Participation of propionate in cholesterol biosynthesis by rat liver

Incorporation of (2¹⁴C) propionate into cholesterol was demonstrated using rat liver slices and homogenates. The incorporation of (2¹⁴C) propionate was greater than that of (2¹⁴C) acetate. Using the same liver homogenate preparation (2 ¹⁴C) succinate and (2¹⁴C) pyruvate were incorporated into cholesterol to a lesser extent than (2¹⁴C) acetate and (2 ¹⁴C) propionate. Addition of unlabeled acetate failed to dilute the incorporation of (2 ¹⁴C) propionate. Incorporation of the 2 and 3 carbon atoms pf propionate were equal; little incorporation of the 1 carbon atom was demonstrable. These results indicate that propionate is an excellent source of 2 carbon units for isoprenoid biosynthesis; the intermediary pathway does not involve a common acetate pool nor can these results be satisfactorily explained by citric acid intermediary metabolism.     

Glycerolipid biosynthesis in isolated rat intestinal epithelial cells.

Intestinal epithelial cells were prepared from fasted rats by dispersion with collagenase (EC 3.4.24.3). The structural and metabolic integrity of the cells was verified by electron microscopy, a high percentage of Trypan Blue exclusion, a low degree of release of lactate dehydrogenase (EC 1.1.1.27) in the medium, and by the retention of sensitivity to agents known to modify metabolic and transport activity in everted sacs of intestinal mucosa. The isolated intestinal epithelial cells were used to study glycerolipid biosynthesis from glucose, glycerol, 2-monoacylglycerol, and free fatty acids. The cells actively incorporated the labeled precursors into glycerolipids without specific cofactor requirements. Addition of fatty acids stimulated the incorporation of both glucose and glycerol into triacylglycerols and glycerophospholipids, the greatest effect being observed with palmitate. The stimulation of monoacylglycerol acylation appeared to depend on both the nature of the monoacylglycerol and fatty acid supplied. Stereospecific analyses of the diacylglycerols formed from 2-monoacylglycerols and free fatty acids showed that 1,2-diacyl-sn-glycerols (62-70%) were the major and that 2,3-diacyl-sn-glycerols (30-38%) the minor intermediates in triacylglycerol biosynthesis. The data indicate that isolated intestinal epithelial cells exhibit a total capacity of glycerolipid synthesis and a stereochemical course of reaction which is comparable to that observed for triacylglycerol formation in everted sacs of intestinal mucosa, but much less specific than that seen in microsomal preparations of intestinal mucosa.     

Correlation of rat liver chromatin-bound free and esterified cholesterol with the circadian rhythm of cholesterol biosynthesis in the rat.

Cholesterol has been shown to be present in rat liver chromatin isolated by methods designed to avoid contamination by membrane fragments. Evidence that the cholesterol was actually a component of chromatin includes (a) the constancy of the amount (1.30 +/- 0.14 mug per mg DNA), (b) the striking difference in the ratio of free (i.e. unesterified) to esterified cholesterol between that in chromatin and that in membrane, and (c) the rapid and marked changes which occurred in this ratio during the circadian cycle in chromatin but not in membranes. Although the total amount of chromatin-bound cholesterol did not change throughout the circadian cycle, the concentration of free cholesterol increased sharply a short time before the peak of cholesterol synthetic activity was reached at about midnight; it reached a basal level about 6 h later at approximately the same time the rate of synthesis returned to its basal level. When labelled cholesterol was administered by stomach tube, it was detectable within 2 h in whole nuclei and in chromatin, indicating that chromatin-bound cholesterol is rapidly exchangeable with that in liver cytoplasm and in blood plasma. Removal of basic proteins from chromatin did not result in the loss of any cholesterol, but removal of most of the acidic as well as the basic proteins resulted in loss of most of the chromatin-bound cholesterol. These results indicate that cholesterol is bound either to the acidic proteins or to both the acidic proteins and DNA. The data are compatible with the hypothesis that cholesterol biosynthesis controlled at the nuclear level and suggest that the relative amounts of free and esterified cholesterol associated with chromatin may play a role.     

Futile cycles in isolated perfused rat liver and in isolated rat liver parenchymal cells.

Isolated livers from fed rats were perfused with a medium containing glucose labeled uniformly with ¹⁴C and specifically with ³H. There was considerable formation of glucose from endogenous sources but simultaneously uptake of about half of the ¹⁴C in glucose. After 2 hours the ${}^3\text{H}{}^{14}\text{C}$ ratios in perfusate glucose decreased by 55–60% with (2-³H, U-¹⁴C), 40–50% with (5-³H, U-¹⁴C), 25–30% with (3-³H or 4-³H, U-¹⁴C) and by 10–15% with (6-³H, U-¹⁴C) glucose. Qualitatively comparable patterns were obtained with rat hepatocytes. These results demonstrate recycling of carbon between glucose and pyruvate. Superimposed upon this there is an extensive futile cycle between glucose and glucose 6-P. There is also futile cycling between fructose 6-P and fructose 1,6 P₂ and to a small extent between phosphoenol pyruvate and pyruvate.     

Measurement of the absolute cell count in the heart and liver. The quantitative preservation of proteins and DNA in isolated cells

Some improvement has been made of the Belov et al. (1975) method of alkaline dissociation of tissues. No cell loss occurs during cell suspension preparation or other procedures. The improved method was shown to be suited for determination of absolute numbers of cardiomyocytes and hepatocytes. Comparison of the living and dissociated hepatocytes revealed preservation of the total cell dry weight and DNA content (classes of ploidy) in the cells treated with formol and concentrated alkali. The method is recommended for studying the cell number, weight, ploidy and mitoses, and for DNA autoradiography.     

