Inhibition of fatty acid synthesis in isolated adipocytes by 5-(tetradecyloxy)-2-furoic acid

The compound 5-(tetradecyloxy)-2-furoic acid (TOFA), a hypolipidemic agent, inhibits fatty acid synthesis, lactate and pyruvate accumulation and CO₂ release in isolated at adipocytes. TOFA stimulates the accumulation of citrate. ATP levels are not lowered by TOFA. In comparison with the natural fatty acid, oleate, TOFA exhibited a much greater inhibitory effect on lipogenesis. TOFyl-CoA formation within intact adipocytes was demonstrated. Although not inhibited by TOFA, acetyl-CoA carboxylase is inhibited by TOFyl-CoA. It is proposed that many of the metabolic effects of TOFA in isolated adipocytes can be explained by TOFyl-CoA inhibition of acetyl-CoA carboxylase. TOFA inhibits glycolysis as a secondary event with the primary event of inhibition of fatty acid synthesis causing an accumulation of citrate which is an inhibitor of phosphofructokinase.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Inhibition of hepatic lipogenesis by 2-tetradecylglycidic acid

2-Tetradecylglycidic acid (TDGA), a hypoglycemic agent, has been found to be a very effective inhibitor of de novo fatty acid synthesis by isolated hepatocytes. A comparison was made between the effectiveness of TDGA and 5-(tetradecyloxy)-2-furoic acid (TOFA), a hypolipidemic agent, on the metabolic processes of isolated hepatocytes. These compounds are structurally related and both inhibit fatty acid synthesis; however, they have opposite effects from each other on the oxidation and esterification of fatty acids. TDGA inhibits whereas TOFA stimulates fatty acid oxidation. TDGA stimulates whereas TOFA inhibits fatty acid esterification.     

Hyperthyroidism affects lipid metabolism in lactating and suckling rats

Two per thousand pregnant women have hyperthyroidism (HT), and although the symptoms are attenuated during pregnancy, they rebound after delivery, affecting infant development. To examine the effects of hyperthyroidism on lactation, we studied lipid metabolism in maternal mammary glands and livers of hyperthyroid rats and their pups. Thyroxine (10 μg/100 g body weight/d) or vehicle-treated rats were made pregnant 2 wk after commencement of treatment and sacrificed on days 7, 14, and 21 of lactation with the litters. Circulating triiodothyronine and tetraiodothyronine concentratins in the HT mothers were increased on all days. Hepatic esterified cholesterol (EC) and free cholesterol (FC) and triglyceride (TG) concentrations were diminished on days 14 and 21. Lipid synthesis, measured by incorporation of [³H]H₂O into EC, FC, and TG, fatty acid synthase, and acetyl CoA carboxylase activities increased at day 14, while incorporation into FC and EC decreased at days 7 and 21, respectively. Mammary FC and TG concentrations were diminished at day 14. Incorporation of [³H]H₂O into TG decreased at days 7 and 21, and incorporation of [³H]H₂O into FC increased at day 14. In the HT pups, growth rate was diminished, tetraiodothyronine concentration rose at days 7 and 14 of lactation, and triiodothyronine increased only at day 14, Liver TG concentrations increased at day 7 and fell at day 14, while FC increased at day 14 and only acetyl CoA carboxylase activity fell at day 14. Thus, hyperthyroidism changed maternal liver and mammary lipid metabolism, with decreased lipid concentration in spite of increased liver rate of synthesis and decreased in mammary synthesis. These changes, along with the mild hyperthyroidism of the litters, may have contributed to their reduced growth rate.     

