Betaine feeding prevents the blood alcohol cycle in rats fed alcohol continuously for 1 month using the rat intragastric tube feeding model

Abstract

Background: Blood alcohol levels (BAL) cycle up and down over a 7-8 day period when ethanol is fed continuously for one month in the intragastric tube feeding rat model (ITFRM) of alcoholic liver disease. The cycling phenomenon is due to an alternating increase and decrease in the metabolic rate. Recently, we found that S-adenosyl-methionine (SAMe) fed with alcohol prevented the BAL cycle.

Method: Using the ITFRM we fed rats betaine (2 g/kg/day) with ethanol for 1 month and recorded the daily 24 h urine ethanol level (UAL) to measure the BAL cycle. UAL is equivalent to BAL because of the constant ethanol infusion. Liver histology, steatosis and BAL were measured terminally after 1 month of treatment. Microarray analysis was done on the mRNA extracted from the liver to determine the effects of betaine and alcohol on changes in gene expression.

Results: Betaine fed with ethanol completely prevented the BAL cycle similar to SAMe. Betaine also significantly reduced the BAL compared to ethanol fed rats without betaine. This was also observed when SAMe was fed with ethanol. The mechanism involved in both cases is that SAMe is required for the conversion of epinephrine from norepinephrine by phenylethanolamine methyltransferase (PNMT). Epinephrine is 5 to 10 fold more potent than norepinephrine in increasing the metabolic rate. The increase in the metabolic rate generates NAD, permitting ADH to increase the oxidation of alcohol. NAD is the rate limiting factor in oxidation of alcohol by alcohol dehydrogenase (ADH). This explains how SAMe and betaine prevented the cycle. Microarray analysis showed that betaine feeding prevented the up regulation of a large number of genes including TLR2/4, Il-1b, Jax3, Sirt3, Fas, Ifngr1, Tgfgr2, Tnfrsf21, Lbp and Stat 3 which could explain how betaine prevented fatty liver.

Conclusion: Betaine feeding lowers the BAL and prevents the BAL cycle by increasing the metabolic rate. This increases the rate of ethanol elimination by generating NAD.

Fig 1

A. There was no significant difference in body weights among the 4 groups of rats at either the beginning or the end of the experiment (ET=ethanol, Bet=betaine, Dex=dextrose). B. All 4 groups gained the same amount of weight. There was a significant increase in the liver weights in the group 1 ethanol fed rats compared to the other 3 groups (Mean ±S.E.M., n=3). C. The liver/body weight ratios were also increased when ethanol alone was fed (Mean ±S.E.M., n=3).

Fig 2

A. There was a significant increase in ALT only in the group 1 ethanol fed rats when compared with the other 3 groups at the end of the experiment (Mean ±S.E.M., n=3). B. The total pathology score was markedly increased in the group 1 ethanol-fed rats compared to the other 3 groups of rats (Mean ±S.E.M., n=3). C. The representative liver histology from the 3 groups of rats fed ethanol or betaine are shown. The control fed dextrose showed the same histology as the control fed betaine (results not shown). Note that the group 1 ethanol fed rat livers showed an increase in macrovesicular fat and inflammation (arrow). H&E. D. Morphometric measurement of fat in histologic sections of the liver of the 4 groups of rats showed a significant increase of fat in the group 1 ethanol-fed rats (Mean ±S.E.M., n=3).

Fig 3

A. The UAL cycle was present when group 1 ethanol fed rats were monitored. However, the group 3 rats fed ethanol with betaine did not develop the UAL cycle. Instead the UAL hovered between 100 and 200 mg%. B. The terminal BAL of group 1 ethanol fed rats was significantly higher when compared with the rats fed betaine with ethanol (Mean ± SEM, n = 3).

Fig 4

A. The heat map of gene expression changes showed that rats fed alcohol alone differed from the other 3 groups of rats (15 fold, p<0.005). (Red is up regulated, blue is down regulated). B. The KEGG functional pathways changed when group 1 rats fed ethanol were compared with group 3 rats fed ethanol plus betaine. Ethanol up regulated (red bars) most functional pathways compared to the group 3 rats fed ethanol plus betaine (red= up regulated, yellow down regulated).

Fig 5

A. Liver tissue levels of choline were significantly increased in group 3 rats fed ethanol and betaine (Mean ±SEM, n=3). B. DMG levels in liver tissue were decreased in group 1 rats fed ethanol and group 3 rats fed ethanol plus betaine compared to group 2 rats fed isocaloric dextrose. (Mean ±SEM, n=3).

Fig 6

A. Betaine with or without ethanol markedly reduced serum choline levels (Mean +SEM, n=3). B. The serum betaine levels were decreased by feeding ethanol and betaine (group 3) compared to isocaloric dextrose with betaine (group 4) (Mean ±SEM, n=3).

Fig 7

The urine DMG levels were increased when group 3 alcohol plus betaine fed rats were compared to group 1 rats fed ethanol alone. (Mean ±SEM, n=3).

Fig 8

A. Urine, choline (A), betaine (B) and DMG (C) levels varied daily up and down over the 14 day feeding period, for ethanol group 1, and ethanol plus betaine, group 3.

Fig 9

SAMe is required for the enzymatic conversion from norepinephrine to epinephrine.