Effects of Parathyroid Hormone on Skeletal Muscle Protein and Amino Acid Metabolism in the Rat

Because prominent skeletal muscle dysfunction and muscle wasting are seen in both chronic uremia and in primary hyperparathyroidism, and because markedly elevated parathyroid hormone levels occur in both disorders, potential effects of parathyroid hormone on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism were studied in vitro using isolated intact rat epitrochlearis skeletal muscle preparations. Intact bovine parathyroid hormone and the synthetic 1-34 fragment of this hormone stimulated the release of alanine and glutamine from muscle of control but not from chronically uremic animals. This stimulation was dependent upon the concentration of parathyroid hormone added: At 10⁵ ng/ml parathyroid hormone increased alanine release 84% and glutamine release 75%. Intracellular levels of alanine and glutamine were not altered by parathyroid hormone. Increasing concentrations of the 1-34 polypeptide decreased [³H]leucine incorporation into protein of muscles from both control and uremic animals. Using muscles from animals given a pulse-chase label of [guanido-¹⁴C]arginine in vivo, parathyroid hormone increased the rate of loss of ¹⁴C label from acid-precipitable protein during incubation and correspondingly increased the rate of appearance of this label in the incubation media. Parathyroid hormone increased muscle cAMP levels by 140% and cGMP levels by 185%, but had no effect on skeletal muscle cyclic nucleotide phosphodiesterase activities as assayed in vitro. Adenylyl cyclase activity in membrane preparations from control but not uremic rats was stimulated by parathyroid hormone in a concentration-dependent fashion. However, no stimulation of guanylyl cyclase activity was noted by parathyroid hormone, although stimulation by sodium azide was present. Incubation of muscles with added parathyroid hormone produced a diminished responsiveness towards epinephrine or serotonin regulation of amino acid release and cAMP formation in the presence compared to the absence of parathyroid hormone. In the absence of parathyroid hormone, detectable inhibition of alanine and glutamine release was produced by 10⁻⁹ M epinephrine, whereas in the presence of parathyroid hormone (1,000 ng/ml) inhibition of alanine and glutamine release required 10⁻⁶ M or greater epinephrine. Resistance to cyclic AMP action as well as inhibition of cyclic AMP formation by parathyroid hormone was found. Preincubation of rat sarcolemma with 1-34 parathyroid hormone produced a decreased activity of the isoproterenol-stimulable adenylyl cyclase activity but there was no apparent change in the concentration of isoproterenol required for one-half maximal and maximal stimulation of the enzyme.     

The Regulation of Skeletal Muscle Alanine and Glutamine Formation and Release in Experimental Chronic Uremia in the Rat: SUBSENSITIVITY OF ADENYLATE CYCLASE AND AMINO ACID RELEASE TO EPINEPHRINE AND SEROTONIN

The mechanism of the increased alanine and glutamine formation and release from skeletal muscle in experimental uremia was investigated using epitrochlearis preparations from control and chronically uremic rats. In uremic muscle, insensitivity to epinephrine or serotonin suppression of alanine and glutamine release was observed. With control muscles, 1 nm or greater, epinephrine inhibited alanine and glutamine release, whereas with uremic muscles, epinephrine concentrations <1 muM did not alter amino acid release. Decreased alanine and glutamine release with 1 nM serotonin was observed in control muscles, but no inhibition was observed with concentrations <1 muM in uremic muscle. Muscle amino acid levels were the same in control and uremic muscles in the presence or absence of epinephrine or serotonin. The reutilization of released alanine by protein synthesis or oxidation to CO(2) was not differentially affected by epinephrine in uremic muscles as compared with control muscle. Dibutyryl-cAMP inhibited amino acid release equally in uremic and control muscles. Epinephrine or serotonin increased cAMP levels two- to four-fold or more in control than in uremic muscle. Basal- and fluoride-stimulated adenylate cyclase activities were equal in uremic and control muscle homogenates and in membrane fractions, but 10 muM epinephrine-stimulated adenylate cyclase was reduced 30-60% with uremia. At any concentration of epinephrine (0.001-100 muM), the stimulation of membrane adenylate cyclase activity was one- to twofold greater with control membranes than with uremic muscle membranes. With either control or uremic muscle, peak adenylate cyclase activity was observed at 1 muM epinephrine.These data indicate that skeletal muscle in chronic uremia acquires an insensitivity to the metabolic action of epinephrine or serotonin. This insensitivity may be attributable in part to the diminished increments in muscle cAMP levels produced by adrenergic and serotonergic agonists. The decreased cAMP levels may derive in turn from a decreased activity or subsensitization of the agonist-stimulated adenylate cyclase in uremic muscle.     

