Renata Z. Christiansen

Active transport of butyrobetaine and carnitine into isolated liver cells

1. The liver cells lose the major part of their carnitine during the commonly used isolation procedure by the collagenase-perfusion method. 2. The cells take up carnitine and the carnitine precursor butyrobetaine when these substances are added to the medium. The carnitine content of isolated liver cells can increase to about 15 mM with no apparent harm to the cells. 3. The data indicate the existence of a common carrier in the plasma membrane which mediates the uphill transport of both carnitine and butyrobetaine. The carrier has a high affinity for butyrobetaine (Km=0.5 mM) and a lower one for carnitine (Km=5.6 mM). 4. The intracellular butyrobetaine is hydroxylated to carnitine with a rate of approximately 0.33 mumol-g wet weight-1-h-1 which is sufficient to cover the turn over of carnitine in the whole rat. Carnitine is effectively esterified in the liver cells to acetylcarnitine and long-chain acylcarnitines. 5. Both carnitine and acetylcarnitine are released from the cells. The release of both compounds is probably physiological since it was found that acetylcarnitine constitutes a similar fraction of the total acid soluble carnitine both in the blood and liver of the intact rat.

The effect of (−)carnitine on the metabolism of palmitate in liver cells isolated from fasted and refed rats

In studies on fatty acid metabolizing enzymes in fasted and fed rats it has been observed that the activity of palmityl-CoA: carnitine palmityltransf&ase (EC 2.3.1.2 1) and acyl-CoA: glycerophosphate acyltransferase (EC 2.3.1.15) in the liver vary in opposite directions depending on the nutritional state of the animal [l] . In fasting the activity of carnitine palmityltransferase increases and the glycerophosphate acyltransferase decreases. Fasting also increases the content of carnitine in the liver [2,3], and in perfusion studies it has been observed that addition of carnitine to the perfusion fluid accelerates ketogenesis in livers from fed rats [3] . In studies on isolated mitochondria Borrebaek [4] found that the acylation of glycerophosphate has a certain preference over the acylation of carnitine when a low concentration of palmitate was added to the incubation system. Both reactions were easily saturated with high palmitate concentrations. These observations seen together suggest that both the relative activities of carnitine palmityltransferase and glycerophosphate acyltransferase and variations in the concentration of carnitine can have directing effects on the metabolic fate of fatty acids in the liver. Recent studies on carnitine in isolated liver cells have shown that these cells have lost 3/4 to 4/5 of their normal carnitine content during the preparation procedure. The cells again take up carnitine when it is added to the medium (Christiansen and Bremer, unpublished results). Glycerol or fructose are known to increase the concentration of glycerophosphate in the liver [5] .

Regulation of palmitate metabolism by carnitine and glucagon in hepatocytes isolated from fasted and carbohydrate refed rats

Abstract 1. 1. Glucagon stimulates the oxidation of palmitate in the liver cells isolated from carbohydrate-refed rats and incubated in a simple salt medium. The maximal stimulation was observed at the concentration of glucagon of 4 · 10 −8 M and was already noticeable after 5 min of incubation. The stimulation of the oxidation was balanced mainly by the inhibition of triacylglycerol synthesis. The extent of stimulation was not dependent on the concentration of intracellular carnitine, but was decreased at higher concentrations of palmitate in the medium. 2. 2. Glucagon did not have any significant effect on palmitate metabolism in the cells isolated from fasted rats. It seems probable that those cells are isolated in the activated state as far as it concerns their capacity to oxidize fatty acids. 3. 3. Under optimal conditions (in the presence of glucagon and an excess of carnitine), the refed cells (cells isolated from livers of rats fasted for 48 h and refed with carbohydrate for 48 h) oxidized palmitate at approximately the same rate as fasted cells (cells isolated from livers of rats fasted for 48 h). However, the refed cells still differed from fasted cells in that they required a much higher intracellular carnitine concentration and a higher palmitate concentration in the medium to reach the maximal level of oxidation. 4. 4. Carnitine decreased the intramitochondrial redox potential ( E = E 0 − 2.3 RT / nF log[red]/[ox] in the refed cells which indicated more specific stimulation of β-oxidation. Glucagon increased the redox potential under all conditions used. 5. 5. Glucagon slightly stimulated carnitine transport into the cells and acetylcarnitine formation, and had a very pronounced stimulatory effect on the synthesis of long-chain acylcarnitines in refed cells. 6. 6. The level of total long-chain acyl-CoA was increased by glucagon in refed cells. Carnitine increased the level of total long-chain acyl-CoA in fasted cells. It seems probable that in the presence of glucagon, the extramitochondrial acyl-CoA level is increased, which may indicate direct inhibition of the triacylglycerol synthesizing enzymes. 7. 7. It is concluded that glucagon acts at least in part at one of the early stages in fatty acid metabolism, i.e., carnitine acyltransferase and/or glycerophosphate acyltransferase.