Measurement of bile acid synthesis in man by release of14CO2 from [26-14C]cholesterol: Comparison to isotope dilution and assessment of optimum reference cholesterol specific activity

Bile acid synthesis can be measured as release of¹⁴CO₂ from [26-¹⁴C]cholesterol divided by cholesterol specific activity, but this method has not been validated in human subjects. We made twelve comparisons of this CO₂ method to standard isotope dilution in six normal subjects and found a mean discrepancy of 6%. Linear regression analysis of one value with respect to the other revealed a correlation coefficient of 0.83 (P<0.001), a Y-intercept close to zero (−4.98) and a slope close to 1 (1.06), suggesting good correspondence between the two methods. To assess the potential for error arising from use of serum cholesterol to estimate specific activity of cholesterol used for bile acid synthesis, we compared synthesis measured using serum free cholesterol specific activity to that measured using bile cholesterol specific activity, which is known to be near isotopic equilibrium with the precursor pool used for bile acid synthesis. Synthesis calculated in these two ways differed by less than 10%. The data indicate that the CO₂ method using either serum or bile cholesterol specific activity provides a valid estimate of bile acid synthesis in man.     

Influence of tramadol [2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanol hydrate] on corrosion inhibition of mild steel in acidic media

Tramadol[2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol hydrate], a drug, was tested as a corrosion inhibitor for mild steel in 0.5 M HCl and 0.25 M H₂SO₄ separately at 300, 310 and 320 K using mass loss and galvanostatic polarization techniques. The percentage protection efficiencies were evaluated at different concentrations of the inhibitor at different temperatures. The protection efficiency increased with increase in inhibitor concentration and decreased with increase in temperature in both the acid solutions. Galvanostatic polarization studies showed that the inhibitor is of mixed type with a slight predominance of cathodic character. The inhibitor was more active in HCl than in H₂SO₄. The maximum protection efficiency approached 82.6% in the presence of 2.16 × 10⁻³ M inhibitor. Some samples of mild steel were examined by scanning electron microscopy. The inhibitor was found to adsorb on the mild steel surface according to the Langmuir adsorption isotherm. The thermodynamic functions of dissolution and adsorption processes were evaluated.     

Lipid metabolism in plasma,liver, and adipose tissue of rats with experimental chronic nephrotic syndrome

Plasma, liver, and adipose tissue lipid composition and synthesis from [1-¹⁴C] acetate were studied three months following induction of nephrotic syndrome in rats by injection of antiglomerular basement membrane protein. Plasma triglyceride concentrations and specific radioactivities were elevated, and the triglycerides contained increased proportions of oleic acid. Plasma cholesterol and phospholipid concentrations were also increased, but free fatty acid levels were not. Liver triglyceride concentrations were decreased and incorporation of [1-¹⁴] acetate into liver triglycerides was also depressed below that of normal controls. Nephrotic rat liver triglycerides contained a higher proportion of oleic acid and lower arachidonic acid than did controls. Incorporation of [1-¹⁴C] acetate into adipose tissue lipids of the nephrotic rats was increased, and the proportion of palmitic acid was decreased. In the chronic nephrotic rat, the major source of the increased plasma triglycerides may be fatty acids mobilized from adipose tissue stores.     

Fatty acid and sterol synthesis by rat small intestine in vitro

Slices of rat jejunum were incubated with [2-¹⁴C]pyruvate, [1-¹⁴C]acetate, or [³H]H₂O to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P≤0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p≤0.05) in lipogenic activity. The highest levels of lipogenesis were observed when [³H]H₂O + glucose at a concentration of 20 mM was used. From such experiments, the absolute rate of fatty acid synthesis in the tissue preparation was calculated: 734±54 nmoles acetyl units incorporated into fatty acids/g tissue/hr.     

Stimulation of hepatic lipogenesis by eicosa-5,8,11,14-tetraynoic acid in mice fed a high linoleate diet

Liver slices, from mice fasted for one day and then refed for three days either a 15% corn oil diet or a 15% corn oil diet containing eicosa-5,8,11,14-tetraynoic acid (TYA), were incubated with [1-¹⁴C] acetate or [³H]H₂O to determine lipogenic capacity. Dietary TYA produced a twofold stimulation in fatty acid and cholesterol synthesis. TYA also caused an increase in the relative proportion of linoleate (C_18∶2) and a decrease in that of arachidonate (C_20∶4) in liver. Thus, (a) despite high levels of C_18∶2, hepatic lipogenesis can be increased, and (b) even short term feeding of TYA can alter the hepatic fatty acid composition presumably by inhibition of arachidonate synthesis from linoleate.     