The Impact of Streptozotocin-induced Diabetes Mellitus on Cyclic Nucleotide Regulation of Skeletal Muscle Amino Acid Metabolism in the Rat

The impact of diabetes on cyclic nucleotide-associated mechanisms regulating skeletal muscle protein and amino acid metabolism was assessed using epitrochlaris preparations from streptozotocin-induced diabetic rats. 1 nM epinephrine inhibited alanine and glutamine release from control preparations, but no inhibition was observed from diabetic preparations with <0.1 mM. 10 nM epinephrine stimulated lactate production from control muscle but stimulation in diabetic preparations was observed only at 0.1 mM. Serotonin inhibited amino acid release and stimulated lactate production equally in control and diabetic muscle. 0.1 mM epinephrine increased cyclic (c)AMP levels by 360% in control muscles, but these levels were increased only 83% in diabetic muscle. Basal-, fluoride-, and serotonin-stimulated adenylyl cyclase activities were equal in membrane preparations of diabetic and control muscle, but epinephrine-stimulated adenylyl cyclase was reduced by 60% in diabetic muscle. Carbamylcholine stimulation of alanine and glutamine release was blunted in diabetic preparations. Carbamylcholine increased cGMP levels in control but not in diabetic muscle. In diabetic muscle, guanylyl cyclase activity was 65% of control and the stimulation of cyclase activity by sodium azide was less in diabetic than control preparations. Added cGMP stimulated alanine and glutamine release from control, but not from diabetic muscle. These data suggest a loss of adrenergic and cholinergic responsiveness in diabetic muscle. Because amino acid release also showed a decreased responsiveness to added cAMP and cGMP, the presence of other derangements in the mechanism(s) of cyclic nucleotide regulation of muscle amino acid metabolism also seems likely.     

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells: SELECTIVE INHIBITION BY CLOFIBRIC ACID

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-¹⁴C]leucine to ¹⁴CO₂ or to the [1-¹⁴C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of ¹⁴CO₂ from [1-¹⁴C]leucine was saturable with a K_m = 6.3 mM and a maximum oxidation rate (V_max) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-¹⁴C]leucine (K_i = 0.85 mM) and [U-¹⁴C]isoleucine, but had no effect on the oxidation of [U-¹⁴C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-¹⁴C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of ¹⁴CO₂ and had relatively little effect on the production of [1-¹⁴C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD⁺ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.     

Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man.

To compare glutamine and alanine as gluconeogenic precursors, we simultaneously measured their systemic turnovers, clearances, and incorporation into plasma glucose, their skeletal muscle uptake and release, and the proportion of their appearance in plasma directly due to their release from protein in postabsorptive normal volunteers. We infused the volunteers with [U-14C] glutamine, [3-13C] alanine, [2H5] phenylalanine, and [6-3H] glucose to isotopic steady state and used the forearm balance technique. We found that glutamine appearance in plasma exceeded that of alanine (5.76 +/- 0.26 vs. 4.40 +/- 0.33 mumol.kg-1.min-1, P < 0.001), while alanine clearance exceeded glutamine clearance (14.7 +/- 1.3 vs. 9.3 +/- 0.8 ml.kg-1.min-1, P < 0.001). Glutamine appearance in plasma directly due to its release from protein was more than double that of alanine (2.45 +/- 0.25 vs. 1.16 +/- 0.12 mumol.kg-1.min-1, P < 0.001). Although overall carbon transfer to glucose from glutamine and alanine was comparable (3.53 +/- 0.24 vs 3.47 +/- 0.32 atoms.kg-1.min-1), nearly twice as much glucose carbon came from protein derived glutamine than alanine (1.48 +/- 0.15 vs 0.88 +/- 0.09 atoms.kg-1.min-1, P < 0.01). Finally, forearm muscle released more glutamine than alanine (0.88 +/- 0.05 vs 0.48 +/- 0.05 mumol.100 ml-1.min-1, P < 0.01). We conclude that in postabsorptive humans glutamine is quantitatively more important than alanine for transporting protein-derived carbon through plasma and adding these carbons to the glucose pool.     

