and abdominal fat mass compared to normal rats. In addition, there was a significant increase in the level of serum triacylglycerol, total cholesterol, LDL-C, liver enzymes and leptin hormone. Malondialdehyde (MDA) and TNF-α concentration of liver homogenate were also significantly increased. It was also observed a significant reduction in serum level of HDL-C. The concentration of SOD and reduced glutathione (GSH) in liver homogenate were reduced. Use of probiotic as proflactive or as treatment return serum lipid profile, liver function enzymes, antioxidant parameters, TNF-α and leptin hormone towards its normal value. These results confirm the beneficial role of probiotic for treatment of obesity.
Biochemical analysis:-Serum total cholesterol level was determined by using of colorimetric method (CHOD-PAP) according to (Richmond, 1973). Serum triacylglycerol and HDL -C levels were determined by colorimetric method according to (Fossati and Prencipe, 1982) and (Burstein et al., 1970) respectively and by using of the commercial diagnostic laboratory kits (Diamond Company, Cairo, Egypt). Serum concentration of LDL-C and VLDL-C were calculated according to the formula of (Friedewald et al., 1972).
The serum activities of ALT and AST were determined colorimetric ally according to (Reitman and Frankel, 1957) and by using Diamond company diagnostic laboratory commercial kits.
The serum activities of ALP and GGT were determined by kinetic method according to (Szasz, 1969) and (Bray, 1974) and by using of Diamond company diagnostic laboratory commercial kits.
Serum leptin level was analyzed by Radio Immune -Assay (RIA) method using RIA kits, Amersham international Ltd Amersham United Kingdom.  Results in (table1) showed a significant increase in body weight (g.) of rats in the HFDS group after 16 weeks of consumption of high fat diet containing 15% palm oil and by adding of sucrose (20%) to the drinking water in comparison to control group and also to the probiotic group. Also body weight (g.) of rats was increased in the HFDS + probiotic group in comparison to control group and probiotic group. Using of probiotic mixture with HFDS from the beginning of the experiment (HFDS + probiotic group) or from the beginning of 8 th week (HFDS then probiotic group) led to non-significant decrease in body weight of rats in comparison to HFDS. Results in the same 1307 table revealed that abdominal fat mass (g.) was significantly increased in HFDS group in comparison to control group. Administration of probiotic mixture led to a significant decrease in abdominal fat mass (g.) in (probiotic group), (HFDS + probiotic group) and (HFDS then probiotic group) in comparison to HFDS group. Results in (table 2) showed an extremely significant increase in serum total cholesterol, triacylglycerol, LDL-C and VLDL-C concentrations in the HFDS group of rats in comparison to control group, while the serum level of HDL-C was significantly decreased. Administration of probiotic led to extremely significant decrease in the serum concentration level of total cholesterol, triacylglycerol, LDL-C and VLDL-C in the (probiotic group), (HFDS + probiotic group) and (HFDS then probiotic group) in comparison to HFDS group, while serum HDL-C level was significantly increased in comparison to HFDS group.  (table 3) revealed an extremely significant increase in serum levels of liver function enzymes (ALT, AST, ALP, GGT) of HFSD group as compared with control group of rats. Probiotic mixture administration represented by (probiotic group), (HFDS + probiotic group) and (HFDS then probiotic group) led to extremely significant decrease in the previous parameters as to HFSD group. Results in (table4) revealed an extremely significant decrease in GSH, SOD and increase in MDA concentrations in liver tissue homogenate of HFSD group as compared to control group. There were no significant changes between probiotic group and control group. Administration of probiotic led to extremely significant increase in GSH, SOD and decrease in MDA concentrations of liver tissue homogenate when compared to HFSD group. Results in (table 5) showed a significant increase in serum leptin concentration in HFDS, (HFDS with probiotic group) and (HFDS then probiotic group) in comparison to control group. Administration of probiotic alone (probiotic group) led to a significant decrease in serum leptin concentration in comparison to HFDS group. Results in the same table revealed that the serum TNF-α concentration was significantly increased in HFDS group in comparison to control group, while administration of probiotic alone (probiotic group) led to a significant decrease in serum TNF-α concentration in comparison to HFDS group.

Results:-
Results in all tables showed that using of probiotic as prophylactic with HFDS from the beginning of the experiment (HFDS with probiotic group) improved the abdominal fat mass, serum lipid profile values, liver function enzymes, antioxidant parameters, serum leptin and serum TNF-α levels much than using of it as treatment (HFDS then 1309 probiotic group), however there was no significant variation between (HFDS with probiotic group) and (HFDS then probiotic group).

Palm oil is extensively used in restaurants, hotels and in preparation of varieties of food products (Basu et al., 2013;
Gulati and Misra, 2017). In our study we induced obesity in male albino rats by adding 15% palm oil to normal diet and 20% sucrose in drinking water. Palm oil is a high saturated oil ( Our results showed that consumption of 15% palm oil and 20% sucrose for 16 weeks lead to a significant increase in body weight and abdominal fat mass (table 1)  Probiotics have been shown to modify the production of satiety hormones when given to rats (Forssten et al., 2013).
Also there was a significant increase in serum triacylglycerol, cholesterol, LDL-C and VLDL-C concentrations (table 2)

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Oxidative stress is a state of imbalance between free radicals production and its degradation by antioxidant systems with increased accumulation of these radicals (Goh and Cooper, 2008; Kao et al., 2010).
In our result (table 4), palm oil &sucrose uptake for 16 weeks lead to a significant decrease in liver homogenate concentration of both GSH and SOD, and a high significant increase in liver homogenate concentration of lipid peroxidation represented by MDA in comparison with that in control (cr group). These results are in agreement with (EL-Gohary and Hussien, 2015) whom explained the oxidative stress induced by obesity by different mechanisms, including the presence of excessive adipose tissue itself as adipocytes has been identified as a source of proinflammatory cytokines, including TNF-α. These cytokines are potent stimulators for the production of reactive oxygen and nitrogen by macrophages and monocytes; therefore, a rise in the concentration of cytokines could be responsible for increased oxidative stress (Fonseca-Alaniz et al., 2007).
Obesity takes place in disorders that affect mitochondrial metabolism, which favors ROS generation and the development of oxidative stress. On the other hand, another mechanism has been proposed that involves an effect of high triglyceride (TG) on the functioning of the mitochondrial respiratory chain, in which intracellular TG, which is also high, inhibits translocation of adenine nucleotides and promotes the generation of superoxide (Monteiro and Azevedo, 2010). In our study we observed a significant increase in ALT and AST activities (table 3)

Conclusion:-
Experimental induction of obesity by using of high fat diet (15% palm oil) and sucrose (20% in drinking water) for 16 weeks led to a significant increase in abdominal fat mass and body weight of rats. Serum levels of TC, TG, LDL-C and VLDL-C were also significantly increased resulted in hypercholesterolemia and hypertriglyceridemia, while serum level of HDL-C was significantly decreased. HFDS has an oxidative stress effect represented by the significant increase in the liver homogenate level of MDA and the significant decrease in the level of both GSH and SOD of liver homogenate of rats. Also the serum level of the proinflammatory cytokines (TNF-α) and leptin were significantly increased in HFDS group of rats. All the previous biochemical parameters in addition to body weight and abdominal fat mass were improved by administration of probiotic mixture either from the beginning of the 1311 experiment or started at the 8 th week of the experiment as probiotic mixture has an antioxidant, hypolipidemic and anti-obesity effects