Leucine and isoleucine as in vitro precursors for lipid synthesis by rat aorta

The in vitro conversion of¹⁴C-labeled leucine, isoleucine, and pyruvate to specific lipids was compared in rat aorta, diaphragm, and fat pad. Total lipid specific radioactivity from all precursors was greatest in aorta. The ratio of label incorporated into polar lipids vs. neutral lipids by aorta was generally several-fold that incorporated by muscle and fat pad. The labeling of sterols in the aorta from¹⁴C-leucine and pyruvate was equivalent. It is concluded that leucine may be a substantial precursor to polar lipids and to sterols in rat aorta. This paper was presented in part at the 1974 Biochemistry and Biophysics Meeting of the Fed. Am. Soc. Exp. Biol.     

Effect of dietary di-2-ethylhexyl phthalate on lipid biosynthesis in selected tissues from the rat,in vitro

Di-2-ethylhexyl phthalate (DEHP), a plasticizer commonly used in the production of polyvinyl chloride plastics, has become an environmental pollutant. At the present time, the biological significance of phthalates in the environment is unknown. In the present studies, we observed that addition of DEHP to a stock diet of rats resulted in marked effects on incorporation of¹⁴C-acetate into lipid by liver and kidney slices; other organs, such as heart, testes, and aorta were unaffected. Incorporation of¹⁴C-acetate into total lipid of liver (dpm/mg wet wt) from rats, fed 0.5% or 1.0% DEHP for 10 or 18 days, respectively, was decreased to ca. 50% of control values. The decreased incorporation into liver lipid is not attributable to any one lipid fraction, inasmuch as incorporation into the phospholipid, sterol+diglyceride, free fatty acid, triglyceride, and sterol ester+hydrocarbon fractions was decreased 30–70% with respect to controls. In addition, the percent distribution of¹⁴C-acetate among the individual phospholipids was ca. 25% lower in phosphatidyl choline of the DEHP-fed rats. In rats fed 0.5% DEHP, incorporation of¹⁴C-acetate into total lipid of kidney was similar to control values, but incorporation into the triglyceride and sterol ester+hydrocarbon fraction was decreased 30–40%, whereas incorporation into the sterol+diglyceride fraction was increased 38%. Livers from DEHP-fed rats were ca. 20% larger than livers from control rats and, at the 0.%% level of DEHP feeding, testes wts were elevated; no significant changes were noted in wts of spleen, heart, aorta, kidney, or body wt gains in rats fed DEHP. These studies emphasize a subtle toxicity of phthalate esters not previously reported and emphasize the need for further biochemical studies to evaluate the effect of phthalates on biological systems.     

Evidence that chlorpromazine inhibits sterologenesis at post-HMGCoA reductase sites in rat liver,in vitro

The mechanism by which chlorpromazine inhibits cholesterogenesis in rat liver was investigated in vitro with the use of [14C] acetate and [14C] mevalonate as sterol precursors. Evidence was obtained that chlorpromazine blocks cholesterogenesis at multiple sites beyond HMGCoA reductase (beta-hydroxy-beta-methylglutarylCoA reductase, EC 1.1.1.34), the rate-limiting step. Squalene synthesis from both labeled acetate and mevalonate is reduced to a similar extent in the presence of chlorpromazine (29-36% at 0.5 mM). The data indicate that there is also an impairment of conversion of squalene to lanosterol, and of lanosterol to cholesterol. Overall inhibition of cholesterogenesis by chlorpromazine reached 65-75% at 0.5 mM and was concentration-dependent over the range 0.15-1.0 mM.     

