Isolation and characterization of a mouse betaine-homocysteine S-methyltransferase gene and pseudogene.

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5'-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.

Exercise training down-regulates hepatic lipogenic enzymes in meal-fed rats: fructose versus complex-carbohydrate diets.

The maximal activity and mRNA abundance of hepatic fatty acid synthase (FAS) and other lipogenic enzymes were investigated in rats meal-fed either a high fructose (F) or a high cornstarch (C) diet. The diet contained 50% F or C (g/100 g), casein (20%), cornstarch (16.13%), corn oil (5%), minerals (5.37%), vitamins (1%) and Solka-floc (2%). Female Sprague-Dawley rats (n = 44) were randomly divided into C or F groups that were meal-fed for 3 h/d; each group was subdivided into exercise-trained (T) and untrained (U) groups. Treadmill training was performed 4 h after the initiation of the meal at 25 m/min, 10% grade for 2 h/d, 5 d/wk, for 10 wk. Rats were killed 9 h after the meal and 27 h after the last training session. F-fed rats had significantly higher activities of all lipogenic enzymes assayed and mRNA abundance of FAS and acetyl-coenzyme A carboxylase (ACC) than C rats (P < 0.05). Concentrations of plasma insulin and glucose and liver pyruvate were not altered by F feeding. Proportions of the fatty acids 18:2 and 20:4 were lower, whereas those of 16:0 and 16:1 were higher, in livers of F than of C rats (P < 0.05). Training decreased FAS activity by 50% (P < 0.05), without affecting FAS mRNA level in C rats; this down-regulation was absent in the F rats. ACC mRNA abundance tended to be lower in CT than in CU rats (P < 0.075). L-Type pyruvate kinase activity was lower in FT than in FU rats (P < 0.05), whereas other lipogenic enzyme activities did not differ between T and U rats of each diet group. We conclude that hepatic lipogenic enzyme induction by high carbohydrate meal feeding may be inhibited by exercise training and that a fructose-rich diet may attenuate this training-induced down-regulation.

Hepatic and renal betaine-homocysteine methyltransferase activity in pigs as affected by dietary intakes of sulfur amino acids, choline, and betaine.

In Exp. 1, young pigs were fed a basal diet containing .17% methionine (Met) (.14% digestible Met), and .48% cystine (.38% digestible cystine) for 14 d (34 to 48 d of age). Treatment additions were .25% DL-Met, .34% betaine, .30% choline, or .25% DL-Met and .34% betaine. Methionine, but not betaine or choline supplementation, increased (P < .05) weight gain and feed efficiency. Hepatic betaine-homocysteine methyltransferase (BHMT) activity was increased (P < .05) by betaine and choline supplementation but was not affected by Met deficiency. Renal BHMT activity was increased (P < .05) by Met deficiency and was further increased (P < .05) by betaine supplementation. In Exp. 2, 10-kg pigs were fed the basal diet from Exp. 1 supplemented with enough DL-Met to bring the total basal Met to .24% (.20% digestible Met). Treatment additions consisted of .20% DL-Met or .34% betaine, and diets were fed for 16 d (34 to 50 d of age). Feed efficiency increased (P < .05) in response to Met, but not to betaine, supplementation. Hepatic BHMT activity increased (P < .05) in response to betaine and Met, but no changes in renal BHMT activity occurred. Although statistically significant changes in hepatic and renal BHMT activity occurred in both experiments, the magnitude of the responses was probably not physiologically important. Therefore, in contrast to previous findings with rats and chicks, it does not seem that hepatic and renal BHMT activity in pigs is influenced substantially by Met deficiency, or by surfeit levels of choline or betaine.

Killing of methicillin-resistant Staphylococcus aureus in vitro using aluminium disulphonated phthalocyanine, a light-activated antimicrobial agent.

The aim of this study was to determine whether 16 epidemic methicillin-resistant strains of Staphylococcus aureus (EMRSA) could be killed by the light-activated antimicrobial agent aluminium disulphonated phthalocyanine (AIPcS2). EMRSA suspensions were irradiated with light from a laser diode in the presence of AIPcS2 and survivors enumerated. All strains were susceptible to killing, the bactericidal effect being dependent on the AIPcS2 concentration and the light dose. AIPcS2 rendered the bacteria light-sensitive almost immediately and killing was unaffected by the growth phase of the organism. Scavengers of singlet oxygen and free radicals protected the bacteria from killing. These results imply that light-activated antimicrobial agents may be useful in eliminating EMRSA from wounds or carriage sites.

Exercise down-regulates hepatic lipogenic enzymes in food-deprived and refed rats.

The effects of an acute bout of prolonged exercise on the activities of several hepatic lipogenic enzymes and the abundance of fatty acid synthase (FAS) mRNA were evaluated using a food deprivation-refeeding protocol in which diets contained 50% of the energy from either fructose or cornstarch. Food was withheld from male rats for 48 h and refed for 0, 4, 8, 12, 24 or 48 h. At each time point, half of each dietary group was subjected to a single bout of treadmill running until exhaustion and killed immediately. The other half of each group rested without food for the same amount of time before being killed. Exercise significantly decreased FAS activity by 57, 46, 10, 26 and 70% at 4, 8, 12, 24 and 48 h of refeeding, respectively, in the fructose-fed rats; and by 70 and 63% at 24 and 48 h of refeeding, respectively, in the cornstarch-fed rats. Activities of L-type pyruvate kinase and glucose 6-phosphate dehydrogenase were significantly decreased after exercise in the fructose-fed, but not cornstarch-fed rats. In rested rats, FAS mRNA abundance increased approximately fourfold above the unfed levels after 8 and 12 h of refeeding. Exercise attenuated the diet-induced increases in FAS mRNA abundance. At 8 h of refeeding, both cornstarch- and fructose-fed exercised rats had 71% (P < 0.05) of the FAS mRNA levels of their rested counterparts; at 12 h, these exercised rats showed only 46 and 27% (P < 0.05) of FAS mRNA levels compared with rested rats fed the same diet. We conclude that dietary induction of FAS activity and mRNA abundance can be inhibited by prolonged exercise, suggesting that exercise may influence FAS transcription and/or mRNA stability.

