Secretion into the circulating medium of lipids synthetized by isolated Wistar rat liver perfused with high concentrations of oleic acid and glycerol

In previously 18-h fasting Wistar rats, the liver is isolated and perfused with [9, 10(-3)H2] oleic acid (346 mumol and [1-14C] glycerol (115 mumol). Then, in a circulating medium, the secretion of triacylglycerols -- synthetized de novo and by esterification of exogenous oleic acid -- and VLDL is inhibited. On the other hand, the secretion of phospholipids is getting away that regulatory process.     

Fatty acid metabolism and lipid secretion by perfused livers from rats fed laboratory stock and sucrose-rich diets

To assess the possible role of altered hepatic processing of free fatty acids in dietary sucrose-induced accumulation of triglyceride in the liver and blood plasma, livers from rats fed commercial laboratory stock and high sucrose diets were perfused both with and without oleic acid substrate. Consumption of the sucrose diet exerted a multiplicity of effects on oleic acid metabolism, characterized by decreased conversion to both ketone bodies and carbon dioxide, increased esterification into liver triglyceride, and increased secretion in triglyceride-rich lipoproteins. During the infusion of oleic acid, livers from sucrose-fed rats also exhibited decreased ketogenesis, and increased secretion of triglyceride from endogenous sources. Since oleic acid uptake from the perfusion medium was identical in both groups, the observed effects of sucrose feeding are ascribed to altered rates of intracellular metabolic processes. Mass and radiochemical analyses of perfusate ketone bodies and triglycerides were indicative of greater mobilization of triglycerides from hepatocellular lipid droplets in the livers from sucrose-fed rats. These livers contained more triglyceride and secreted more triglyceride even in the absence of infused oleic acid. In summary, the sucrose-rich diet increased the esterification:oxidation ratio of intracellular free fatty acids derived from both the circulation and endogenous sources within the hepatocyte. In response, secretion of triglyceride-rich lipoproteins by the liver and deposition of triglyceride within the liver were promoted. It is concluded that alterations in the processing of free fatty acids by the liver contribute significantly to the liver and plasma triglyceride accumulation following sucrose consumption.     

Oxidation and esterification of cis- and trans-isomers of octadecenoic and octadecadienoic acids in isolated rat liver

The metabolism of 9-octadecenoic and 9,12-octadecadienoic acids with different geometrical configurations was compared in isolated perfused rat liver. More ketone bodies were produced when the trans-isomers were infused. In contrast, only the cis-isomer augmented the triacylglycerol secretion almost entirely as very-low-density lipoprotein (VLDL). Although these responses were independent of the difference in the degree of unsaturation in both the cis- and trans-isomers, the trans-monoenic acid compared to the trans-dienic acid was incorporated more readily into perfusate and hepatic lipids. Quantitative information was obtained with radioactive tracer experiments. The hepatic uptakes of 9-[10-14C]octadecenoic acids were comparable in the cis- and trans-isomers. The trans-octadecenoic acid compared to the cis counterpart was oxidized more readily and incorporated more into liver phospholipid but less into perfusate and liver triacylglycerol. These reciprocal responses counterbalanced each other. The lower rates of triacylglycerol synthesis and secretion in the liver perfused with the trans-octadecenoic acid was confirmed using [2- 3H]glycerol as a tracer. The marked difference in the channelling of cis- and trans-fatty acids in the pathways of oxidation and esterification seems to modify the VLDL secretion in perfused rat liver. Present observations indicate a considerable difference in the fate of unsaturated fatty acids with different configurations. trans-Fatty acids are expected to be an efficient energy source in animal tissues and may not be hyperlipidemic.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

Early effects of dietary orotic acid upon liver lipid synthesis and bile cholesterol secretion in rats

Dietary orotic acid is known to cause impaired fatty acid synthesis and increased cholesterol synthesis in rats. We found that the impaired fatty acid synthesis occurs during the first day of orotic acid feeding and, in studies with albumin-bound [1-14C]palmitic acid, an associated decrease in the rate of esterification of this fatty acid into triacylglycerol, phospholipid, and cholesteryl ester was observed. These changes may result from the known decreases in liver levels of adenine nucleotides or, as reported here, from decreased liver CoASH levels in orotic acid-fed rats. The increase in hepatic cholesterol synthesis occurred during the second day of orotic acid feeding. It was detected by increased incorporation of [1,2-14C]acetate into cholesterol by liver slices and by a 7-fold increase in HMG-CoA reductase activity. At the same time the biliary output of cholesterol was increased 2-fold and studies using 3H2O revealed that the output of newly synthesized cholesterol in bile was increased 5-fold. The content of cholesteryl ester in hepatic microsomes decreased during orotic acid feeding but free cholesterol was unchanged. The findings are interpreted to suggest that the increased bile cholesterol secretion caused by orotic acid is a result of impaired hepatic cholesterol esterification and that the increase in HMG-CoA reductase activity is a result of diminished negative feedback due to the depleted content of cholesteryl ester in the hepatic microsomes.     

