Borgar Borrebaek

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 The cells again take up carnitine when it is added to the medium (Christiansen and Bremer, unpublished results).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] .

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.

Formation of creatine phosphate from creatine and 32P-labelled ATP by isolated rabbit heart mitochondria.

Abstract With ATP [γ- 32 P] we have demonstrated directly that mitochondrial creatine phosphokinase catalyzes the formation of large amounts of creatine phosphate with mitochondria generated ATP as substrate rather than added extramitochondrial ATP.

Fatty acid products of peroxisomal β-oxidation

Abstract 1. 1. Fatty acid products from the peroxisomal oxidation of [U- 46 C]palmitoyl-CoA and [10- 14 C]oleoyl-CoA have been detected. Products of the first 2–3 β-oxidation cycles predominate. 2. 2. Although NAD is associated with peroxisomal fatty acid oxidation as an obligatory cofactor, it is shown that also NADP can support some peroxisomal oxidation with these two acyl-CoA esters. 3. 3. Peroxisomal preparations have been found to contain appreciable palmitoyl-CoA hydrolase (EC 3.1.2.-) activity, as well as hydrolase activities towards other acyl-CoA esters. Unlike β-oxidation, peroxisomal hydrolase activities are not influenced by clofibrate treatment. A possible role of these hydrolases in controlling the extent of peroxisomal acyl-CoA oxidation is discussed.

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.

In vivo induction of 4-enoyl-CoA reductase by clofibrate in liver mitochondria and its effect on pent-4-enoate metabolism

Abstract Feeding of clofibrate to male rats leads to a 4–7 fold increase in the activity of the 4-enoyl-CoA reductase in the liver. Concomitantly the inhibition of fatty acid oxidation by pent-4-enoate is abolished, and an increased glucose formation in the presence of pent-4-enoate is observed. It is suggested that pent-4-enoate is converted to propionyl-CoA via the reaction sequence pent-4-enoyl-CoA→pent-2,4-dienoyl-CoA→pent-2-enoyl-CoA→propionyl-CoA + acetyl-CoA.

Adaptable hexokinase activity in epididymal adipose tissue studied in vivo and in vitro.

Abstract 1. 1. The effect of dietary carbohydrate (glucose) on rat epididymal adipose tissue hexokinase activity was studied. When fasted rats were refed a high carbohydrate diet ad libitum , hexokinase activity increased slowly until a maximal value was reached about 36 h after the start of refeeding. This increase in enzyme activity was blocked in animals receiving intraperitoneal injections of puromycin. 2. 2. Increased hexokinase activity was observed during incubations of epididymal fat pads in vitro when either glucose or insulin was present in the incubation medium. Additive effects were observed when glucose and insulin were present together in the incubation medium. The effect in vitro of glucose as well as that of insulin could also be obtained in fat pads from alloxan-diabetic rats. It is concluded that glucose and insulin separately initiate an increase in hexokinase activity. 3. 3. The effects of glucose and insulin on hexokinase activity in vitro were blocked by the addition of either puromycin, actinomycin D, or p -fluorophenylalanine to the incubation medium. This indicates that these effects are the results of an increased net balance between hexokinase synthesis and degradation. It was observed that glucose, but not insulin, protects the hexokinase activity in situ .

Regulation of palmitate esterification/oxidation by glucagon in isolated hepatocytes: The role of α-glycerophosphate concentration

Lipolysis was measured as the disappearance of [3H]glycerol previously incorporated into triacylglycerol, diacylglycerol and phosphatidic acid. There was no effect of glucagon on the lipolysis of any of these lipids. A transient increase in cellular alpha-glycerophosphate was induced by addition of glycerol during incubation. This resulted in an immediate and temporary decrease in oxidation and increase in esterification of palmitate while the uptake of palmitate from the incubation medium was unchanged. The change in alpha-glycerophosphate was also correlated with a transient drop in acyl-CoA and acylcarnitine. The lactate/pyruvate ratio was increased by the glycerol addition, but was still elevated for some while after the transient change in alpha-glycerophosphate. Similar effects were obtained by addition of dihydroxyacetone instead of glycerol. It is concluded that fatty acid esterification/oxidation can be changed by variations in the concentration of alpha-glycerophosphate, and that glucagon acts on lipid metabolism by decreasing the level of this metabolite.

Determination of malonyl-coenzyme A in rat heart, kidney, and liver: a comparison between acetyl-coenzyme A and butyryl-coenzyme A as fatty acid synthase primers in the assay procedure

The malonyl-CoA assay was nonlinear at low malonyl-CoA concentrations when labeled acetyl-CoA was used as fatty acid synthase primer. Linearity was obtained with low concentrations of both fatty acid synthase and labeled acetyl-CoA, but then the assay was disturbed by the diluting effect of endogenous acetyl-CoA. The problems of nonlinearity and dilution of radioactivity by endogenous compounds were absent when labeled butyryl-CoA was used as primer. The levels of malonyl-CoA in rat heart, kidney, and liver were determined. The use of butyryl-CoA gave higher values of malonyl-CoA.

Arsenite inhibits β-oxidation in isolated rat liver mitochondria

Abstract A partial inhibition of acylcarnitine oxidation by arsenite in rat liver mitochondria has been studied. This inhibition is confined to the thiolase(s). The inhibition was observed also in the presence of malate, indicating no selective effect on ketogenesis. Ketogenesis from acetyl-CoA was also inhibited by arenite. Mitochondrial CoA was acylated by acylcarnitine nearly as rapidly in the presence of arsenite as in its absence. Thus, arsenite did not interfere with the availibility of CoA in the mitochondria. No effect of arsenite on enzymes of β-oxidation other than the thiolase(s) was observed. When arsenite and acylcarnitine were added simultaneously to mitochondria, there was a delay before maximal inhibition of oxygen uptake occurred. When the mitochondria were preincubated with arsenite before addition of acylcarnitine, the inhibitory effect on oxygen utilization was initially large, but then partially repealed. Similar time delays were observed in the activity of acetoacetyl-CoA thiolase of disrupted mitochondria depending on the sequence of arsenite and acetoacetyl-CoA addition. It is suggested that substrate and arsenite compete for the reactive sulfhydryl group at the active site of the thiolase(s).