Effects Of Betaine On Lipid Metabolism, Gene Expression Of Key Enzymes And Approach To The Mechanism In Finishing Pigs

The study was conducted to investigate the effects of betaine on lipid metabolism offinishing pigs by determining related enzymes activities and gene expression of keyemzymes (fatty acid synthase, carnitine palmitoyltransferaseâ… ), growth hormone (GH)pulsatile secretion and other related hormone, as well as physiological and biochemicalparameters. The object of this study was to approach to the mechanism that betaine maypossibly improve on carcass traits of pigs.The special primers for fatty acid synthase(FAS), liver-type carnitine palmitoyltransferaseâ… (L-CPTâ… ), muscle-type carnitine palmitoyltransferaseâ… (M-CPTâ… ) andβ-actin weredesigned respectively according to the gene sequences reported previously in Genebank.Then total RNA was extracted from subcutaneous adipose tissue, liver and longissimusmuscle of pigs and the target gene mRNA amplified through RT-PCR with special primers.The cDNA fragments of FAS, L-CPTâ… , M-CPTâ… andβ-actin were obtained. The PCRproduct was cloned into pUCm-T Vector and sequenced. Sequence analysis suggested thatthe gene fragment gained in this study shared 98.77ï¼…,99.59ï¼…,99.61ï¼…and 99.27ï¼…identity with the reported cDNA sequence (AY183428, AF288789, AY181062 and U07786)of pig in Genebank, respectively. Based on the gene cloning, the suitable annealingtempreture, Mg²⁺ concentration, and cycles in PCR system were discussed and the optimalsemi-quantitative RT-PCR methods were constructed to determine the effect of betaine onmRNA expression of FAS and CPTâ… in finishing pigs.A total of 48 crossbred barrows and gilts [Duroc boar×Seghers hybrid sow (15 line boar×12/36 line sow)] weighing about 55 kg were divided into two groups on the basis of weight,ancestry and gender, each with three replicates of eight pigs (four barrows and four gilts)per replicate, and fed corn-soybean meal basal diets supplemented with betaine at 0 or1250 mg/kg feed. The feeding experiment lasted 42 days after a 7-day of adaptation period.Feed and water were provided for ad libitum consumption throughout the experiment. Atthe end of the feeding trial, 12pigs (six pigs from each dietary treatment with one barrowand one gilt per replicate) weighing about 90 kg were selected to collect blood samples for the determination of GH pulsatile secretion. After 1 h of feed removal, blood samples wereobtained through jugular catheters every 15 min for 3 h (from 13:00 to 16:00 h). Pigs werethen slaughtered for carcass evaluation. Samples of serum, liver, longissimus muscle andsubcutenous adipose tissue were collected for subsequent analysis.In vitro cell culture, primary porcine hepatocytes were cultured in medium containing 0,1 mmol/L canitine, 2 mmol/L betaine, 4 mmol/L betaine, 1 mmol/L carnitne+2 mmol/Lbetaine, 1 mmol/L carnitne+4 mmol/L betaine, respectively, each with three replicates,and CPT I activity was measured after hepatocytes were cultured for 48 h and 96 h.The results of feeding experiment showed that adding 1250 m/kg betaine to diets offinishing pigs resulted in 5.45% (P＜0.05) increase in average daily gain, whereas averagedaily feed intake was not markedly affected (P＞0.05), and feed to gain ratio tended todecrease with betaine supplementation (P＞0.05).The results of carcass evaluation indicated that betaine supplementation significantlyimproved carcass characteristics of finishing pigs. Carcass lean percentage and loinmuscle area in pigs fed betaine were increased by 5.2% (P＜0.01) and 17.9% (P＜0.01),respectively, whereas carcass fat percentage and average backfat thickness were decreasedby 13.1% (P＜0.01) and 10.3 % (P＜0.05), respectively. There was no remarkable influenceof betaine on dressing percentage, carcass skin percentage, carcass bone percentage, leaffat weight (P＞0.05).Analysis of liver and longissimus muscle showed:â‘ Ether extract content inlongissimus muscle of pigs receiving betaine was 23.61% higher (P＜0.05) than that ofcontrols, whereas hepatic ether extract content tended to decrease (P＞0.05). No apparentdifferences in crude protein content of liver and longissimus muscle were obseveredbetween treatment groups (P＞0.05).