Cholesterol ester turnover in isolated liver cells. Effects of cholesterol feeding

Ionic currents through sodium channels in nodal membranes were measured under voltage clamp conditions both at normal and at low (4.8-4.9) external solution pH. The measurements of so-called 'instantaneous' currents were used to distinguish between the proton blockage in open channels and the influence of low pH on channel gating processes. It is shown that the amount of the proton blockage in open channels decreases as membrane potential becomes more positive. This result suggests that at least one of the acid groups accessible from the outside is located within the conducting pore. The influence of the other group(s) on the degree of potential-dependence of proton blockage is discussed.     

The effect of phosphorylation of the EF-2 isolated from rat liver cells on protein biosynthesis in vitro.

The activity of EF-2 was distinctly decreased after phosphorylation catalysed by a partly purified calmodulin and Ca2+ dependent protein kinase III. At the same time 32P from [gamma-32P]ATP was incorporated into EF-2 molecule. After dephosphorylation of EF-2 catalysed by alkaline phosphatase the activity of this factor was increased. This suggests that the phosphorylation-dephosphorylation of EF-2 is the regulatory process in the elongation step of the translation. Preliminary purification of the kinase III from rat liver resulted in 8-fold purified enzyme with a recovery of 60%.     

Induction of cadmium-thionein in isolated rat liver cells.

The uptake of cadmium by isolated liver cells was linearly related to the cadmium concentration to which the cells were exposed in the medium. Cadmium-treated cells synthesized proteins de novo with the characteristics of cadmium-thionein induced in the liver of cadmium-treated animals. Thionein from liver cells incorporated cadmium and [35S]cysteine, had a Ve/Vo (Sephadex G-50) of 1.8-1.9, and was separated into two subfractions by DEAE-cellulose ion-exchange chromatography. Cycloheximide and actinomycin D when added after a cadmium exposure prevented the synthesis of thionein. However, addition of actinomycin D after synthesis had started only decreased the total amount of thionein synthesized. The concentration of cadmium to which the cells were exposed affected the amount of cadmium-thionein synthesized in 6h. The maximum response occurred when cells were exposed to 0.5 microgram of cadmium/ml; at higher metal concentrations the total amount of cadmium-thionein synthesized declined. The system described in the present paper can be used to study the mode of metal toxicity and the mechanism of cadmium-thionein synthesis.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

Cholesterol 7 alpha-hydroxylase in isolated rat liver cells.

1. The activity of cholesterol 7 alpha-hydroxylase found in the 10000 x g supernatant prepared from isolated rat liver cells was comparable to that found with microsomal fractions from whole liver. 2. The activity of cholesterol 7 alpha-hydroxylase from cells prepared from livers of rats fed the bile salt sequestering agent cholestyramine was 2--3 fold higher than the activity of this enzyme found in cells isolated from animals on a control diet. 3. On incubation of hepatocytes in a suitable medium at 37 degrees C, cholesterol 7 alpha-hydroxylase activity declined to about 50% of its original value after three hours despite the fact that the cells retained a high level of viability over 5--6 h as measured by various sensitive criteria. 4. The decrease in cholesterol 7 alpha-hydroxylase activity was observed whether cholestyramine was included in the diet or excluded from the diet of the animals used as sources of the liver cells. 5. The change in cholesterol 7 alpha-hydroxylase activity seen on incubation of the cells was not affected by including in the incubation medium additional nutrients such as amino acids, the glucocorticoid cortisol, phospholipid dispersions, or sodium taurocholate. 6. Changing the incubation medium in which the cells were suspended at regular intervals during the three-hour experiments failed to prevent this decline in the cholesterol 7 alpha-hydroxylase activity during the incubation of these cells. 7. Although isolated liver cells have been shown to lose glutathione on incubation, addition of physiological levels of this compound did not prevent the decline in cholesterol 7 alpha-hydroxylase activity. 8. Cycloheximide addition to the incubation medium accelerated the decrease in cholesterol 7 alpha-hydroxylase activity. This suggests that some protein synthesis associated with cholesterol 7 alpha-hydroxylase activity occurs during the incubation and inhibition of such protein synthesis accelerated the decrease in this enzyme activity. 9. The cytochrome P-450 content of the 10000 x g supernatant prepared from hepatocytes declined slowly to about 65% of its original value after four hours of incubation at 37 degrees C. This decline in the 10000 x g supernatant cytochrome P-450 content may partly explain the observed loss of cholesterol 7 alpha-hydroxylase activity during incubations in vitro. 10. Isolated hepatocytes rapidly take up radioactively labelled sodium cholate. Subsequent excretion of the radioactivity was also very rapid even in the presence of large amounts of this bile salt in the medium.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes.

The dehydrogenation reaction of cholest-7-en-3beta-ol (I) to cholesta-5,7-dien-3beta-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3alpha-3H]cholest-7-en-3beta-ol. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3beta-ol. The conversion of cholest-7-en-3beta-ol to cholesta-5,7-dien-3beta-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b5 is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b5 were elicited in rabbits. The anticytochrome b5 immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome b5 was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b5 immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b5 in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7-en-3beta-ol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.