Skeletal muscle glutamine production in thermally injured rats

1. The effect of thermal injury (33-35% of body surface area) on the regulation of glutamine metabolism was studied in skeletal muscles of rats 7 days after injury. 2. Injury increased the rates of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 3. Injury produced decreases in the concentrations of skeletal muscle glutamine (36%, P less than 0.001), glutamate (39%, P less than 0.001), alanine (24%, P less than 0.001), pyruvate (35%, P less than 0.001), 2-oxoglutarate (51%, P less than 0.001) and adenosine 5'-triphosphate (38%, P less than 0.001). The concentrations of ammonia (42%, P less than 0.001) and inosine 5'-phosphate (430%, P less than 0.001) were increased. 4. The maximal activity of glutamine synthetase was increased (22-40%, P less than 0.001) in muscles of injured rats, whereas that of glutaminase was unchanged. 5. Hindlimb blood flow decreased by approximately 15% in injured rats, which was accompanied by an enhanced net release of glutamine (80%, P less than 0.001) and alanine (44%, P less than 0.001). 6. It is concluded that there is an enhanced rate of release of both glutamine and alanine from skeletal muscle of thermally injured rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.     

Inhibition of muscle glutamine formation in hypercatabolic patients.

Glutamine is synthesized primarily in skeletal muscle, and enables transfer of nitrogen to the liver, as well as serving other functions. There is increasing evidence for beneficial clinical effects of glutamine supplementation in critically ill patients. However, the response of endogenous glutamine formation to severe stress is poorly understood. The rates of net protein balance, leucine oxidative decarboxylation, and alanine and glutamine synthesis de novo were determined in leg skeletal muscle of 20 severely burned patients and 19 normal controls in the post-absorptive state. Patients were studied at 14+/-5 days post-burn, and their mean burn size was 66+/-18% of total body surface area. Methods were based on the leg arteriovenous balance technique in combination with biopsies of the vastus lateralis muscle. In the post-absorptive state, patients with severe burns, as compared with healthy control subjects, exhibited accelerated muscle loss (+150%) (i.e. proteolysis minus synthesis) and leucine oxidative decarboxylation (+117%), and depletion of the intramuscular free glutamine pool (-63%). The average rate of glutamine synthesis de novo was decreased by 48%, whereas net alanine synthesis de novo was increased by 174%, in skeletal muscle of burned patients. In conclusion, in severely hypercatabolic burned patients, muscle glutamine formation was suppressed, whereas alanine was the major vehicle for inter-organ nitrogen transport. These changes account for a decreased glutamine availability during prolonged severe stress.     

Branched-chain Amino Acid Nitrogen Transfer to Alanine In Vivo in Dogs: DIRECT ISOTOPIC DETERMINATION WITH [15N]LEUCINE

To investigate the contribution of branched-chain amino acids as a nitrogen source for alanine in vivo, dogs were infused with l-[¹⁵N]leucine, l-[U-¹⁴C]leucine, l-[2,3,3,3-²H₄]alanine, and d-[6,6-²H₂]-glucose. ¹⁴C and ¹⁵N isotopic equilibrium in plasma leucine, and deuterium enrichment in arterial and femoral plasma glucose and alanine were achieved within 3 h of initiation of the respective isotope infusion in all animals. The average flux of leucine determined by [¹⁵N]leucine was 5.4 μmol·kg⁻¹·min⁻¹, whereas using [¹⁴C]leucine it was 3.7 μmol·kg⁻¹·min⁻¹. Turnover rates for alanine and glucose were 11.0 and 17.2 μmol·kg⁻¹·min⁻¹, respectively.     