Effects of acute exercise on hepatic lipogenic enzymes in fasted and refed rats.

The effects of an acute bout of prolonged exhaustive exercise on the activities of hepatic lipogenic enzymes have been investigated. Male Sprague-Dawley rats were randomly divided into three groups: fasted for 48 h without refeeding (FA) and fasted for 48 h and refed a diet high in fructose (RF) or in cornstarch (RC). One-half of each group of rats exercised on a treadmill at 20 m/min, 5% grade, until exhaustion and the other half rested for the same amount of time without food. Dietary intakes during refeeding were kept equal between the exercised and rested control animals. Activities of all hepatic lipogenic enzymes measured, i.e., fatty acid synthase (FAS), L-type pyruvate kinase (L-PK), ATP citrate lyase, malic enzyme, and glucose-6-phosphate dehydrogenase, were induced dramatically by fasting-refeeding and were significantly higher in the RF than in the RC rats (P < 0.05). FAS activity was increased 19- and 39-fold, respectively, in the RC and RF rats compared with the FA rats. Exercise decreased FAS activity to approximately one-third of the resting control value in both RC and RF rats (P < 0.05) but not in FA rats. L-PK activity was elevated by 55% in RC and 100% in RF rats compared with FA rats (P < 0.05). FA and RF rats also showed a reduction of L-PK activity with exercise. No significant alteration of other lipogenic enzymes was observed after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of hepatic VLDL secretion in vivo in the growing turkey (Meleagris gallopavo) and chicken (Gallus domesticus).

Hepatic secretion of VLDL was compared in young turkeys and chickens (8 and 4 weeks of age, respectively) and older birds (11 and 8 weeks of age, respectively) reared together under the same nutritional conditions. VLDL, VLDL-TG and total TG secretion rates were higher in chickens than in turkeys. The cholesteryl ester content of turkey VLDL was higher than that of chicken. Differences in the fatty acid composition of the VLDL lipids were observed between the species: the proportion of linoleic acid was greater in turkeys, whereas monounsaturated fatty acids were more abundant in chickens. These results are consistent with the hypothesis of a positive relationship between hepatic lipogenesis, delta-9-desaturation, VLDL secretion and fattening in turkeys and chickens.

Liver stearyl-CoA desaturase activity and fatness in birds. In vitro studies in the growing turkey and chicken.

1. Liver stearyl-CoA desaturase activity and total body fat were measured in 4-, 8- and 12-week-old turkeys and chickens reared together and fed either of two isocaloric diets differing in their protein content. 2. Chickens showed higher desaturase activity and body fatness as compared to turkeys. 3. These results have been discussed with special emphasis on the role of liver stearyl-CoA desaturase activity upon lipid transport and deposition in birds.

Effects of exercise training on diet-induced lipogenic enzymes and body composition in rats.

It is well known that a carbohydrate-rich diet promotes lipogenesis via induction of hepatic lipogenic enzymes, whereas chronic physical exercise reduces body fat. The purpose of this study was to investigate whether exercise training can decrease diet-induced hepatic lipogenic enzymes and total body fat composition. Female Sprague-Dawley rats were randomly divided into exercise trained (T) and sedentary (S) groups. Training was performed on a treadmill for 8 weeks for up to 25 m/minute, 15% grade for 90 minutes/day, 5 days/week. The T and S groups were further divided into four dietary treatments. Fifty percent of the calories for each diet were from a basal ingredient and the other 50% were from either cornstarch (C), glucose (G), fructose (Fr), or fat (Fa). T significantly decreased body fat and increased body water content in all dietary groups (p < 0.01), but had no effect on body weight. Fr rats had significantly heavier liver weight than did the remaining groups. Activities of hepatic fatty acid synthase, ATP-citrate lyase (CL), malic enzyme (ME), pyruvate kinase, and glucose-6-phosphate dehydrogenase were significantly higher in Fr and lower in Fa compared with C and G. Training significantly increased activities of CL and ME in Fa, but had little effect on these enzymes in other groups. These data suggest that the fat-reducing effect of conventional exercise training is elicited by regulatory mechanisms other than by a reduction of hepatic lipogenic enzymes.

Hepatic lipogenesis in genetically lean and fat chickens. In vitro studies.

1. Acetyl-coenzyme A carboxylase, malic enzyme, glucose 6-phosphate dehydrogenase and delta 9-desaturase activity was measured in liver extracts from 5- to 11-week-old genetically lean or fat chickens. 2. A significant difference between the two lines of chickens was shown as concerns desaturating activity only, which was 45% higher in the fat animals than in the lean ones. 3. This result is consistent with the hypothesis of a higher rate of lipoprotein processing and secretion in the liver of the fat line chickens.

Lipid biosynthesis and deposition in genetically lean and fat chickens. Comparative in vivo studies with 14C acetate.

Following intravenous administration of 14C-acetate to genetically lean or fat chickens, the radioactivity in their liver, plasma, striated muscle and abdominal adipose tissue lipids and that in the breathed out CO2 were studied in function of time. As compared to the lean ones, the fat chickens showed a higher labelling of their triglyceride fatty acids in the plasma and adipose tissue, but not in the liver. Thus a higher rate of triglyceride secretion from the liver would be responsible for the higher abdominal fat weight in the fat line chickens.