The stimulation of erucate metabolism in isolated rat hepatocytes by rapeseed oil and hydrogenated marine oil-containing diets

1. The metabolism of palmitate and especially of erucate was studied in hepatocytes isolated from rats fed for 3 weeks a diet containing peanut oil (diet, 1), rapeseed oil (diet 2) and partially hydrogenated marine oil (diet 3). 2. The metabolism of palmitate was not significantly influenced by the diet. The rapeseed oil diet caused 1.4 fold and 1.3 fold increase and marine oil diet 3 fold and 2.2 fold increase in the oxidation and chain-shortening respectively of [14-14C]erucic acid in isolated hepatocytes. 3. Cyanide and antimycin A did not inhibit the chain-shortening of erucate in liver cells of rats fed rapeseed oil and peanut oil. The high capacity of the chain-shortening system in hepatocytes of marine oil-fed rats was partially inhibited. 4. Inhibition of the transfer of fatty acids into the mitochondria by lowering the intracellular carnitine concentration and/or by addition of (+)-decanoyl-carnitine resulted in a very pronounced apparent stimulation of the chain-shortening of erucic acid. It is suggested that the chain-shortening system may be virtually independent of the mitochondria, unless the availability of the extramitochondria NAD+ and/or NADP+ is rate-limiting under conditions of extremely low redox potential of the mitochondria. 5. Feeding marine oil or rapeseed oil to the rats induced a 30% increase in catalase activity, a 25--30% increase in urate oxidase activity and a 50% increase in the total CoA in the liver compared to rats fed peanut oil. 6. It is suggested that the increased metabolism of erucate in hepatocytes of marine oil and rapeseed oil-fed rats may be due to the increase in ther peroxisomal beta-oxidation.

The effect of clofibrate-feeding on hepatic fatty acid metabolism

Abstract 1. 1. The hepatocytes isolated from clofibrate-fed rats oxidized palmitate to ketone bodies and CO2 more rapidly than did hepatocytes from control rats. Glucagon stimulated the oxidation of palmitate further. The extent of stimulation was approximately the same in cells from control and clofibrate-fed animals. The esterification of palmitate was decreased by clofibrate-feeding. 2. 2. Clofibrate stimulated the oxidation, chain shortening and esterification of erucate in isolated hepatocytes. The oxidation of erucate was not stimulated by glucagon. The increase in the esterification seemed to depend on the availability of the chain-shortened fatty acids derived from [14-14C]erucic acid. 3. 3. The pattern of chain-shortened fatty acids changed towards longer fatty acids (C20) with increasing concentration of erucic acid in the medium, suggesting a competition between erucate and shorter fatty acids for the limited capacity of the chain-shortening system. 4. 4. The chain-shortened fatty acids derived from [14-14C]erucate were found mainly in phospholipids and triacylglycerol. Relatively more unchanged erucate was found in the cellular lipids at higher concentrations of erucate in the medium. 5. 5. (+)-Decanoylcarnitine had a much smaller inhibitory effect on the oxidation of palmitate and erucate in cells from clofibrate-fed rats. The inhibitor had a small stimulatory effect on the chain-shortening of erucate. 6. 6. It is concluded that both oxidation and esterification of very long chain fatty acids are limited by the capacity of the chain-shortening system which is localized extramitochondrially, most probably in peroxisomes. The peroxisomal oxidation system may also contribute to the oxidation of palmitate especially when carnitine palmitoyltransferase is rate-limiting.