Lipid synthesis by rat lung in vitro

Oxidation and lipogenesis in isolated rat lung tissue were studied in vitro. The minced tissue was incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) with 1-¹⁴C-acetate, 2-¹⁴C-pyruvate, U-¹⁴C-D-glucose, 1,5-¹⁴C-citrate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, 1-¹⁴C-linoleate. The lung tissue readily oxidized all of these substrates to¹⁴CO₂ and incorporated them into¹⁴C-lipids with the exception of 1,5-¹⁴C-citrate, for which there was no significant incorporation into¹⁴C-lipids. Most of the lipid¹⁴C was recovered in phospholipids, more specifically phosphatidyl choline. Twenty-eight per cent of glucose carbons was incorporated into the fatty acid moiety of phospholipids, while more than 90% of the carbons of other substrates was found in phospholipid fatty acids. The main fatty acid of the phospholipid fraction synthesized from acetate, pyruvate or glucose was palmitic acid. The oxidation of fatty acids was apparently influenced both by the carbon chain length and number of double bonds. Accumulation of¹⁴C-fatty acids in the tissue was observed when fatty acids were used as substrates; this finding suggests that the rate limiting step was not in the uptake of fatty acids. Chemical degradation of¹⁴C-myristic and palmitic acids obtained by hydrolysis of phospholipids biosynthesized from 1-¹⁴C-laurate indicated that the phospholipid fatty acids were synthesized via the de novo synthesis pathway. Presented at the AOCS-ISF World Congress, Chicago, September 1970.     

The effect of beta blocking drugs on lipid peroxidation in rat heartin vitro

Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetolol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetolol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 μM FeSO₄, 5 μMt-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO₄ (liposomes) plus O₂. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetolol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation event at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.     

Lung lipid synthesis from acetoacetate and glucose in developing rats in vitro

Acetoacetate (AcAc) and glucose were compared as energy sources and as precursors for lipid synthesis in the lungs of developing rats. Minced lung tissue was incubated with [3-14C]AcAc or [U-14C]glucose and the oxidation of each substrate to CO2 or its incorporation into tissue lipids was quantified. The highest rates of oxidation were obtained during the first 5 days for AcAc and the first 2 days of life for glucose and oxidation of AcAc was 3-4 times greater than that of glucose at all ages. Throughout postnatal development, the rates of nonsaponifiable lipid, fatty acid and hence total lipid (chloroform/methanol extractable) synthesis from AcAc were 2-3 times those of glucose. The highest rates of total lipid synthesis from AcAc and glucose were observed at birth. Glucose was utilized for glyceride-glycerol synthesis at a higher rate than AcAc. Similar patterns of incorporation of AcAc and glucose into various lipid classes were noted. Of the total lipids synthesized from AcAc and glucose, respectively, phospholipid plus monoglyceride accounted for 64% and 77%, triglyceride 13% and 13%, diglyceride plus cholesterol 11% and 4%, fatty acids 9% and 4%, and cholesteryl esters 3% and 1%. AT birth, the specific activities of all lipids except triglyceride derived from AcAc were greater than those from glucose. Rates of synthesis of all complex lipids declined with age. The results of these experiments demonstrate that AcAc is utilized more readily than glucose for energy production and lipid synthesis in developing rat lungs.     

Effects of renal factors on in vitro hepatic cholesterol synthesis in the rat

Two products derived from rat renal tissue have been shown to affect in vitro hepatic cholesterol synthesis. A premevalonate inhibitor of hepatic cholesterol synthesis is associated with the membranes of the renal endoplasmic reticulum. It is stable at −75 C and at 4 C but is heat labile. A pre-mevalonate stimulator of in vitro hepatic cholesterol synthesis is located within renal lysosomes and can be prepared in a nonsedimentable form by extraction with hypotonic buffer. While it is stable at −75 C, it loses activity at 4 C. Both of these products appear to have a molecular weight in excess of 150,000 as determined by gel filtration. Deceased.     

Effect of soy sterols on cholesterol synthesis in the rat

Soy sterols increased the incorporation of acetate into cholesterol by rat liver slices and prevented the depression of cholesterogenesis caused by dietary cholesterol. Acetate incorporation into cholesterol by intestinal slices was not affected by soy sterols but was decreased by cholesterol. The results with liver slices agree with the view that soy sterols, by interfering with the intestinal absorption of cholesterol, prevent the feedback inhibition of hepatic cholesterogenesis.     