Enzyme studies on the esterification of vitamin D in rat tissues

1. The mechanism of vitamin D esterification in the rat was studied with liver, small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and [1-(3)H]cholecalciferol, [U-(14)C]ergocalciferol and [4-(14)C]cholesterol as substrates. 2. No esterification of vitamin D could be detected with liver preparations nor with homogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreatic juice esterified [1-(3)H]cholecalciferol with oleic acid, and specificity studies indicated that a cholesterol-esterifying enzyme was using vitamin D as substrate. 4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The specificity of the esterification reaction is discussed in relation to (a) the molecular structure of the substrates and (b) their availability, in a micellar solution, to the enzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin D in vivo during absorption from the small intestine and while it is transported in blood.     

Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts

The objective of this study was to determine the contribution of myocardial triglycerides to overall ATP production in isolated working rat hearts. Endogenous lipid pools were initially prelabeled (pulsed) by perfusing hearts for 60 min with Krebs-Henseleit buffer containing 1.2 mM [1-14C]palmitate. During a subsequent 60-min period (chase), hearts were perfused with either no fat, low fat (0.4 mM [9,10-3H] palmitate), or high fat (1.2 mM [9,10-3H]palmitate). All buffers contained 11 mM glucose. During the "chase," 14CO2 production (a measure of endogenous fatty acid oxidation) and 3H2O production (a measure of exogenous fatty acid oxidation) were determined. Oxidative rates of endogenous fatty acids during the chase were 279 +/- 50, 88 +/- 14, and 88 +/- 8 nmol of [14C]palmitate oxidized per g dry weight.min in the no fat, low fat, and high fat groups, respectively, compared to exogenous palmitate oxidation rates of 0, 361 +/- 68, and 633 +/- 60 nmol of [3H]palmitate/g dry weight.min, in the no fat, low fat, and high fat groups, respectively. Endogenous [14C]palmitate oxidation rates were matched by loss of [14C]palmitate from endogenous myocardial triglycerides. Overall triglyceride content decreased during the no fat and low fat chase perfusion but did not change during the high fat chase. Loss of triglyceride [14C]palmitate during the high fat chase was matched by incorporation of exogenous [3H]palmitate in triglycerides. In a second series of perfusions, three groups of hearts were perfused under similar conditions, except that unlabeled palmitate was used during the "pulse" and that 11 mM [2-3H/U-14C]glucose and unlabeled palmitate was present during the chase. During the chase, both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured. Rates of glucose oxidation were inversely related to the fatty acid concentration in the perfusate (1257 +/- 158, 366 +/- 40, and 124 +/- 26 nmol of glucose oxidized per min.g dry weight in the no fat, low fat, and high fat groups, respectively), while rates of glycolysis were not significantly different between these groups. Calculation of overall ATP production from both oxidative and glycolytic sources determined that even in the presence of high concentrations of fatty acids, myocardial triglyceride turnover can provide over 11% of steady state ATP production in the aerobically perfused heart. In the absence of fatty acids, myocardial triglyceride fatty acids can become the major energy substrate of the heart.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of triglyceride and ketogenesis by livers from spontaneous diabetic BB Wistar rats

Livers from male and female BB Wistar spontaneously diabetic rats were perfused in vitro to determine the effects of spontaneously occurring insulin-dependent diabetes on the metabolism of fatty acid. The secretion of triglyceride and the incorporation of [1-14C] oleic acid into perfusate and hepatic triglyceride was reduced by the diabetic state, whereas beta-hydroxybutyrate production and output of total ketone bodies were increased. The spontaneous diabetic Wistar rat clearly is a suitable model to study the derangements induced in lipid/plasma lipoprotein metabolism by the insulin-dependent diabetic state; the data obtained with this model confirm our earlier observations on experimental insulin deficiency induced with alloxan, streptozotocin, and anti-insulin serum.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

The immediate effects of insulin and fructose on the metabolism of the perfused liver. Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism

