V. Arce

Effect of dietary cholesterol on mevalonate metabolism by sterol and nonsterol pathways

Results in the present communication demonstrate for the first time that the shunt pathway of mevalonate not leading to sterols is regulated by cholesterol feeding in a reverse fashion to the sterol pathway. Mevalonate incorporation into nonsaponifiable lipids by liver slices was inhibited by cholesterol feeding while the shunt pathway was clearly enhanced. Moreover, inhibition of renal sterologenesis by dietary cholesterol is also reported. These changes in the mevalonate metabolism are closely correlated with the increase observed in the esterified cholesterol content in neonatal chick liver and kidneys after 10 days of 2% cholesterol supplementation of the diet.

Postnatal Development of the Sterol and Nonsterol Mevalonate Metabolism in Chick Liver and Kidneys

The effect of 2% cholesterol feeding on changes throughout postnatal development of total, free, and esterified cholesterol in neonatal chick liver and kidneys was studied. The increase observed in the hepatic cholesterol content after supplementation of the diet with 2% cholesterol was mainly due to the accumulation of esterified cholesterol. Small but significant differences were also found in the esterified cholesterol content in kidneys between control and cholesterol-fed animals. In normally fed chicks, the hepatic percentage of squalene synthesized from mevalonate decreased during the first days of independent life while cholesterol percentage increased. On the contrary, the percentage of squalene recovered in kidneys immediately after hatching was minimal, increasing during postnatal development. Addition of 2% cholesterol to the diet produced a clear inhibition in the mevalonate incorporation into nonsaponifiable lipids by liver slices, especially from 4 days onwards. This inhibition was particularly clear in the percentage of cholesterol synthesized. Mevalonate incorporation by kidney slices was higher than in liver, although cholesterol supplementation had little influence on the percentage of each nonsaponifiable lipid formed. In normally fed chicks, kidneys metabolized mevalonate by the shunt pathway not leading to sterols at a rate more than 50 times that of liver. Cholesterol feeding produced a clear enhancement of the hepatic shunt pathway while in kidneys it had practically no effect.

Studies of the in vivo metabolism of mevalonic acid in the neonatal chick

After 4 hr of the intraperitoneal injection of different doses of (R)-[5-14C]mevalonic acid (MVA), its incorporation into nonsaponifiable and saponifiable lipids was maximal in neonatal chick kidneys and liver, and minimal in brain, spinal cord and skin. Using 14CO2 production from [5-14C]MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that about 11% of MVA was in vivo metabolized by this pathway in nonmammalian species. Kidneys presented the maximal ability to incorporate MVA into nonsaponifiable and saponifiable lipids at any time considered (15-750 min). The percentage of radioactivity recovered as saponifiable lipids in liver and kidney decreased after 12 hr the injection of MVA. Although the absolute amounts of 14C incorporated in both derivatives were much less in brain, spinal cord and skin than in liver and kidneys, the relative percentages found in the saponifiable fraction were clearly higher in the former tissues, especially in the spinal cord.

Regulation of hepatic cholesterogenesis by polar steroids accumulated after cholesterol feeding

The incorporation of mevalonate into nonsaponifiable lipids by chick liverin vivo strongly increased between 1–18 days after hatching. Cholesterol feeding (2%) inhibited this. Synthesis of cholesterol was strongly inhibited, whereas the intermediates isolated by TLC accumulated. Most of the polar nonsaponifiable lipids that accumulated in liver 90 minutes after mevalonate administration to 18-day-old cholesterol-fed chicks were identified as lanosterol derivative. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, as well as acetate and mevalonate incorporation into nonsaponifiable lipids, was inhibited by the presence of these compounds. To our knowledge, this is the first report of such inhibition; this confirms the physiological function of polar steroids in the regulation of cholesterogenesisin vivo.

Incorporation of mevalonate into squalene, lanosterol and cholesterol by different neonatal chick tissues

The role of neonatal chick liver and kidneys in the incorporation of mevalonic acid into squalene, lanosterol and cholesterol was studied. Differences between the synthesizing ability of these and other tissues and the influence of the in vivo or in vitro conditions were also examined. In the in vivo experiments, distribution of radioactivity among the nonsaponifiable lipids was not dependent of the doses of mevalonic acid injected. About 80-95% of radioactivity was recovered as cholesterol in liver and brain, whereas in kidneys this percentage was only about 35%. Squalene and lanosterol were formed by kidneys in a high percentage, higher than in liver and other tissues. 12 hr after mevalonate injection, the percentage of cholesterol formed by kidneys increased until more than 50%. In the in vitro experiments carried out in the presence of 0.045-4.0 mM mevalonate, cholesterol was also the main nonsaponifiable identified, but in a lesser percentage than in vivo. In the same conditions, the incorporation of mevalonic acid by kidneys was maximal into squalene. After in vitro incubations for 2 hr, the percentage of cholesterol in kidneys also increased.