Glutamine Transport in Rat Kidney Mitochondria in Metabolic Acidosis

In order to study factors regulating renal ammoniagenesis, the transport and metabolism of L-glutamine were studied in mitochondria from kidneys of control and acidotic rats. On incubation in 1 mM [¹⁴C]glutamine, there was production and accumulation of [¹⁴C]glutamate within the matrix space. However no [¹⁴C]glutamine was detected in the matrix space, even with 10 mM [¹⁴C]glutamine as substrate or with inhibition of glutamine deamidation (low temperature, p-chloromercuribenzoate, mersalyl). These results suggest that glutamine crosses the inner membrane by a carrier-mediated step and that this step is rate-limiting in glutamine deamidation.     

Concentration of Myo-inositol in Skeletal Muscle of the Rat Occurs without Active Transport

The cellular uptake of nonphosphorylated myo-inositol (MI) and its incorporation into phosphoinositide in the rat epitrochlearis muscle was measured. Cellular uptake of [2-³H]MI was determined by the difference between total uptake and [2-³H]MI present in the extracellular fluid determined with [1-¹⁴C]mannitol. Cellular uptake was parabolic and directly proportional to medium MI concentrations between 25 and 3,200 μM. Saturation of a MI carrier was not evident. Moreover, uptake was not inhibited by 2 mM ouabain, 0.3 mM 2,4-dinitrophenol, or 22 mM glucose. Insulin, 100 mU/ml, was without effect on either cellular uptake of [2-³H]MI or its incorporation into phosphoinositides. In muscles that were preloaded with [2-³H]MI and then incubated in media that contained a constant amount of MI but no [2-³H]MI, 44.3% of the [2-³H]MI was released after 10 min increasing to 62.5% by 120 min. Cellular MI concentrations were 0.18 μmol/g wet tissue (four times plasma levels) in rapidly isolated and frozen epitrochlearis muscle. When muscle was incubated without MI, 48% of endogenous MI was lost rapidly. Restoration of cellular MI in 50 μM MI media occurred in two phases, a rapid uptake phase lasting 10 min and a subsequent slow phase of MI uptake.     

The separate and combined effect of leucine and insulin on muscle free amino acids

Summary. The effect of insulin and leucine on amino acid and protein metabolism in muscle is not fully understood. To characterize their separate and combined effects on free amino acids in muscle and plasma, 11 volunteers received an infusion of either leucine (1 g h^-1, Group 1) or glucose (20 g h^-1, Group 2) for 2 h followed by a combination of the two infusions for an additional 2-h period. In muscle both the leucine infusion and the leucine plus glucose infusion increased the concentration of free leucine significantly, while the sum of the other branched chain amino acids (BCAA), of the aromatic amino acids and of the basic amino acids decreased. Glucose infusion alone decreased the sum of the essential amino acids, the BCAA and the aromatic amino acids. The combination of leucine and glucose augmented the decreases, while the concentrations of glutamate, glutamine and alanine were unaffected. In plasma the leucine infusion doubled the leucine concentration and decreased alanine, valine, methionine, tyrosine, phenylalanine and the sum of the aromatic amino acids. Glucose infusion decreased methionine, serine, isoleucine and the sum of the essential amino acids and of the BCAA. The combination of leucine infusion and hyperinsulinaemia augmented the decreases. The plasma concentrations of the keto acids of valine and isoleucine decreased by the leucine infusion while the concentrations of the keto acid of leucine and isoleucine decreased by glucose infusion. The combination of leucine and glucose had an additive effect. These effects are attributed to a specific effect of leucine on the other two BCAA and a depression of muscle proteolysis by both leucine and insulin, resulting from glucose infusion.     

Leucine. A possible regulator of protein turnover in muscle.