Ketone bodies serve as important precursors of brain lipids in the developing rat

Ketone bodies are readily oxidized for energy by extrahepatic tissues. Since oxidation of ketone bodies produces acetyl coenzyme A (AcCoA), and hence could be an important source of immediate precursors for fatty acid synthesis, we investigated, in whole-brain homogenates of developing rats, the preferential utilization of [3-¹⁴C]acetoacetate (AcAc), [3-¹⁴C]β-hydroxybutyrate (β-OHB), and [U-¹⁴C]glucose for production of CO₂ and lipids, including phospholipids, glycerides, cholesterol, and free fatty acids. Throughout the postnatal period, the rate of AcAc oxidation was 2–3 and 2–6 times the rate for β-OHB and glucose, respectively. The eynthesis of lipids from AcAc was 7- to 11-fold higher than from glucose. The brain’s capacity for lipid synthesis from β-OHB was similar to that from AcAc during the first 8 days of life; however, during the next 10 days, the synthesis of lipids from β-OHB decreased to 60% of AcAc-dependent synthesis. The high rate of lipid synthesis from ketone bodies was accompanied by increased activities of cytoplasmic acetoacetyl CoA synthetase and acetoacetyl CoA thiolase in the developing brain. During the entire postnatal development, the proportion of radioactivity claimed by lipids vs. CO₂ from [3-¹⁴C]AcAc was 44–62% vs. 38–56%; from [3-¹⁴C]β-OHB, 50–81% vs. 19–50%; and from [U-¹⁴C]glucose, 14–43% vs. 57–86%. Phospholipids accounted for more than two-thirds of total lipids synthesized from either ketone bodies or glucose, while diglycerides plus cholesterol and free fatty acids accounted for most of the remainder. Addition of glucose to the incubation medium did not alter lipid production from AcAc throughout the suckling period, but moderately depressed energy production in the brain of 16- to 20-day-old rats. It is clear that in cell-free preparations from the brain of developing rats, ketone bodies are preferred over glucose as precursors for both energy and lipids, mainly phospholipids. These results suggest that ketone bodies are important for the growth and development of the brain.     

Metabolism of erucic acid in adipocytes isolated from rat epididymal fat

The metabolism of [14-¹⁴C]erucic acid and [U-¹⁴C]palmitic acid has been investigated in adipocytes isolated from rat epididymal fat. The rate of acylation of [¹⁴C]erucic acid in cellular lipids and oxidation to CO₂ and acid-soluble activity was ca. 1/3 of the rate with [¹⁴C]palmitic acid as substrate. A maximal incorporation of fatty acids in triacylglycerol was found at a fatty acid concentration of 0.8 mM in the medium, both with [¹⁴C]erucic acid and [¹⁴C]palmitic acid as substrate. Glucose added to the medium increased the esterification and decreased the oxidation of both fatty acids. No significant chain-shortening of [¹⁴C]erucic acid to shorter monoenes was identified in the fat cells. Increasing concentrations of unlabeled palmitic acid in the incubation medium markedly inhibited the esterification of [¹⁴C]erucic acid, whereas unlabeled erucic acid had little effect on the rate of esterification of [¹⁴C]palmitic acid.     

Modulation by ketone bodies of the rate of fatty acid synthesis in mammary gland slices from lactating rats

The absolute rate of fatty acid synthesis was measured in slices of mammary gland from lactating rats by incubation in [³H]₂O-labeled medium containing 10 mM D-3-hydroxybutyrate or aceto-acetate alone and paired in combination with 10 mM glucose, lactate, or pyruvate. When compared with our previous studies, the ketone bodies alone supported significant fatty acid synthesis; the rate of synthesis from either ketone body and lactate was higher than that from pyruvate and lactate, and that from pyruvate and glucose; the rate of synthesis from D-3-hydroxybutyrate and lactate was the highest we have observed in the absence of an exogenous substrate for the hexose monophosphate pathway. This study confirms our previous contention that, in rat mammary gland, substrates formed in the mitochondria can be utilized in the cytosol to provide some of the NADPH necessary for fatty acid synthesis.     