Effect of acute dietary alteration upon intestinal lipid synthesis

The specific activities of three enzymes engaged in complex lipid synthesis, diglyceride acyltransferase, cholinephosphotransferase, and lysolecithin acyltransferase were studied in intestinal mucosa of hamsters fed either saline, hydrolyzed casein, or corn oil for 9 1/2 hr. In the most proximal intestine, saline feeding was associated with a reduced specific activity in villous tips with all three enzymes studied when compared with the two caloric supplemented groups. In the most distal intestine, oil feeding increased the activity of lysolecithin acyltransferase and choline phosphotransferase twofold as compared to casein fed hamsters; diglyceride acyltransferase was increased one- and one-half-fold. The response of lysolecithin acyltransferase and diglyceride acyltransferase to fat feeding was incomplete when compared to hamsters fed a fat supplemented diet for 7 days, suggesting that their pattern of response to dietary substrate was similar to the disaccharidases. By contrast, the response of cholinephosphotransferase to fat feeding was complete at 24 hr, suggesting that it responds in a manner similar to the glycolytic enzymes.     

Effect of age on plasma bile acids and lipid components in the rat

With increasing age, total plasma bile acid contents increased in rats over a period of 11 months, and also total plasma cholesterol and carcass fat contents increased in the same manner. Plasma showing high bile acid levels at 11 months was found by means of high performance liquid chromatography to contain cholic acid as one of the major components, chenodeoxycholic acid and trace deoxycholic acid. These results suggest that there are close relationships between the plasma bile acids and age-dependent changes of lipid components in the rat.     

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.     

Effect of hypothyroidism on the lipid composition of rat plasma and erythrocyte membranes

The effect of hydrothyroidism on plasma and erythrocyte membrane lipid components has been investigated. This pathological state is accompanied by a) a cholesterol increase of about 60% in plasma, and at the same time a 22% reduction in erythroycte membranes; b) 44% and 30% phospholipid level decreases in both plasma and red cell membranes, respectively; and c) almost unaffected phospholipid and fatty acid compositions of both plasma and erythrocyte membranes. All changes were corrected by treatment of the hypothyroid rats with triiodothyronine for two days. These findings suggest that in hypothyroid rats a reduced transfer of cholesterol from plasma to erythrocyte membrane probably takes place. This could explain, at least in part, the increased hematic cholesterol level observed in hypothyroid animals. In red cell membranes, the simultaneous decrease in cholesterol and phospholipid levels does not alter the cholesterol/phospholipid molar ratio, thus avoiding their abnormal function.     

Effect of lipid derivatives on invasion in vitro and on surface glycoproteins of three rodent cell types

The antiinvasive activity on MO₄ mouse cells of the following lipid derivatives was tested in vitro: an alkyllysophospholipid derivative (BM 41.440), an alkyl-linked lipoidal amine (CP-46,665) and a naturally occurring ester-linked phospholipid (2-LPC). In this test, BM 41.440 had the same antiinvasive potency as ET-18-OCH₃, whereas CP-46,665 and 2-LPC had no effect on invasion. Comparison of the antiinvasive effect of ET-18-OCH₃ on three types of cells showed the following ranking: 12R1C-RK rat kidney adenovirus type 12 transfected cells>MO₄ mouse cells>LLC-H61 Lewis lung carcinoma cells. This ranking was not reflected in ET-18-OCH₃-induced changes of cell surface exposed glycopeptides derived from the three types of cells metabolically labeled with radioactive fucose. The present and previous experiments suggested that changes in invasion caused by lipid derivatives depended upon relative cell surface fucosylglycopeptide alterations in both the invasive cells and the normal tissue.     

