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.     

INTERACTION OF FATTY ACID AND GLUCOSE OXIDATION BY CULTURED HEART CELLS

Chick embryo heart cells in tissue culture actively oxidize [1-¹⁴C]palmitate to ¹⁴CO₂. Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30–70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in ¹⁴CO₂ production from [1-¹⁴C]palmitate. Conversely, palmitate had a similar sparing effect on [¹⁴C]glucose oxidation to ¹⁴CO₂. Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.     

The Effect of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) on Photosynthesis, Glycolate Excretion ('Photorespiration') and Amino Acid Metabolism in the Blue-Green Alga Anacystis

When photosynthesis of the blue-green alga Anacystis nidulans was measured as ¹⁴CO₂-fixation, the inhibitory effect of DCMU at low concentrations was greatest when mainly Photosystem 1 (PS 1) (excitation at 446 or 687 nm) was operative. At concentrations above 10-⁶M the inhibition on ¹⁴CO₂-fixation was greatest when mainly Photosystem 2 (PS 2) was operative (excitation at 619). During excitation of PS 1, the excretion of glycolate was stimulated at low concentrations of DCMU (5 × 10^-8M and lower), while higher concentrations inhibited excretion. All concentrations of DCMU inhibited glycolate excretion when mainly PS 2 was excited. The curves showing the relative effect of DCMU on the two photosystems, measured as PS 1/PS 2, had opposite shapes for ¹⁴CO₂-fixation and glycolate excretion. An increase in ¹⁴CO₂-fixation coincided with a decrease in glycolate excretion and vice versa. It appears that the increased rate of photosynthesis when mainly PS 1 was operative relative to that when mainly PS 2 was excited, increases the consumption of glycolate in an oxidation process associated with the excitation of PS 1, resulting in less excretion of glycolate to the medium. The influence of DCMU inhibition on labelled amino acid pools connected to the glycolate pathway (glycine-serine) is quite similar to that for ¹⁴CO₂-fixation. At concentrations below 10^-6M DCMU, inhibition of ¹⁴CO₂- incorporation into the amino acids was greatest when PS 1 was excited, while at the higher concentrations tested, inhibition was greater when PS 2 was excited. We conclude that the metabolism of glycine and serine is closely connected to the rate of photosynthesis.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Bicarbonate kinetics in Indian males

Measurement of rates ofin vivo substrate oxidation such as that of glucose, fatty acids and amino acids, are based on tracer (¹⁴C or¹³C) data, and often depend on the isotopic content of expired CO₂. The recovery of tracer-labelled CO₂ generated from the oxidation of¹³C labelled substrates may not be 100% over short term. This can lead to underestimation of oxidation rate of substrates, and consequently a correction for the incomplete recovery of tracer has to be applied by the determination of the recovery of¹³CO₂ in the breath during tracer bicarbonate infusions. We have studied the recovery of tracer-labelled bicarbonate using a bolus administration model, and further characterized kinetics of bicarbonate using a three-compartment model, to assess which compartmental fluxes changed during the change from a fasted state to fed state. Recovery of bicarbonate was lower at 69% and 67% (fasted and fed state) than the value of 71% and 74% found during earlier longer term of continuous infusions. During feeding, there was a 20-fold increase in the flux of bicarbonate between the central compartment and the compartment that was equivalent to the viscera. This study shows that the difference between the fasted and fed state recovery of tracer bicarbonate similar to that obtained with continuous infusions, and that bicarbonate fluxes show large changes between different compartments in the body depending on metabolic state.     

Comparison of oxidative metabolism in starved, fat-fed and carbohydrate-fed rats

1. Rats were starved for 48hr. or fed for 1 week on a high-fat or a high-carbohydrate diet. The effects of these dietary alterations on the rate of production of ¹⁴CO₂ from trace amounts of [U-¹⁴C]glucose, [1-¹⁴C]palmitate or [1-¹⁴C]acetate administered intravenously were studied. 2. The oxidation of [¹⁴C]glucose was most rapid in the carbohydrate-fed condition and was decreased significantly and to the same extent after starvation and after feeding with fat. 3. Under all dietary regimes studied the maximum rate of elimination of ¹⁴CO₂ from [¹⁴C]palmitate occurred within a few minutes after injection, but considerably more was oxidized after starvation and feeding with fat than after feeding with carbohydrate. 4. Alterations in diet had no effect on the oxidation and high recovery of administered [¹⁴C]acetate as ¹⁴CO₂. 5. Graphical analysis showed the presence of several exponential components in the ¹⁴CO₂-elimination curves. 6. In all studies a marked similarity in oxidative pattern was noted between the starved and the fat-fed rat.     