Arylsulfonate esters of fatty alcohols: IV. Effects on cholesterol catabolism

Hypercholesterolemic rats, fed 1% cholesterol and 0.5% glycocholate, were treated with arylsulfonates in various ways to observe the pattern of cholesterol elimination. Dietary linoleyl p-toluene-sulfonate (LTS) hastened return to normocholesterolemia and lowered hepatic cholesterol either with or without continued cholesterol feeding. LTS administered via the portal vein significantly lowered plasma cholesterol in 48 hr; ethyl linoleate and monoolein produced no lowering. LTS administered via the portal vein to glycocholate-infused rats increased the biliary excretions of label from [4-¹⁴C]cholesterol administered intracardially and also increased total bile acid excretion 21% without increased bile volume when compared to similar injection of ethyl linoleate. No change in biliary excretion of cholesterol was seen. Bile acid kinetics were studied by using isotopic dilution techniques. Cholate turnover was enhanced by feeding oleyl p-toluenesulfonate (OTS) and oleylp-(n-decyl)-benzenesulfonate (ODS) as suggested by a 16–35% decrease in half-life in both normal and hypercholesterolemic rats. Rats consuming a grain-based colony diet had a 54% increase in cholate synthesis when OTS was included in the diet. The composition of bile was changed when either OTS or ODS was fed; an increase in chenodeoxycholate was noted. This change was gradual with OTS but rapid with ODS and paralleled enhanced decay of chenodeoxycholate specific radioactivity in response to treatment. ODS and OTS also increased¹⁴CO₂ expiration from oral [26-¹⁴C] cholesterol in hypercholesterolemic rats. Dietary OTS and ODS elevated hepatic free cholesterol in hypercholesterolemic rats; ODS also elevated plasma free cholesterol and increased cholesteryl ester hydrolase activity in the liver. The data suggest that arylsulfonates stimulate cholesterol catabolism, in addition to the reported inhibition of cholesterol absorption [Lipids 12:819 (1977)]. Published as Journal Paper No. 6699, A.E.S., Purdue University.     

Cholesterol-lowering effects of guar gum: Changes in bile acid pools and intestinal reabsorption

Soluble fibers such as guar gum (GG) may exert cholesterol-lowering effects. It is generally accepted that bile acid (BA) reabsorption in portal blood is reduced, thus limiting the capacity of BA to down-regulate liver cholesterol 7α-hydroxylase, the rate-limiting enzyme of BA synthesis. In the present work, rats were adapted to fiber-free (FF) or 5% GG diets (supplemented or not with 0.25% cholesterol), to investigate various aspects of enterohepatic BA cycling. GG in the diet at a level of 5% elicited a significant lowering of plasma cholesterol during the absorptive period, in cholesterol-free (−13%) or 0.25% cholesterol (−20%) diet conditions. In rats adapted to the GG diets, the small intestinal and cecal BA pools and the ileal vein-artery difference for BA were markedly enhanced; reabsorption in the cecal vein was also enhanced in these rats. [¹⁴C]Taurocholate absorption, determined in perfused ileal segments, was not significantly different in rats adapted to the FF or GG diet, suggesting that a greater flux of BA in the ileum might support a greater ileal BA reabsorption in rats adapted to the GG diet. In contrast, capacities for [¹⁴C]cholate absorption from the cecum at pH 6.5 were higher in rats adapted to the GG diet than to the FF diet. Acidification of the bulk medium in isolated cecum (from pH 7.1 down to pH 6.5 or 5.8) or addition of 100 mM volatile fatty acids was also found to stimulate cecal [¹⁴C]cholate absorption. These factors could contribute to accelerated cecal BA absorption in rats fed the GG diet. The effects of GG on steroid fecal excretion thus appear to accompany a greater intestinal BA absorption and portal flux to the liver. These results suggest that some mechanisms invoked to explain cholesterol-lowering effect of fibers should be reconsidered.     