Effect of calcium on absorption of fatty acid by rat jejunum in vitro

The effect of Ca⁺⁺ on jejunal osmiophilic particles was studied in a recirculating system which was not contaminated with plasma lipoproteins. An isolated, infused segment of rat jejunum was suspended in a bath of liquid paraffin. Transudate, containing osmiophilic particles, appeared like beads of sweat on the serosal surface, and fell to the bottom of the bath. In the range of 25–38 C, 30 C proved to be optimal for histological preservation of villous architecture. Production of transudate, 20 mg/min/g of jejunum, and transport of [¹⁴C] oleate proceeded nearly linearly after the first 30 min. Necrosis of mid-villus and crypt cells became obvious by light microscopy after one hour. Therefore, transudate was collected between the period of 30–60 min. Shadow casting of transudates, produced when saline was infused, revealed that 86±9 (SD) % of osmiophilic particles was <800 Å in diameter; 13±8% was 800–1000 Å; 0.4±0.5% was 1000–2000 Å. Corresponding values were 58±10, 25±5, and 16±5% when 5 mM [¹⁴C] oleate+2.5 mM monoolein was infused; 75% of the transported [¹⁴C] appeared in triglyceride. Adding 2 mM Ca⁺⁺ to the infusion doubled the transport of [¹⁴C] triglyceride without increasing particle size further. We conclude that luminal Ca⁺⁺ increases the absorption of luminal fatty acid by rat jejunum in vitro.     

Effect of Lactobacillus plantarum P‐8 on lipid metabolism in hyperlipidemic rat model

Probiotics have been reported to play an important role in the prevention of metabolic disorders. We recently identified a novel probiotic strain Lactobacillus plantarum (L. plantarum) P‐8. The objective of this study was to determine the effects of L. plantarum P‐8 on lipid metabolism of rats fed with high fat diet. All experimental rats were divided into three groups: control group, model group, and L. plantarum P‐8 group. Changes in serum lipid levels, hepatic lipid deposition, serum oxidative stress‐related parameters, activities of liver function marker enzymes, organ indices, gut microflora, and fecal lipids were assessed. Compared with model group, L. plantarum P‐8 exhibited hypolipidemic effects by lowering serum total cholesterol (TC), triglyceride (TG), and low‐density lipoprotein cholesterol levels, accompanied with elevation of high‐density lipoprotein cholesterol level. L. plantarum P‐8 also exerted beneficial effects against high‐fat diet‐induced oxidative stress, curtailed the accumulation of liver lipids and protected healthy liver function. Moreover, L. plantarum P‐8 was able to regulate intestinal bacteria and enhance the fecal excretion of TC, TG, and bile acid. These findings indicate that L. plantarum P‐8 may represent a potential therapeutic agent for controlling hyperlipidemia.     

The effect of dihydroergotoxine on lipid peroxidation in vitro

Dihydroergotoxine mesylate (DHET), an ergot alkaloid derivative, is widely used to treat senile cerebral vascular insufficiency. Aspects of this age-related phenomenon may be due to deterioration by lipid oxidation of cellular membranes. DHET stabilizes EEG alpha frequencies, increases cerebral blood flow and oxygen uptake and accumulates in lipid-rich structures of the brain. The effect of DHET was studied on iron-catalyzed peroxidation of liposomes as measured by the thiobarbituric acid assay. It was found that DHET inhibits peroxidation in vitro in a dose-dependent manner. These results suggest that DHET acts in part as a lipid antioxidant when used to treat senile cerebral vascular insufficiency.     

Effect of dietary vitamin E on sulfolipid synthesis in rat submandibular salivary gland

The effect of feeding diets containing various levels of vitamin E for 6 months on the in vitro synthesis of sulfolipids in rat submandibular salivary glands (SMG) was elucidated. The incorporation of [³⁵S]sulfate into sulfolipid of SMG from rats on deficient or “normal” vitamin E diets was quite similar, however, the uptake was significantly increased in glands from rats on diets high in vitamin E. Whereas, in many instances, antioxidants can mimic the effect of vitamin E, in the present study, the antioxidant N,N′-diphenyl-ρ-phenylene diamine (DPPD) was actually found significantly to depress sulfolipid-labeling below that noted in SMG from all other diets. The results suggest that in the synthesis of SMG sulfolipid the action of vitamin E may be more than that of a simple antioxidant.