1. When livers from fed rats were perfused with blood containing elevated concentrations of rat insulin or blood to which fructose was added, the oxidation of free fatty acids was depressed and their esterification was increased. 2. Raised concentrations of insulin or addition of fructose increased secretion of triglyceride in very-low-density lipoproteins, but only insulin caused more of the free fatty acids taken up by the liver to be incorporated into very-low-density lipoproteins. 3. When insulin and fructose were added together the combined effect on oxidation and esterification of free fatty acids and on secretion of very-low-density lipoproteins was equal to the sum of the effects of either alone. No statistically significant interaction between the effects of fructose and insulin was found for any of the parameters investigated. 4. Bovine insulin had similar effects, in most respects, to comparable studies with raised concentrations of rat insulin. 5. Lipogenesis was increased in the livers treated with fructose plus bovine insulin. 6. A significant proportion of the fatty acids in very-low-density lipoproteins were derived either from the liver triglyceride pool or from lipogenesis. This fraction was increased both by treatment with insulin or fructose, and was augmented further when both insulin and fructose were present together. 7. The uptake of fructose by the perfused liver was similar to that found in vivo. It was unaffected by the presence of insulin. 8. Addition of fructose to the perfused liver caused perfusate lactate concentrations to increase, as a result of diminished hepatic uptake of lactate. 9. The uptake of free fatty acids by the perfused liver was unaffected by the addition of either insulin or fructose. 10. The distribution among the various lipid classes in plasma lipoproteins of label arising from the hepatic uptake of [(14)C]oleate was unaltered by the addition of either fructose or insulin. 11. It is suggested that the effects described are due principally to control of the balance between esterification of fatty acids and lipolysis of the ensuing triglyceride, fructose enhancing esterification and insulin inhibiting lipolysis.     

Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglyceride by livers of carbohydrate-fed rats

High carbohydrate diets enhance the hepatic output of very low density lipoprotein triglycerides. The fatty acids of these triglycerides could come from exogenous sources (i.e., diet or adipose tissue) or from de novo fatty acid synthesis in the liver. The role of exogenous free fatty acids was evaluated in rats fed Purina Chow or diets containing 10% fructose for up to 14 wk. In carbohydrate-fed rats, serum triglycerides were twice normal, and VLDL accounted for about 60% of the increases. Pre-beta-lipoprotein was increased and alpha- and beta-lipoprotein were decreased. Phospholipid and cholesterol levels were unchanged. Livers were perfused with glucose and free fatty acids. Perfusate free fatty acids rose from 180 to 1800 micro eq/liter as the infused acids increased from 0 to 992 micro eq/3 hr; simultaneously, net free fatty acid uptake rose from < 1 to 18 micro eq/g/hr and triglyceride output by the liver doubled. However, rates of secretion of triglyceride became constant, and triglyceride accumulated in liver at uptakes of free fatty acids > 13 micro eq/g/hr. More lauric and myristic acid appeared in the perfusate than was infused, suggesting the hepatic discharge of free fatty acids. Livers of fructose-fed rats secreted twice as much oleate-(14)C-labeled triglyceride as controls at all levels of free fatty acid uptake. The ratios of the specific activities of perfusate triglyceride to free oleate-(14)C were unaffected by diet and were about 0.6 and 1.0 at low and high triglyceride secretion rates, respectively. Thus, carbohydrate feeding did not result in altered uptakes of free fatty acids or preferential secretion of triglycerides containing endogenously synthesized fatty acid. Instead, the increased secretion of triglyceride was accomplished by enhanced formation of VLDL triglyceride from exogenous free fatty acids.     

Multiple,independently regulated pathways of cholesterol transport across the intestinal epithelial cells

The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.     

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system.

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.     

Effect of fish oil diet on hepatic lipid metabolism in nonhuman primates: lowering of secretion of hepatic triglyceride but not apoB

African green monkeys were fed diets containing either 11% (by weight) fish oil or lard for 2.5 yr. To test the hypothesis that fish oil decreases hepatic secretion of triglyceride (TG) and apoB, livers from these animals were perfused with a fatty acid mixture [85% (w/w) oleate containing [14C]oleate and 15% n-3 containing [3H]eicosapentaenoic acid (EPA)] at a rate of 0.1 mumol fatty acid/min per g liver. Liver perfusate was sampled every 30 min during 4 h of recirculating perfusion. The concentration of triglyceride was similar for livers of animals of both groups and there was no difference between groups in the extent of incorporation of [3H]EPA or [14C]oleate into hepatic TG. While the secretion rate for the mass of TG was less in the fish oil-fed group (8.3 +/- 2.5 vs 18.3 +/- 4.4 mg/h per 100 g liver, P less than 0.05), the apoB secretion rate was similar (0.92 +/- 0.15 vs 1.01 +/- 0.13 mg/h per 100 g liver). Significantly less [3H]EPA was incorporated into secreted TG in the fish oil group (0.4 +/- 0.1 vs 1.0 +/- 0.1% infused dose/h; P less than 0.01). The rate of secretion of [14C]TG was similar for both groups (1.3 +/- 0.3 vs 1.4 +/- 0.1% infused dose/h for fish oil and lard groups, respectively). No significant diet-related differences in [3H]TG or [14C]TG fatty acid specific activity were observed for perfusate TG or hepatic TG. After perfusion, livers from fish oil-fed monkeys contained significantly more [3H]EPA in hepatic phospholipid than livers from lard-fed monkeys (19.5 +/- 1.8 vs 11.4 +/- 1.7% infused dose; P less than 0.01) although hepatic phospholipid mass concentrations were similar. The liver phospholipids of the fish oil group were enriched in n-3 fatty acid mass and were relatively depleted of oleate and linoleate. We conclude that although apoB secretion was unaffected, dietary fish oil significantly decreased hepatic TG secretion through relatively poor utilization of EPA for the synthesis of TG destined for secretion in VLDL; at the same time, increased incorporation of [3H]EPA into hepatic phospholipid accompanied the decreased incorporation into secreted TG and these events may be coupled.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the ethanol-induced decrease of fatty acid oxidation in rat and human liver slices