Metabolism of [4-14C]levulinic acid by etiolated and greening leaves of Hordeum vulgare

When etiolated barley (Hordeum vulgare L. var. Larker) shoots are incubated with [4-¹⁴C]levulinic acid, ¹⁴CO₂ is evolved, and amino and organic acids are labelled. Respiratory inhibitors and short-chain fatty acids, similar in size to levulinic acid, reduce the production of ¹⁴CO₂ from [4-¹⁴C]levulinic acid, while δ-aminolevulinic acid treatment or illuminating the tissue increase ¹⁴CO₂ evolution. The contribution of levulinic acid metabolism to α-aminolevulinic acid biosynthesis is no greater than that of a general cellular metabolite. The data suggest that fatty acid oxidation and the citric acid cycle are involved in levulinic acid metabolism.     

In vivo utilization ofd(−)-3-hydroxybutyrate by chick brain and spinal cord

The in vivo utilization ofd-3-hydroxy[3-¹⁴C]butyrate for oxidation in the whole animal and for lipid and amino acid synthesis in brain and spinal cord of overnight-fasted 15-day-old chicks has been measured. Appreciable amounts of injected 3-hydroxy[3-¹⁴C]butyrate were expired as¹⁴CO₂ one hour after injection, the total amount of which increased with increasing dosages. Lipid synthesis was high in both brain and spinal cord. Free, cholesterol and phospholipids were the main lipids labeled in both, tissues, increasing with time after injection up to 120 min. The incorporation of radioactivity into triglycerides, esterified cholesterol and free fatty acids was not time-dependent. Increased concentrations of 3-hydroxybutyrate gave rise to higher synthetic rates both in brain and spinal cord The rate of amino acid synthesis was slightly higher in brain than in spinal cord. Glutamate was always the major amino acid formed.     

Metabolism of Leucine and Alanine in Growing Rats Fed the Diets with Various Protein to Energy Ratios

In order to clarify the nutritional significance of metabolism of the carbon skeleton of individual amino acids, the metabolic fates of l-leucine-U-¹⁴C and l-alanine-U-¹⁴C were investigated in growing rats fed the diets with various protein calories percents (PC%) at 410 kcal of metabolizable energy.

The incorporation of ¹⁴C into body protein in 12 hr after the injection of leucine-¹⁴C was about 73% of the dose in the 0 and 5 PC% groups, though it decreased with increasing the levels of dietary protein from 10 to 30 PC%. The value of ¹⁴C recovery in body protein almost agreed with the net protein utilization (NPU) determined for the whole egg protein in a similar experimental condition. The ¹⁴C recovery in expired CO₂ and body lipid suggested that the carbon skeleton of leucine is well utilized as an energy source when the dietary carbohydrate is extensively replaced by protein.

While, the incorporation of ¹⁴C into body protein from alanine-¹⁴C was less than about 11% of the dose in all the dietary groups, and the majority of ¹⁴C was recovered in expired CO₂ and body lipid in a remarked contrast to leucine.

A similar pattern in urinary excretion of ¹⁴C was obtained for these amino acids, and the refracted rise of ¹⁴C from 10 PC% may give an indication for minimum protein requirements.     

Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Influence of Temperature and Seed Ripening on the in-vivo Incorporation of CO(2) into the Lipids of Oat Grains (Avena sativa L.)

To elucidate the influence of growth temperature and of stage of maturity on lipid synthesis in seeds, oat plants (Avena sativa nuda L., variety NOS) were fed with ¹⁴CO₂ at different stages after flowering, and the ¹⁴C-incorporation into the grain lipids was determined at 2, 24, and 48 hours after the end of ¹⁴CO₂-application. By changing growth temperature from 12 C to 28 C after the application of ¹⁴CO₂ to intact plants, a higher ¹⁴C-labeling of saturated fatty acids was found at the higher temperature. At 28 C, palmitic and stearic acids contained 23% and 9% respectively of total fatty acid-¹⁴C shortly after the ¹⁴CO₂-application, whereas at 12 C the corresponding values were 19% and 4%, respectively. Within 2 days ¹⁴C-activity of saturated fatty acids decreased at both temperatures, but to a lesser degree at 28 C. The higher ¹⁴C-labeling of saturated fatty acids and its lower decrease within 2 days at 28 C clearly show a direct influence of temperature on fatty acid biosynthesis in oat grains.     