â‘¡Adding 1250 m/kg betaine to diets of finishing pigsimproved free carnitine content in liver and acid-insoluble carnitine content in longissimusmuscle by 19.5% (P＜0.05) and by 45.6% (P＜0.01), respectively. Furthermore, Betainesupplementation decreased M-CPT I activity and M-CPT I mRNA expression by 11.1%(P＜0.05) and 14.6%(P＜0.05), respectively. No obvious effect of betaine was found onL-CPT I activity and L-CPT I mRNA expression (P＞0.05). There was a good correlationbetween enzyme activity and mRNA expression level for both L-CPT I and M-CPT I (P＜0.05; r=0.67 vs. r=0.72 for L-CPT I and M-CPT I, respectively).Analysis of subcutenous adipose tissue indicated that adding 1250 mg/kg betaine todiets markedly changed the activities of lipogenic enzymes, hormone-sensitive lipase andlipoprotein lipase in subcutaneous adipose tissue of finishing pigs. Hormone-sensitivelipase activity in subcutaneous adipose tissue of pigs treated with betaine was increased by15.1%(P＜0.05), while the activities of FAS, acetyl-CoA carboxylase, malic dehydrogenaseand lipoprotein lipase were decreased by 18.8% (P＜0.05), 18.0% (P＜0.01), 14.5% (P＜0.05)and 8.9% (P＜0.05), respectively, and the activities of glucose-6-phosphate dehydrogenaseand isocitrate dehydrogenase tended to reduce(P＞0.05).Moreover, betaine supplementationresulted in 25.0% (P＜0.05) decrease in FAS mRNA expressiom, and there was a significantcorrelation between mRNA expression level and enzyme activity for FAS (r=0.93, P＜0.01).Betaine also enhanced cAMP level in subcutaneous adipose tissue of pigsDetermination of GH pulsatile secretion showed that serum basal GH level, mean GHlevel and GH pulse amplitude were elevated by 41.8%(P＜0.01), 49.0%(P＜0.01) and 35.1%(P＜0.05) respectively with betaine supplementation, but GH pulse frequency and pulseduration remained unchanged (P＞0.05).Analysis of serum sample indicated that adding 1250 mg/kg betaine to diets offinishing pigs improved the concentration of insulin-like growth factor-â… , insulin, freetriiodothyronine, free thyroxine and thyroid-stimulating hormone in serum by 55.5%(P＜0.01), 42.3% (P＜0.05), 58.0% (P＜0.01), 51.8% (P＜0.01) and 49.7% (P＜0.01),respectively. Meanwhile, the level of serum total protein, free fatty acids and lipase activitywere increased by 8.8% (P＜0.05), 25.7% (P＜0.01) and 22.6% (P＜0.01) respectively withbetaine, supplementation, whereas serun urea nitrogen concentration was decrease by21.6% (P＜0.01). There was no apparent effect of betain on other parameters in finishingpigs.The results of cell culture showed that treatment with 2 mmol/L or 4 mmol/L betaine hadno significant effect on CPT I activity of primary porcine hepatocytes cultured for either 48hours or 96 hours compared with control (P＞0.05), whereas 1 mmol/L camitine andtreatment with both betaine and carnitine resulted in a remarkable increase in CPT Iactivity (P＜0.01). No marked difference in CPT I activity was observed between 1 mmol/L carntine and treatment with both betaine and carnitine (P＞0.05). Furthermore, CPT Iactivities at 96 h were higher than those at 48 h in all treatment except for control (P＜0.05).The results of the present study implicated:â‘ Betaine could increase weight gain andimprove carcass characteristics of finishing pigs.â‘¡Betaine could increase serum GHlevel in pigs and and affect GH pulsatile secretion.â‘¢Betaine could influence the responseof GH, insulin-like growth factor-â… , free triiodothyronine, free thyroxine,thyroid-stimulating hormone and insulin in finshing pigs, and therefore inhibit fat synthesisthrough reducing the activities and gene expression of lipogenic enzymes (as evidenced bythe decrease in FAS mRNA expression) and promote fat degradation by increasinghormone sensitive lipase activity. Meanwhile, betaine could improve free camitine contentin liver and acid-insoluble carnitine content in muscle via transmethylation, and hence leadto a decrease in the activity and mRNA expression of M-CPT I, with a resultant increase inintramuscular fat content. Betaine could alter lipid partitioning in finishing pigs such thatadipose tissue deposition is reduced and intramuscular fat accretion is increased.