Effects of a single ingestion of sodium taurocholate on esterified cholesterol concentration in liver and cholesterol turnover in the rat

The overall response of the rat’s cholesterol metabolism to a single ingestion of taurocholate (80 mg) was studied with the isotopic equilibrium method. The bile acid production, measured by the daily¹⁴CO₂ output of rats in isotopic equilibrium of [26-¹⁴C]-cholesterol, initially decreased and then increased. Conversely, the hepatic concentration of esterified cholesterol first increased and then decreased. Moreover, the ingestion of taurocholate increasing the intestinal absorption coefficient of dietary cholesterol increased the abosprtion and decreased the fecal excretion and the intestinal biosynthesis of cholesterol. The balance of these last effects is an excess cholesterol inflow. The classical hypothesis of negative feedback regulation of bile acid production fails to explain the observed biphasic effect of taurocholate. This compound, when its origin is exogenous, appears to stimulate the storage of esterified cholesterol in the liver, at the expense of bile acid synthesis. This accumulation rate takes into account not only the decrease in cholesterol transformation into bile acids but also the excess inflow of cholesterol. As the exogenous taurocholate was eliminated from the body, cholesteryl ester hydrolysis occurred and provided a supplementary source of free cholesterol for bile acid synthesis.     

Stearic acid unlike shorter-chain saturated fatty acids is poorly utilized for triacylglycerol synthesis and β-oxidation in cultured rat hepatocytes

Utilization of stearate as compared to various saturated fatty acids for cholesterol and lipid synthesis and β-oxidation was determined in primary culture of rat hepatocytes. At 0.5 mmol/L in the medium, stearate (18:0) adequately solubilized by albumin was less inhibitory to cholesterol synthesis from [2-¹⁴C] acetate than myristate (14:0) and palmitate (16:0) (68% vs. 91 and 88% inhibition, respectively). The rate of incorporation into cholesterol from [1-¹⁴C] stearate (3.0±0.6 nmol/mg protein/4 h) was 37-, 1.8-, and 7.8-fold of that from myristate, palmitate, and oleate, respectively. Conversely, the rate of [1-¹⁴C] stearate incorporation into total glycerolipids was 88–90% lower than that of labeled palmitate, myristate, and oleate. The rate of [1-¹⁴C] stearate incorporation into triacylglycerol (3.6±0.4 nmol/mg protein/4 h) was 6–8% of that from myristate, palmitate, oleate, and linoleate. The rate of stearate incorporation into phospholipids was the lowest among tested fatty acids, whereas the rate of mono- and diacylglycerol synthesis was the highest with stearate treatment. The rate of β-oxidation as measured by CO₂ and acid soluble metabolite production was also the lowest with [1-¹⁴C] stearate treatment at 22.7 nmol/mg protein/4 h, which was 35–40% of those from other [1-¹⁴C] labeled fatty acids. A greater proportion of stearate than other fatty acids taken up by the hepatocytes remained free and was not metabolized. Clearly, stearate as compared to shorter-chain saturated fatty acids was less efficiently oxidized and esterified to triacylglycerol in cultured rat hepatocytes.     