The In vitro metabolism of mevalonate by sterol and nonsterol pathways in neonatal chick

1. Incorporation of mevalonate into nonsaponifiable lipids by neonatal chick liver and kidney slices was studied as a function of weight of tissue and incubation time. In the same conditions, more nonsaponifiable lipids were produced in kidney than in liver. 2. CO2 production from mevalonate increased with the weight of tissue and with the incubation time. Over 80% of CO2 produced from mevalonate resulted from the shunt pathway in kidney, while in liver this route was quantitatively insignificant. 3. 2-14C from mevalonate was also incorporated into saponifiable (acidic) fraction by kidney slices. 4. Incorporation into nonsaponifiable lipids and total CO2 increased with the concentration of mevalonate. This increase was more pronounced in kidney than in liver especially at low mevalonate concentrations. In the presence of 0.1-8.0 mM mevalonate over 80% of the CO2 produced from this substrate resulted from the shunt pathway, while liver was practically ineffective whatever be the concentration of mevalonate. 5. The percentage of mevalonate metabolized in the kidney by shunt pathway increased with the incubation time and reached over 15% in the presence of 0.1-8.0 mM mevalonate.

A comparative study of cholesterogenic activities from acetate of neonatal chick liver, kidney and intestinal mucosa.

1. Acetate incorporation into nonsaponifiable lipids by liver and kidney slices and intestinal mucosa scrapes from neonatal chick was linear up to 100-150 mg of tissue. CO2 production from [1-14C]acetate was also studied as a function of weight of tissues. 2. Rate of acetate incorporation by the three mentioned tissues was essentially linear with respect to the incubation time assayed (0-120 min), CO2 production was also linear up to 60 min in kidney and 120 min in liver and intestinal mucosa. 3. In the conditions assayed the formation of nonsaponifiable lipids by liver slices reached constant values at acetate concentration of approximately 6 mM, while acetate incorporation by kidney and intestinal mucosa in the same conditions increased in nearly linear relationship to the concentration of available substrate. 4. Using the optimal assay conditions, acetate incorporation into nonsaponifiable lipids was maximal in neonatal chick liver, whereas kidney and intestinal mucosa only showed about 10% of the hepatic cholesterogenic activity. The percentage of acetate utilization for cholesterogenesis was also maximal in neonatal liver (about 10%). In the intestinal mucosa this percentage was also important (2-3%) being minimal in kidney slices (0.3%).

Age-related changes in the mevalonate metabolism in vivo in chick kidneys

The mevalonate incorporation in vivo into total nonsaponifiable lipids by chick kidneys drastically increased after hatching, reaching similar levels to those previously observed in liver. Cholesterol was the major sterol formed from mevalonate from 11 days onward, while a fraction of polar nonsaponifiable lipid(s) was observed as the major compound(s) synthesized at 5-8 days. Relative percentages of squalene, squalene oxide(s) and lanosterol synthesized from mevalonate also increased between 11-18 days after hatching. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipid(s) identified as lanosterol derivatives and cholesterol precursors formed by kidneys from [5-14C]mevalonate in experiments carried out in vivo, as well as their evolution during postnatal period.

Studies on the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of mevalonate-activating enzymes.

Both in vivo and in vitro incorporation of mevalonic acid into nonsaponifiable lipids by 17-day-old chick liver and kidney did not show diurnal rhythm. Using 14CO2 production from MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that there is no diurnal rhythm in this pathway. No significant differences were found in the specific activities of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase from chick liver and kidney throughout a period of 24 hr, using [1-14C]mevalonate as substrate. The absence of diurnal rhythm in the decarboxylase activity was corroborated by further experiments carried out using [2-14C]mevalonate-5-pyrophosphate as specific substrate of this enzyme.

Diurnal rhythm of the in vivo acetate metabolism to CO2 and nonsaponifiable lipids by neonatal chick

The in vivo incorporation of acetate into nonsaponifiable lipids was studied in different tissues from 14-day-old chick. Total nonsaponifiable lipids (nmol/30 min/g tissue) were mainly synthesized in testicles and liver. The in vivo CO2 production from acetate by 1-day-old chick did not exhibit diurnal variations. However, in 14-day-old chick, a maximal value was observed in the middle of the light period, while a minimal value was found 9 h after the start of the dark period. No significant diurnal differences were detected in the in vivo acetate incorporation into nonsaponifiable lipids by liver and duodenal mucosa from 1-day-old chick. Nevertheless, a clear diurnal rhythm was found in liver and duodenal mucosa from 14-day-old chick, but not in brain and kidney from animals of the same age. Distribution of radioactivity from (1-14C)acetate among the different constituents of the nonsaponifiable fraction has been also studied at 3-h intervals. Cholesterol was the major sterol formed from acetate by chick liver at any time of day. In duodenal mucosa and kidney, maximal values in the percentage of cholesterol synthesized were observed during the light period.