Some characteristics of monocarboxylic acid transfer across the cell membrane of epithelial cells from rat small intestine.

1. The translocation of monovalent organic anions (pyruvate, propionate, acetate and butyrate) across the cell membrane of isolated epithelial cells from rat small intestine was studied by measuring competitive inhibition kinetics, exchange diffusion and temperature dependence of the efflux rate. A possible function of a monocarboxylate carrier in intestine will be discussed. 2. Earlier studies on the inhibition of pyruvate transport of fatty acids were extended to propionate and found to show the same characteristics. The kinetics, however, appeared to be more complex by the contribution of several diffusion pathways for propionate. 3. The mechanism of countertransport was most compatible with an "accelerated exchange diffusion" and could be studied at both sides of the membrane. This exchange diffusion exhibited saturation kinetics. It is proposed that different monocarboxylate anions may have different affinities for a common carrier. 4. Temperature dependence of the efflux of pyruvate and propionate was studied. Arrhenius plots obtained were not found to be linear between 0 and 5 degrees C. Between 5 and 15 degrees C activation energies for pyruvate and propionate efflux rates were found to be 19.6 and 12.6 kcal/mol, respectively.     

On the effects of propionate and other short-chain fatty acids on sodium transport by the toad bladder.

1. Propionate and other unbranched short-chain fatty acids, butyrate, pentanoate, hexanoate and octanoate were found to both stimulate and inhibit active sodium transport by the toad bladder, as measured by the short-circuit current (s.c.c.). 2. Stimulation alone followed addition of low concentrations of fatty acids (0.1-1.0 mM) to either the serosal or mucosal bathing medium; stimulation was also seen after an initial period of inhibition in response to higher concentrations (approx. 5 mM) of some compounds. 3. Inhibition alone followed addition of high concentrations (5-20 mM) of these compounds. The duration and magnitude of the inhibition varied with increasing concentration and chain length of the fatty acid, and was greater following mucosal addition than serosal addition. 4. The inhibitory effect of mucosal propionate increased with decreasing pH of the mucosal bathing medium. 5. Inhibition by the fatty acids was completely reversed upon removing the compound from the bathing medium, and stimulation characteristically followed. 6. In studies designed to evaluate the role of metabolism of the fatty acids in their mucosal inhibitory effects it was found that 14-c-labelled propionate, when added to the mucosal surface of the bladder, was converted to 14-CO2, and mucosal succinate and alpha-oxoglutaric acid at 20 mM inhibited the s.c.c. slightly. However, malonate did not interfere with inhibition by mucosal propionate and two non-metabolizable acids, dimethylpropionate and benzoate, induced inhibition (and no stimulation) of the s.c.c. 7. In the presence of an inhibitory concentration of fatty acid, the ability of the bladder to respond to added pyruvate was reduced in proportion to the reduction in the level of the s.c.c., whereas the natriferic response to vasopressin was largely intact. 8. We conclude that stimulation of sodium transport by propionate and other short-chain fatty acids is due to metabolism of the compounds and provision of energy to the sodium transport mechanism. The basis of the inhibition appears complex. It may in part depend on metabolism of the fatty acids and/or uncoupling of oxidative phosphorylation, with resultant reduction in net ATP production for the sodium transport mechanism. However, the inhibition may also be caused in part by a direct effect on the mucosal entry of sodium into the transporting epithelial cells.     

The effect of fatty acids and starvation on the metabolism of gluconeogenic precursors by isolated sheep liver cells.

Isolated liver cells prepared from fed sheep synthesize glucose from propionate at twice the rate observed with cells from starved animals. Addition of palmitate or palmitate + carnitine to incubations of liver cells from starved animals inhibited the rate of glucose synthesis with lactate as a precursor, but had little effect when propionate and pyruvate were substrates. Liver cells from fed and starved sheep synthesized lactate and pyruvate when incubated with propionate. Fatty acids inhibited this formation of lactate and pyruvate from propionate. It is proposed that the different responses of gluconeogenic precursors to fatty acids can be explained by the effect of reducing equivalents on the transport of carbon atoms across the mitochondrial membrane.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Development and Characterization of Pantothenic Acid Transport in Brain

In vitro, the transport of [3H]pantothenic acid into and from rabbit brain slices was studied. In newborn rabbits and throughout development, forebrain and cerebellar slices were able to accumulate and phosphorylate [3H]pantothenic acid comparably to slices from adults. The accumulation and phosphorylation of [3H]pantothenic acid by adult forebrain slices were not decreased by substitution of LiCl for NaCl in the artificial CSF or by addition of short-chain fuels (e.g., 5 mM pyruvate or acetoacetate) to the medium. However, probenecid and ouabain (both 1 mM) and medium-chain fatty acids (e.g., 0.1 mM octanoate, nonanoate, and decanoate) profoundly inhibited [3H]pantothenic acid accumulation by forebrain slices but not intracellular phosphorylation and conversion to [3H]CoA. There in vitro results suggest that brain slices accumulate pantothenic acid by a saturable system (probably facilitated diffusion) that is sensitive to inhibition by probenecid and medium-chain fatty acids.     