The effects of clofibrate feeding on the metabolism of palmitate and erucate in isolated hepatocytes

The metabolism of palmitate and erucate has been investigated in hepatocytes isolated from control rats and from rats fed 0.3% clofibrate. Clofibrate increased the oxidation of [1-14C]palmitate 1.5 to 2-fold while the esterification was decreased. At a high concentration of palmitate (1.5 mM), the total rate of fatty acid metabolism was stimulated. Clofibrate stimulated both the oxidation (3.5 to 5-fold) and the esterification (1.7-fold) of [14-14C]erucate. Erucate undergoes chain-shortening in isolated liver cells. This chain-shortening was stimulated at least 2-fold by clofibrate feedings. The isolated mitochondrial fraction from clofibrate-fed rats showed an increased capacity for oxidation of short-chain acylcarnitines (including acetylcarnitine), while the oxidation of palmitoyl- and erucoylcarnitine showed little change. It is suggested that erucate is shortened by the recently detected β-oxidation system of peroxisomes.

Acetylation of Tris(hydroxymethyl)aminomethane (Tris) and Tris derivatives by carnitine acetyltransferase

The most sensitive method for determining carnitine is based on the separation of [r4C]acetylcamitine from [ l14C] acetyl-CoA on a small Dowex2-Cl column after incubation of the carnitine with excess [lr4C]acetyl-CoA and carnitineacetyl-transferase [ 1,2] . This method has been used for studies of camitine uptake and release in isolated liver cells [3] . We observed erratic high camitine values in the cells, especially when Tricine was used in the cell suspension medium, but also Tris, Tes and phosphate interfered in the carnitine analysis. We have studied Tris and Tris derivatives in this assay more closely. It turns out that these buffers, especially Tricine, can act as acetyl acceptors for carnitine acetyltransferase, thus interfering with the assay of carnitine .

The glycerophosphateacyltransferases and their function in the metabolism of fatty acids.

Summary1From different studies on the cellular localization, positional specificity, and regulatory properties of acyl-CoA: glycerophosphate acyltransferase (EC 2,3,1.15) and acyl-CoA: 1-acylglycerophosphate acyltransferase (EC 2,3,1⋯.) the following conclusions can be drawn:2The glycerophosphate acyltransferase is localized in the endoplasmatic reticulum (microsomes) and in the outer membrane of the mitochondria of the animal cell. Its reaction product is 1-acylglycerophosphate (1-lysophosphatidic acid). The mitochondrial enzyme shows a high preference for saturated fatty acids while the microsomal enzyme is less specific (alternatively the microsomes contain more than one glycerophosphate acyltransferase).The 1-acylglycerophosphate acyltransferase is localized in the endoplasmatic reticulum (microsomes) in the animal cell. Possibly a minor fraction of this enzyme is localized to the outer membrane of the mitochondria. This enzyme shows a strong preference for unsaturated fatty acids.3Both the microsomal and the mitochondrial dihydroxyacetonephosphate acyltransferase show similar fatty acid specificity as the corresponding glycerophosphate acyltransferases. It cannot be excluded that dihydroxyacetonephosphate and glycerophosphate are acylated by the same enzymes.4The activity of the glycerophosphate acyltransferase(s) in the liver decreases in fasting or fat feeding and increases upon feeding of carbohydrate. The activity of carnitine palmityltransferase varies exactly opposit. These enzymes do not show dietary variations in heart and adipose tissue.5Under otherwise identical conditions the rate of carnitine acylation in isolated mitochondria decreases more than the rate of glycerophosphate acylation when the concentration of palmityl-CoA is reduced.6In isolated liver cells (which has lost most of their carnitine) addition of carnitine increases the rate of fatty acid oxidation and decreases the rate of triglyceride formation.7Glycerol and fructose lower the rate of fatty acid oxidation, probably by lowering the levels of acyl-CoA and acyl-carnitine in the cells.8It is concluded that the relative activities of glycerophosphate acyltransferase and carnitine palmityltransferase probably influence the fate of fatty acids in the cell.