Metabolism of endogenous lipid by the perfused rat kidney

It has been demonstrated that in vivo, exogenous [¹⁴C] palmitate is rapidly taken up and incorporated into phospholipid, neutral lipid and free fatty acid fractions of the kidney. During subsequent perfusion in an in vitro system, the amount of isotope decreases most rapidly in the neutral lipid (triglyceride) fraction. Net loss of chemical fatty acids cannot be detected after 2 hr. perfusion. The primary source of ¹⁴CO₂ produced appears to be fatty acids from either neutral lipid or phospholipids. Since loss of ¹⁴C from neutral lipids is independent of O₂ and substrates, regulation of fatty acid oxidation must be beyond triglyceride lipase.     

Energy metabolism of swimming trout (Salmo gairdneri)

Summary The oxidation of 1-¹⁴C palmitate, 2-¹⁴C glucose, 1-¹⁴C lactate, 1-¹⁴C alanine, 1-¹⁴C leucine and 1-¹⁴C glutamate, injected via a cannula into the dorsal aorta, was determined in trout, either at rest, or during swimming at 80% of the maximum sustained speed. The oxygen consumption and the excretion rates of¹⁴CO₂ as well as CO₂ were measured.While the oxygen consumption of swimming trout was about twice as high as of resting trout, the oxidation rates of the injected tracers increased by up to 9 time. Despite the increased importance of blood borne substrates, the estimated contribution to total CO₂ production is about 6% for the resting and 17% for the active trout. The majority of the oxidisable substrates must therefore be endogenous.The mobilization and oxidation rates of lactate, palmitate and leucine were particularly increased during swimming. During rest, palmitate and leucine oxidation rates are low. While oxidation rates of alanine and glutamate are intermediate, those of glucose were found to be extremely low, both at rest and during swimming. The measured RQ values for resting and swimming trout were 0.91 and 0.96 respectively, indicating that protein is the major fuel, since glucose oxidation seems of minor importance.Abbreviations and symbols SA specific activity - tt transit time - decay (time) constant - flux (in % of injected dose per hour) - U_crit critical velocity of sustained swimming - TCO₂ total CO₂     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Mevalonate metabolism: Effect of K2Cr2O7, a renal tubular toxin

K₂Cr₂O₇ was given to one member of 10 pairs of rats 2.5 hours before injection of $\text{RS}\text{-[5-}{}^{14}\text{C]}$mevalonate. The poisoned rats expired 30.8 ± 3.5% less ¹⁴CO₂ than their unpoisoned controls in the 2.5 hours after mevalonate injection (p < .001) and incorporated 16.3 ± 7.1% less label into renal (p<.05) and 63.7 ± 12.0% more label into hepatic unsaponifiable lipids (p < .001). K₂Cr₂O₇ had no effect on the oxidation of Na[1-¹⁴C]octanoate. These changes occurred at least 14 hours before any previously demonstrated effect of K₂Cr₂O₇ on the kidney and represent an early biochemical expression of renal tubular damage.     

Meal fatty acid uptake in human adipose tissue: technical and experimental design issues

The adipose tissue uptake of dietary fat has been studied using fatty acid radiotracers incorporated into a meal, followed by adipose tissue biopsies. A number of experimental design issues, including the use of isotopic tracers to measure meal fatty acid oxidation and plasma appearance of tracer, as well as the heterogeneity of adipose tissue fatty acid uptake, have been addressed. We examined these questions in a study of 24 volunteers (12 men and 12 women) who consumed a meal containing [(3)H]triolein and [(14)C]triolein. Slight differences in the purity of [(3)H]triolein vs. [(14)C]triolein were found, which could affect the apparent adipose tissue uptake of meal fatty acids. The adipose tissue triglyceride specific activity from bilateral biopsy sites agreed well, implying that a unilateral biopsy is satisfactory for measuring tracer uptake. Meal fatty acid oxidation measured using [(3)H]triolein and [(14)C]triolein was well correlated (r = 0.79, P < 0.0001). The peak tracer appearance in plasma chylomicrons occurred 1 h after the ingestion of a second, unlabeled meal. Our findings have implications for the experimental design of future meal fatty acid tracer/adipose tissue biopsy studies.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.