Earl Shrago

The inhibition of adenine nucleotide translocase activity by oleoyl CoA and its reversal in rat liver mitochondria.

The inhibitory effect of oleic acid on the 32Pi-ATP exchange activity and 14C-ADP translocation through the mitochondrial membrane can be reversed by substrates preferentially utilizing CoA and thereby preventing acylation of the fatty acid. By contrast the inhibition produced by oleoyl CoA can be reversed only when carnitine is added to augment oxidation of the CoA ester. The slow rate of respiration and sluggish response to ADP in oleoyl CoA treated mitochondria is overcome by the uncoupler, salicyl-anilide XIII. Inhibition of translocation of adenine nucleotides through the inner mitochondrial membrane by oleoyl CoA could regulate metabolism by inducing a transition from state 3 to state 4 respiration.

Comparative aspects of lipogenesis in mammalian tissues

Abstract Human adipose tissue, unlike that of the rat, has a poor capacity to synthesize fatty acids de novo. The citrate cleavage enzyme is essentially absent from human adipose tissue, and other related lipogenic enzymatic activities are considerably lower and less adaptive than in rat epididymal fat. Studies carried out with monkey liver indicate, indirectly at least, a closer relationship of primate and rat liver. Comparison of lipogenesis in a number of animals indicates that the rat may be a poor experimental model to study the carbon pathway and regulation of lipogenesis.

Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography

Long-chain acyl-CoA esters were extracted from freeze-clamped livers of fed and fasted rats according to the method of Mancha et al. [M. Mancha, G. B. Stokes, and P. K. Stumpf (1975) Anal. Biochem. 68, 600-608] and analyzed on a radially compressed C18, 5 microns, reverse-phase column using a gradient system consisting of acetonitrile and 25 mM KH2PO4, pH 5.3, at 254 nm. Total analysis time was 25 min. Eight peaks in the extract with carbon chain lengths of 12 to 18, which subsequently disappeared on alkaline hydrolysis, were identified. The major acyl-CoA peaks in the extract in order of increasing retention times were 14:0, 16:1, 18:2, 16:0, 18:1, and 18:0. Total liver long-chain acyl-CoA esters were 108 +/- 11 and 248 +/- 19 nmol/g protein for fed and fasted rats, respectively. On fasting (48 h) the levels of 18:2, 16:0, and 18:1 increased two-to threefold and that of 18:0 sixfold. The advantages of this method are that it not only provides a more direct determination of total tissue long-chain acyl-CoA esters, in that no decomposition of the CoA ester is involved, but it also detects the constituent molecular species.

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO2 and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.

Age-related changes in respiration coupled to phosphorylation. II. cardiac mitochondria☆

Age-related changes in mitochondrial adenine nucleotide metabolism may underlie the progressive decline in cardiac function. Oxidase activity coupled with phosphorylation, adenine nucleotide translocase (AdNT) activity, adenine nucleotide pool size and membrane lipid composition were determined using cardiac mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. While an age-associated 15% decrease in respiratory activity was not significant, AdNT activity of the aged rat was 20% lower (P less than 0.05) than that of the young rat. The exchangeable matrix adenine nucleotide pool (ATP + ADP) tended to decrease with age. In comparison to the young, membrane lipids of cardiac mitochondria from aged rat had a 43% higher (P less than 0.01) cholesterol/phospholipid-Pi ratio and a significantly lower (P less than 0.01) phosphatidyl ethanolamine/phosphatidyl choline ratio. The overall change in the fatty acid pattern of mitochondrial membrane lipids resulted in a significant (P less than 0.01) decrease in the n-6/n-3 fatty acid ratio. All values obtained for the mature rat fell between those of the young and aged rats. These data suggest that the reduced cardiac AdNT activity in the aged rat is a consequence of both a diminished pool of exchangeable adenine nucleotides and a lower AdNT velocity. Age-related changes in the lipid components of the membrane matrix in which the AdNT is embedded may underlie the decrease in respiratory activity.