Mechanisms Underlying Decreased Hepatic Triacylglycerol and Cholesterol by Dietary Bitter Melon Extract in the Rat

In these studies, we focused on finding the mechanism(s) underlying the bitter melon (Momordica charantia L.) methanol fraction (MF)-dependent reduction in the concentration of hepatic triacylglycerol (TAG) and cholesterol in the rat. Rats were fed diets containing low (5 %) fat for 2 weeks (experiment 1), or low (5 %) and high (15 %) fat for a longer period of 8 weeks (experiment 2). MF was supplemented at 1 % level in both experiments. After feeding, rats were sacrificed, and their livers were prepared as slices and hepatocytes, followed by incubation with [1(2)-¹⁴C] acetate or [1-¹⁴C] oleic acid (18:1 n-6). Under these conditions, we found that rats fed diets containing MF, as compared to those without MF, showed: (1) no adverse effects on food intake and growth, (2) a decreased hepatic TAG and total cholesterol, irrespective of the difference in dietary fat level or feeding period, and (3) a decreased incorporation of [1(2)-¹⁴C] acetate and [1-¹⁴C] oleic acid into TAG of liver slices and hepatocytes. MF-supplemented rats also showed no altered incorporation of labeled acetate into cholesterol and cholesterol ester, an increased fecal excretion of neutral steroids, but not of acidic steroids, and an enhanced mRNA abundance of carnitine palmitoylacyltransferase I, which is the rate-limiting enzyme for fatty acid oxidation. These results suggest that dietary MF decreases hepatic TAG synthesis while enhancing fatty acid oxidation, thereby reducing the concentration of hepatic TAG. The liver cholesterol-lowering effect of MF, however, is probably mediated through an increased fecal excretion of neutral steroids, without an effect on cholesterogenesis.     

Garlic reduces plasma lipids by inhibiting hepatic cholesterol and triacylglycerol synthesis

Prompted by the reported hypolipidemic activity of garlic, the present study was undertaken to elucidate the mechanism(s) underlying the cholesterol-lowering effects of garlic. Rat hepatocytes in primary culture were used to determine the short-term effects of garlic preparations on [1-¹⁴C]acetate and [2-³H]glycerol incorporation into cholesterol, fatty acids and glycerol lipids. When compared with the control group, cells treated with a high concentration of garlic extracts [i.e., petroleum ether- (PEF), methanol- (MEF) and water-extractable (WEF) fractions from fresh garlic] showed decreased rates of [1-¹⁴C]acetate incorporation into cholesterol (by 37–64%) and into fatty acids (by 28–64%). Kyolic containingS-allyl cysteine and organosulfur compounds inhibited cholesterogenesis in a concentration dependent manner with a maximum inhibition of 87% at 0.4 mM. At this concentration, Kyolic decreased [1-¹⁴C]acetate incorporation into fatty acids by 67%.S-allyl cysteine at 2.0 and 4.0 mM inhibited cholesterogenesis by 20–25%. PEF, MEF and WEF depressed the rates of [2-³H]glycerol incorporation into triacylglycerol, diacylglycerol and phospholipids in the presence of acetate, but not in the presence of oleate. The results suggest that the hypocholesterolemic effect of garlic stems, in part, from decreased hepatic cholesterogenesis, whereas the triacylglycerol-lowering effect appears to be due to inhibition of fatty acid synthesis. Primary hepatocyte cultures as used in the present study have been proven useful as tools for screening the anticholesterogenic properties of garlic principles.     

Effect of chronic ethanol administration on cholesterol and bile acid synthesis in vivo

Chronic administration of ethanol failed to stimulate the hepatic rate of cholesterol synthesis in meal-fed rats. In contrast, chronic ethanol feeding caused a 50% inhibition in the rate of incorporation of [4-¹⁴C] cholesterol to bile acids in the bile-duct cannulated rats. It is, therefore, suggested that the decreased rate of cholesterol degradation to bile acids may play an important role in ethanol-induced accumulation of cholesterol in liver.     

Effect of tibric acid on hepatic cholesterol synthesis in rats

Male albino rats were administered various oral doses of tibric acid daily for 1 week. Serum cholesterol and triglyceride levels were reduced, but total liver content of cholesterol, phospholipids, and triglycerides was increased. Tibric acid treatment suppressed the incorporation of both [¹⁴C] acetate and [³H] mevalonate into cholesterol by liver homogenates.