Incorporation of radiolabeled precursors into muscle proteins was studied in isolated rat hemidiaphragms. A mixture of three branched-chain amino acids (0.3 mM each) added to media containing glucose stimulated the incorporation of [14C]lysine into proteins. When tested separately, valine was ineffective, isoleucine was inhibitory, but 0.5 mM leucine increased the specific activity of muscle proteins during incubation with [14C]lysine or [14C]acetate in hemidiaphragms from fed or fasted rats incubated with or without insulin. Preincubation with 0.5 mM leucine increased the specific activity of muscle proteins during a subsequent 30- or 60-min incubation with [14C]lysine or [14C]pyruvate without leucine. Preincubation with other amino acids (glutamate, histidine, methionine, phenylalanine, or tryptophan) did not exert this effect. When hemidiaphragms were incubated with a mixture of amino acids at concentrations found in rat serum and a [14C]lysine tracer, the specific activity of muscle proteins increased when leucine in the medium was raised from 0.1 to 0.5 mM. Experiments with actinomycin D and cycloheximide suggested that neither RNA synthesis nor protein synthesis are required for the initiation of the leucine effect. Leucine was not effective when added after 1 h preincubation without leucine. The concentration of lysine in the tissue water of diaphragms decreased during incubation with 0.5 mM leucine in the presence or absence of cycloheximide, suggesting that leucine inhibited protein degradation. During incubation with [3h]tyrosine (0.35 mM) the addition of 0.5 mM leucine increased the specific activity of muscle proteins, while the specific activity of intracellular tyrosine remained constant and its concentration decreased, suggesting that leucine also promoted protein synthesis. The concentration of leucine in muscle cells or a compartment thereof may play a role in regulating the turnover of muscle proteins and influence the transition to negative nitrogen balance during fasting, uncontrolled diabetes, and the posttraumatic state. Leucine may play a pivotal role in the protein-sparing effect of amino aicds.     

Leucine Oxidation and Protein Turnover in Clofibrate-induced Muscle Protein Degradation in Rats

Treatment of hyperlipidemia with clofibrate may result in development of a muscular syndrome. Our previous investigation (1979. J. Clin. Invest.64: 405.) showed that chronic administration of clofibrate to rats causes myotonia and decreases glucose and fatty acid oxidation and total protein of skeletal muscle. In the present experiments we have investigated amino acid and protein metabolism in these rats. Clofibrate administration decreased the concentration of all three branched-chain amino acids without affecting those of others in muscle. Studies to examine the mechanism of decreases in muscle concentrations of branched-chain amino acids showed the following: (a) Plasma concentration of leucine was decreased, whereas there was no significant change in the concentration of isoleucine and valine. (b) Liver concentrations of all three branched-chain amino acids remained unaltered. (c) The uptake of cycloleucine (a nonmetabolizable analogue of leucine) by both muscle and liver was unaffected. (d) The percentage of a trace amount of injected [1-¹⁴C]leucine expired as ¹⁴CO₂ in 1 h was significantly increased. (e) The capacity of muscle homogenate for α-decarboxylation of leucine was enhanced, whereas that of liver was unaffected. (f) The activity of leucine transaminase was unaffected, whereas that of α-ketoisocaproate dehydrogenase was increased in muscle.     

The Dyggve-Melchior-Clausen syndrome.

Metabolism of mucopolysaccharides and glycoproteins in the lymphocytes of three patients suffering from the Dyggve-Melchior-Clausen syndrome was studied using radioactive precursors. [³⁵S] Sulphate, d-[1-¹⁴C]galactosamine, l-[U-¹⁴-C]leucine and l-[1-¹⁴C]fucose were continuously followed for incorporation in lymphocytes of the patients. The activity of the latter three precursors rose steadily in the patients' lymphocytes up to a period of 92 h unlike normal lymphocytes where equilibrium between incorporation and degradation was reached within 20–40 h. The sulphate incorporation in patients' lymphocytes, however, reached an equilibrium state after 48 h. Furthermore the incorporation of l-[U-¹⁴C]leucine in mixed lymphocyte culture (equal number of patients' and normal lymphocytes) was parallel to that in normal lymphocytes. These results may indicate a defect in glycoprotein-mucopolysaccharide metabolism in the patients which was corrected in mixed lymphocyte cultures.Urine samples from the patients were found to contain higher amounts of acid mucopolysaccharides (AMPS) compared with normal subjects. Patients urinary AMPS were rich in protein, sialic acid, hexosamine, N-sulphated hexosamine and reducing sugar while their sulphate content was comparable to that from normal subjects. Fractionation of urinary AMPS on Dowex columns revealed that they were not enriched in any single mucopolysaccharide. Excess protein was found to be present in hyaLuronic acid, chondroitin sulphate and keratan sulphate fractions. Furthermore, heparan sulphate fractions were undersulphated.Considering the present data, deficiency of a specific sulphatase and/or a protease responsible for breaking glycoprotein-mucopolysaccharide linkage of proteoglycans may be suggested as the cause of the Dyggve-Melchior-Clausen syndrome.     