Impact of lemongrass oil, an essential oil, on serum cholesterol

To test the hypothesis that non-sterol mevalonate pathway end products lower serum cholesterol levels, we asked 22 hypercholesterolemic subjects (315±9 mg cholesterol/dl) to take a daily capsule containing 140 mg of lemongrass oil, an essential oil rich in geraniol and citral. The paired difference in serum cholesterol levels of subjects completing the 90-day study approached significance (P<0.06, 2-tailed t-test). The subjects segregated into two groups, one consisting of 14 subjects resistant to the protocol and the other consisting of 8 subjects who responded. Paired differences in cholesterol level at 30, 60 and 90 d for resistant subjects were +2±6, +2±7 and −1±6 mg/dl; paired differences for the responding subjects were −25±10 (p<0.05), −33±8 (p<0.01) and −38±10 (p<0.025), respectively. The paired difference (+8±4) in the cholesterol levels of six responders 90 days after the discontinuation of lemongrass oil was not significant.

Characterization and purification of fatty acid-binding protein in rat and human adipose tissue.

A protein with properties similar to fatty acid-binding protein has been isolated from rat and human adipose tissue. Comparison of fatty acid-binding protein from rat liver and adipose tissue and human adipose tissue shows that all have approximately similar molecular weights. Immunologically, rat liver fatty acid-binding protein is similar to the protein characterized from rat adipose tissue. In isolated rat fat cells the fatty acid-binding protein was demonstrated to be involved in the uptake and esterification of long-chain fatty acids. These observations constitute evidence for a potential role of this protein in the fatty acid metabolism of adipocytes.

Studies on pyruvate carboxylase in rat and human liver

Abstract Pyruvate carboxylase in rat and human liver was assayed by a sensitive and specific [2-14C]pyruvate-oxaloacetate exchange reaction. The final product [2-14C]-aspartate was identified by high-voltage electrophoresis and autoradiography. Studies on the subcellular distribution of pyruvate carboxylase indicated that the majority, if not the entire enzyme activity, was located in the mitochondrial fraction of both rat and human liver. Phosphoenolpyruvate carboxykinase was identified in both the mitochondrial and cytosol fractions of human liver which is in contrast to the specific cytosol location in rat liver. The activity of pyruvate carboxylase in rat-liver mitochondria and cytosol was not influenced by the production of alloxan diabetes.

Decreases in serum high-density-lipoprotein cholesterol and total cholesterol resulting from naturally produced and recombinant DNA-derived leukocyte interferons☆

A three-phase study was conducted to examine the effect of leukocyte interferon administration on serum high-density-lipoprotein (HDL) cholesterol and total cholesterol levels. In the initial phase, human leukocyte interferon decreased HDL cholesterol (P less than 0.05) and total cholesterol (P less than 0.05) levels in patients with breast carcinoma. Decreases began with initiation of the interferon administration, were sustained throughout the period of treatment, and increased toward pretreatment values with discontinuation of interferon. In the second phase of the study, in neither of two comparison groups of women receiving cytotoxic chemotherapy, excluding interferon, did any similar decrease in HDL cholesterol occur. In a third comparison group of women being treated for metastatic breast carcinoma, a predicted and significant (P less than 0.01) drop in HDL cholesterol level without a concomitant lowering of total cholesterol level occurred immediately following the initiation of androgen therapy. To confirm that the observed cholesterol level decreases were associated with interferon rather than a contaminant thereof, analyses were also carried out on samples from a study utilizing interferon rather than a contaminant thereof, analyses were also carried out on samples from a study utilizing interferon produced by recombinant DNA techniques and purified to homogeneity. A similar decrease in HDL cholesterol (P less than 0.05) and total cholesterol (P less than 0.01) was observed. A definite relationship therefore appears to exist between the administration of human leukocyte interferon and decreased plasma levels of HDL cholesterol and total cholesterol.