The control of fatty acid metabolism in liver cells from fed and starved sheep.

Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Multifunctional role for fetuin (fetal protein) in lipid transport.

Recent studies from this laboratory have shown that fetuin 1) is nearly 50-fold more efficient than albumin in incorporating exogenous fatty acids into cultured cells, (JBC, 265: 5883, 1990), and 2) is associated with a lipoprotein-like particle (FASEB J. 3: 2075-2080, 1989). In the present study, this lipid-containing fraction (FLP) was isolated by ultracentrifugation, and its effect on cholesterol efflux from cultured human skin fibroblasts and Hep-G2 cells prelabeled with [14C]cholesterol was investigated. FLP fraction caused a significant efflux of [14C]cholesterol from cells, the same in magnitude as HDL. This effect of fetuin supranatant fraction increased proportionately with concentration and time. Similar results were observed with Hep-G2 cells. This ability to induce efflux of cholesterol was confirmed by a decrease in cholesterol mass of cells after 24 h incubation with FLP. The ultracentrifugal bottom (infranatant) fraction of fetuin (FI) was ineffective in this regard. However, FI was more effective in the incorporation of exogenous fatty acids into cellular triglycerides. These studies suggest that the fetuin molecule is a multifunctional protein (delivery of fatty acids to cells and cholesterol efflux from cells) which may play a role in lipid transport during fetal life.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.     

Lipolysis in heart and adipose tissue: effects of inhibition of glycogenolysis and uncoupling of oxidative phosphorylation.

In the Langendorff heart, lipolysis is arrested when glycogenolysis is inhibited by the addition of 5-gluconolactone. Glucose partially overcomes the inhibition as well as uncoupling of oxidative phosphorylation by dinitrophenol. In isolated fat cells hormone-sensitive lipolysis is also inhibited by glycogenolysis inhibition and in these cells also, glucose addition overcomes the inhibition. In fat cells, uncoupling of oxidative phosphorylation does not stimulate lipolysis, probably because of the relatively low concentration of mitochondria in white adipose tissue. The data are interpreted that both in heart and adipose tissue cells, the removal of fatty acids produced by the endogenous lipase is the main stimulus for lipolysis. Attempts to generate in fat cells glycerol-3-phosphate by glycerogenesis from pyruvate or lactate led to the observation that not only these latter anions, but also propionate and acetate strongly stimulate lipolysis. It suggests that long-chain fatty acid removal from fat cells may be stimulated by anion exchange.     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Effects of octanoate and acetate upon hepatic glycolysis and lipogenesis

Octanoate and N6,O2'-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) cause a marked inhibition of net glucose utilization and lactate and pyruvate accumulation by hepatocytes isolated from meal-fed rats. Acetate is much less effective as an inhibitor of glycolysis. Fatty acid synthesis, as measured by tritiated water incorporation, is inhibited by dibutyryl cyclic AMP, whereas it is stimulated by 10 mM acetate and 1 mM octanoate. Stimulation of fatty acid synthesis by 1 mM octanoate, however, is lost paradoxically at higher concentrations of octanoate. Rates of fatty acid synthesis estimated by [1-14C]octanoate incorporation were consistently higher than rates calculated on the basis of tritiated water incorporation, raising the question as to which is the better index of the rate of de novo fatty acid synthesis. The effects of octanoate were studied because it was reasoned that this fatty acid should not inhibit acetyl-CoA carboxylase but should inhibit glycolysis and supply acetyl-CoA for lipogenesis. This was found to be the case, proving that glycolytic activity is not necessary for rapid rates of de novo fatty acid synthesis by liver.     

Fatty acid effects on gluconeogenesis in goat, calf and guinea pig hepatocytes

1. Regulation of hepatic gluconeogenesis by fatty acid was studied in goat, calf and guinea pig hepatocytes. 2. Fatty acid effects on gluconeogenesis were dependent upon species; fatty acid and gluconeogenic substrate. 3. Oleate and octanoate inhibited gluconeogenesis from propionate in guinea pig hepatocytes and stimulated it in goat hepatocytes. 4. Oleate and octanoate markedly inhibited gluconeogenesis from lactate in guinea pig hepatocytes whereas octanoate, but not oleate, decreased glucose production from lactate in goat hepatocytes. 5. Effects of fatty acids on gluconeogenesis in calf hepatocytes were similar to goat hepatocytes suggesting control of gluconeogenesis is similar among ruminant species but differs from guinea pigs.