Aging affects cellular zinc and protein synthesis in the femoral diaphysis of rats

To clarify the effect of aging on bone metabolism, alteration of the cellular zinc content and protein synthesis was examined in the femoral diaphysis of 3- and 30-week-old male rats. The cellular zinc content in bone tissue markedly decreased in 30-week-old compared to 3-week-old rats. When the bone tissue from older rats were cultured with [³H]leucine, incorporation of [³H]leucine into the acid-insoluble residues was less than for weanling rats. This decrease was partly restored by the oral administration of zinc sulfate (0.5, 1.0, and 2.0 mg Zn/100 g body weight) to elderly rats for 3 days. An increase of in vitro [³H]leucine incorporation by bone tissues obtained from the rats that had received zinc (2.0 mg/100 g) was blocked by cycloheximide (10⁻⁶ M) or dipicolinate (10⁻³ M), a chelator of zinc. These results suggest that bone protein synthesis declines with age, and that this decline may be based partly on the decrease in bone cellular zinc.     

Leucine meal increases glutamine and total nitrogen release from forearm muscle.

To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals (14.7 g) were consumed by six normal postabsorptive individuals. Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal. Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3.1 +/- 0.4 to 5.2 +/- 0.9 ml/100 ml forearm per minute (mean +/- SEM, P less than 0.005). Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise. Less marked changes (significant only at 90 min) in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted. Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles. More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle.     

Monounsaturated trans fatty acids,elaidic acid and trans-vaccenic acid,metabolism and incorporation in phospholipid molecular species in hepatocytes

The incorporation of [¹⁴C]elaidic acid (trans18 : 1(n-9)) in phosphatidylcholine and phosphatidylethanolamine molecular species in isolated rat liver cells has been studied, and the results compared with the incorporation, previously published (B. Woldseth et al. Biochim Biophys Acta 1993;1167:296-302), of [¹⁴C]palmitic acid (16 : 0) and [¹⁴C]stearic acid (18 : 0) and with that of [¹⁴C]oleic acid (cis18 : 1(n-9)). The pattern of incorporation in phospholipid molecular species is similar to that of [¹⁴C]stearic acid and different from that of [¹⁴C]palmitic acid. In phosphatidylcholine [¹⁴C]trans18 : 1-18 : 2 and [¹⁴C]trans18 : 1-20 : 4 were the most abundant species, and in phosphatidylethanolamine [¹⁴C]trans18 : 1-20 : 4 was the predominant species. With increasing concentration of [¹⁴C]elaidic acid increasing amounts of [¹⁴C]trans18 : 1-[¹⁴C]trans18 : 1 were found. The total incorporation in phospholipids was less than that of [¹⁴C]stearic acid, but more than that of [¹⁴C]palmitic acid. The distribution in percent of [¹⁴C]elaidic acid in phospholipid classes was 8.8% in phosphatidylinositol, 1.8% in phosphatidylserine, 59.1% in phosphatidylcholine and 30.3% in phosphatidylethanolamine with 0.1 & mmol l^-1 substrate concentration. More [¹⁴C]elaidic acid than [¹⁴C]palmitic acid or [¹⁴C]stearic acid was oxidized. The incorporation in phospholipids of [¹⁴C]elaidic acid was very different from that of [¹⁴C]oleic acid. The main species with [¹⁴C]oleic acid were 16 : 0-[¹⁴C]cis18 : 1 in phosphatidylcholine, and [¹⁴C]cis18 : 1-20 : 4 in phosphatidylethanolamine. In some experiments [¹⁴C]18 : 2(n-6) was incubated together with unlabelled elaidic or unlabelled trans-vaccenic acid (trans18 : 1(n-7)). In these experiments, more trans18 : 1-18 : 2 was formed from elaidic acid than from trans-vaccenic acid, especially in phosphatidylethanolamine.     

