Avian fatty acid synthetase: Evidence for short-term regulation of activity

Liver biopsies were performed on starved chicks at 0 and 4 h after refeeding a fat-free diet. Fatty acid synthetase activity increased after refeeding, and administration of cycloheximide did not prevent the rise of enzyme activity. Incorporation of [carboxyl-¹⁴C]leucine into fatty acid synthetase was measured in enzyme purified from the livers of starved chicks, starved-refed (4 h) chicks, and starved-refed chicks injected with cycloheximide. The data suggest that the synthesis of enzyme protein was inhibited in starved and cycloheximide-treated refed chicks in comparison with refed chicks. Liver cytosol from fed or starved chicks was filtered through centrifuge ultrafiltration membranes and the residues were suspended in the same or opposite filtrates. Fatty acid synthetase activity in residues from starved chicks was stimulated when suspended in filtrates from fed chicks. The evidence is consistent with the hypothesis that a portion of the fatty acid synthetase in the liver of starved chicks is present as an inactive form which can be activated upon refeeding.     

Developmental and nutritional regulation of the messenger RNAs for fatty acid synthase,malic enzyme and albumin in the livers of embryonic and newly-hatched chicks

Summary The mRNAs for fatty acid synthase and malic enzyme were almost undetectable in total RNA extracted from the livers of 16-day old chick embryos. Both mRNAs increased in abundance between the 16th day of incubation and the day of hatching. In neonates, fatty acid synthase mRNA level was dependent on nutritional status, increasing slowly if the chicks were starved and rapidly if they were fed. The abundance of malic enzyme mRNA decreased in starved neonatal chicks and increased in fed ones. When neonates were first fed and then starved, starvation caused a large decrease in the abundance of both mRNAs. Conversely, feeding, after a period of starvation, resulted in a substantial increase in both mRNAs. The relative abundances of fatty acid synthase and malic enzyme mRNAs correlated positively with relative rates of enzyme synthesis. Thus, nutritional and hormonal regulation of the synthesis of these two lipogenic enzymes is exerted primarily at a pre-translational level.The abundance of albumin mRNA decreased significantly between the 16th day of incubation and the day of hatching but did not change thereafter in fed or starved chicks. The relative stability of albumin mRNA levels after hatching attests to the selectivity of the nutritional regulation of fatty acid synthase and malic enzyme mRNAs. The decrease in albumin mRNA which occurred between 16 days of incubation and hatching contrasts with the increase in albumin mRNA sequences which occurred during late gestation in the fetal rat (20). High levels of albumin in the chick embryo may be related to the lack of an analogue of mammalian alpha-fetoprotein in birds.Abbreviations PIPES piperazine-N,N-bis (2 ethanesulfonic acid) - SDS sodium dodecyl sulfate Postdoctoral Fellow of the Medical Research Council of Canada.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis.     

Levels of mRNAs coding for lipogenic enzymes in rat lung upon fasting and refeeding and during perinatal development

The relative amounts of mRNAs coding for fatty-acid synthase (EC 2.3.1.85), acetyl-CoA carboxylase (EC 6.4.1.2), ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) were determined in lungs and livers of adult rats that were normally fed, starved for 48 h or starved for 48 h and subsequently refed for 72 h with a carbohydrate-rich, fat-free diet. In the liver, starvation caused a small decrease in the relative abundance of the mRNAs which was not statistically significant. Subsequent refeeding caused a statistically significant increase in mRNAs for all of the enzymes studied. In the lung, no significant changes were found, indicating that the regulation of the abundance of mRNAs encoding the lipogenic enzymes in the lung differs from that in the liver. In the developing rat lung, mRNA for fatty-acid synthase increased 3-fold in abundance between fetal days 18 and 20 and decreased directly after birth (at day 22 of gestation). A similar pattern was observed for ATP citrate lyase mRNA. The level of acetyl-CoA carboxylase mRNA decreased significantly after birth. These observations indicate that in perinatal rat lungs, pretranslational regulation is involved in the control of the synthesis of these enzymes. The abundance of acetyl-CoA carboxylase mRNA did not change in the prenatal period, a time during which the specific activity of this enzyme increases. This lack of correlation between the specific activity of acetyl-CoA carboxylase and the abundance of its mRNA may indicate that translational regulation of the synthesis of the enzyme or post-synthetic regulatory effects on enzyme molecules are involved in the control of this enzyme in the prenatal period. No changes in the abundance of lung malic enzyme mRNAs were observed throughout the perinatal period.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

mRNA levels of SREBP-1c do not coincide with the changes in adipose lipogenic gene expression

Physiological differences in lipid metabolism exist according to adipose sites. To delineate at which step such gene regulation could occur, mRNA levels of various proteins involved in the overall lipogenic process were determined in subcutaneous (SC) and retroperitoneal (RP) adipose tissues. Fatty acid synthase, malic enzyme, ATP citrate lyase, insulin-sensitive glucose transporter, and glucose-6-phosphate dehydrogenase mRNA levels were coordinately reduced (by up to 50-fold) during fasting in RP and in SC relative to fed rats, and restored or overexpressed (by up to 5- to 6-fold) during refeeding. The response was most often delayed and lower in SC compared to RP. This could contribute to site-specific differences. Interestingly, SREBP-1c mRNA levels were markedly decreased by fasting in SC but remained unchanged in RP. Refeeding tended to restore levels close to fed group values. We conclude that mRNA levels of SREBP-1c do not coincide with the expected changes in adipose lipogenic gene expression of fasted/refed rats.