In order to elucidate the mechanisms involved in the acute ethanol-induced liver triglyceride accumulation, the oxidation, esterification and β-keto acid formation have been studied in rat and human liver slices after incubation with albumin bound, long chain fatty acids (palmitic. oleic and linoleic acids).The addition of alcohol to rat and human liver slices depressed the formation of ¹⁴CO₂ from palmitic acid-1-¹⁴C, oleic acid-1-¹⁴C and linoleic acid-1-¹⁴C. The esterification to triglycerides and phospholipids was increased and the formation of β-keto acids was decreased by alcohol.Addition of 4-methylpyrazole, an inhibitor of liver alcohol dehydrogenase, almost prevented the alcohol effect on the lipid metabolism of the liver slices. The oxidation of alcohol is thus obligatory for the decreased β-oxidation of fatty acids, the increased esterification and for the decreased formation of β-keto acids. The results suggest that ethanol oxidation inhibits β-oxidation of fatty acids and that this primary effect leads to accumulation of liver triglycerides by increased esterification of plasma free fatty acids.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Normal cholesterol absorption in rats deficient in intestinal acyl coenzyme A:cholesterol acyltransferase activity

Acyl coenzyme A:cholesterol acyl transferase and/or cholesterol esterase may regulate the esterification and absorption of exogenous cholesterol. To assess this, mucosal acyl coenzyme A:cholesterol acyl transferase activity was inhibited selectively with three different drugs [Sandoz #58-035, inhibitor 1; Lederle inhibitor 2 and inhibitor 3] and the effect upon the absorption of a [4-14C]cholesterol meal was studied in the lymph fistula rat. Compared to control rats, ACAT activity measured in mucosal homogenates from the drug-treated rats was reduced 80-90%, 40%, and 30%, respectively, during the predicted time-frame for maximum mucosal esterification of cholesterol (i.e., after cholesterol is fed and before it appears in lymph). In contrast, [14C]cholesterol absorption in the drug-treated animals was unchanged from controls [5.7 +/- 1.2 (inhibitor 1) vs. 5.4 +/- 1.6 mumol/6 hr (control); 6.1 +/- 2.1 (inhibitor 2) and 5.2 +/- 1.5 (inhibitor 3) vs. 4.1 +/- 1.3 mumol/6 hr (control)]. Of the absorbed [14C]cholesterol, approximately 75% was esterified in all groups. Cholesterol esterase activity measured in the drug-treated rats was unchanged compared to controls nor did the drugs inhibit this enzyme in vitro. Under the conditions of this study, drugs causing substantial inhibition of acyl coenzyme A:cholesterol acyl transferase activity had no effect on the absorption of exogenous cholesterol.     

The synthesis of bile acids in perfused rat liver subjected to chronic biliary drainage

1. Isolated rat liver was perfused with heparinized whole blood under physiological pressure resulting in the secretion of bile at about the rate observed in vivo. 2. The preparation remained metabolically active for 4h and was apparently normal in function and microscopic appearance. 3. When the perfusate plasma and liver cholesterol pool was labelled by the introduction of [2-(14)C]mevalonic acid the specific radioactivity of the perfusate cholesterol increased. The biliary acids (cholic acid and chenodeoxycholic acid) were labelled and had the same specific radioactivity. 4. Livers removed from rats immediately after, and 40h after, the start of total biliary drainage, were perfused; increased excretion rates of both cholic acid and chenodeoxycholic acid were found when the liver donors had been subjected to biliary drainage. 5. The incorporation of [2-(14)C]mevalonic acid or rat lipoprotein labelled with [(14)C]cholesterol into bile acids was studied. 6. A dissociation between the mass of bile acid excreted and the rate of incorporation of (14)C was found. This was attributed to the changing specific radioactivity of the cholesterol pool acting as the immediate bile acid precursor.