A Standardized Direct Method for Total Cholesterol Determination in Serum with a Combined Reagent

Abstract

A clonal strain of rat hepatoma cells (MH₁C₁) known to take up and conjugate bilirubin was incubated in media containing bilirubin and albumin in different molar ratios at a constant bilirubin concentration. The highest rate of bilirubin conjugation was found at a molar ratio of 1/1. Excess bilirubin markedly reduced both the conjugation of bilirubin and the incorporation of [¹⁴C]alanine, indicating a toxic effect on the cells. Excess albumin also depressed the formation of bilirubin conjugates, but incorporation of [¹⁴C]alanine proceeded at a normal rate, indicating that the reduced conjugation is probably due to stronger binding of bilirubin to albumin and reduced cellular uptake of bilirubin. Bilirubin bound to albumin at a molar ratio of 1/1 had no toxic effects on the cells as judged by cell morphology and conjugation capacity, even at a total bilirubin concentration as high as 680 μmol/l.     

Effect of Al-chlorophenoxyisobutyrate (Alufibrate) on pyruvate metabolism and on the fate of very low density lipoprotein lipids in hyperlipidemic patients

he exhalation of total CO₂ and ¹⁴ CQ₂ after an intravenous injection of either [1-¹⁴C]- or [2-¹⁴C]pyruvate was measured in six male patients with type IV hyperlipidemia who had previously been receiving treatment with alufibrate or placebo. Considerably more ¹⁴ CO₂ was exhaled after the injection of [1-¹⁴C]pyruvate than after the injection of [2-¹⁴C] pyruvate. Treatment with alufibrate only slightly diminished the oxidation of [1-¹⁴C]-pyruvate, but increased the oxidation of [2-¹⁴C] pyruvate to ¹⁴CO₂ by 25%. The total concentrations of [2-¹⁴C] pyruvate and its incorporation into the lipids of lipoprotein fractions were measured over 24 h. Alufibrate treatment decreased the serum concentration of VLDL by 53%, by reducing the turnover rate by 25% and increasing the net removal rate by about 30%. No effects on LDL and HDL lipid fractions were detected.     

Effects of Antibiotics on Metabolism of Peptidoglycan,Protein, and Lipids in Bifidobacterium bifidum subsp. pennsylvanicus

The formation of cell envelope components of Bifidobacterium bifidum subsp. pennsylvanicus was studied by measuring the incorporation of [³H]glycine, ¹⁴C-labeled fatty acids, and N-benzoyl-[¹⁴C]glucosamine into the membrane protein, membrane lipids, and cell wall peptidoglycan, respectively. Inhibition of peptidoglycan synthesis by antibiotics (penicillin G, vancomycin, d-cycloserine, and bacitracin) and by the omission of glucosamine-containing growth factors caused a marked decrease in glycine incorporation into cellular as well as membrane protein, which was accompanied by a considerable enhancement of fatty acid incorporation. The uncoupling of protein and lipid synthesis led to the release of marked amounts of lipids from the cell under these conditions. Arrestment of protein synthesis by antibiotics (chloramphenicol, tetracycline, and actinomycin D) decreased peptidoglycan and lipid synthesis only partially, but did not lead to lipid release. Mg²⁺ deficiency of the medium caused about 60% inhibition of growth and lipid synthesis, but protein synthesis and especially peptidoglycan synthesis were much less inhibited. Staphylococcin 1580 arrested the growth and also the synthesis of protein and peptidoglycan. However, the synthesis and turnover of lipids were considerably increased and a release of large amounts of lipids was observed. Peptidoglycan and cellular protein did not show any turnover either during normal growth or after the inhibition of cell wall and protein synthesis.