Studies on the mechanism of the inhibitory effects of erucylcarnitine in rat heart mitochondria.

1. The mechanism of the inhibitory effect of erucylcarnitine on palmityl-carnitine oxidation in rat heart mitochondria was studied. 2. Erucylcarnitine inhibited in the same time the oxidation of [U-14-C]-palmitylcarnitine and the total rate of oxygen uptake. Other acylcarnitines competed as well for the oxidation with radioactive palmitylcarnitine, but they were well oxidized themselves, so that the total oxygen uptake did not decrease. 3. The presence of erucylcarnitine did not change the distribution pattern of Krebs cycle intermediates derived from [U-minus 14 C] palmitylcarnitine except that succinate/malate ratio increased. 4. The presence of erucylcarnitine did not lead to the formation of any beta-oxidation cycle intermediates from [U-minus 14 C] palymitylcarnitine. The formation of beta-hydroxy-palmityl derivative when rotenon was included into the incubation medium, decreased in the presence of erucylcarnitine. 5. It is postulated, that the inhibited entrance of palmityl groups into the beta-oxidation cycle is due to the fact that erucylcarnitine and palmitylcarnitine behave as substrate-competitive inhibitors for long chain acyl-CoA dehydrogenase. 6. There was observed a latency of 1-2 min in the effect of erucylcarnitine on the palmitylcarnitine oxidation, which seems to correspond to the time required for the formation of high amounts of intramitochondrial erucyl-CoA. 7. Erucylcarnitine inhibited the total oxygen uptake with long, medium and short chain acylcarnitines, pyruvate and alpha-ketoglutarate as substrates, while the oxidation of succinate was not affected. 8. Sequestration of free CoA in the form of very slowly metabolized erucyl-CoA is proposed as the partial explanation of the observed inhibitory effects of erucylcarnitine on the oxidation of CoA-dependent substrates (alternatively to the inhibition at the level of acyl-CoA dehydrogenases in case of acylcarnitines).

Metabolism of erucic acid in perfused rat liver: Increased chain shortening after feeding partially hydrogenated marine oil and rapeseed oil

The metabolism of [14-14C] erucic acid was studied in perfused livers from rats fed on diets containing partially hydrogenated marine oil or rapeseed oil for three days or three weeks. Control rats were given groundnut oil. Chain-shortening of erucic acid, mainly to 18∶1, was found in all dietary groups. In the marine oil and rapeseed oil groups, the percentage of chain-shortened fatty acids in very low density lipoproteins-triacylglycerols (VLDL-TG) exported from the liver increased after prolonged feeding. A similar increase was found in liver TG only with partially hydrogenated marine oil. This oil, rich intrans fatty acids, thus seemed to be more effective in promoting chain-shortening. The fatty acid composition of the secreted and stored TG differed both with respect to total fatty acids and radioactively labeled fatty acids, indicating that at least 2 different pools of TG exist in the liver. The lack of lipidosis in livers from rats fed dietary oils rich in 22∶1 fatty acids is discussed in relation to these findings. In conclusion, a discussion is presented expressing the view that the reversal of the acute lipidosis in the hearts of rats fed rapeseed oil or partially hydrogenated marine oils is, to a large extent, derived from the increased chain-shortening capacity of erucic acid in liver.