Kou Yi Tserng

Direct Inhibition of Mitochondrial Respiratory Chain Complex III by Cell-permeable Ceramide

Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor α and other agents on cell growth and differentiation. Ceramide is believed to actvia activation of protein phosphatase, proline-directed protein kinase, or protein kinase C. Tumor necrosis factor α-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C2-ceramide with half-maximum effect at 5–7 μm. In contrast,N-acetylsphinganine (C2-dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.

Metabolism of urea and glucose in normal and diabetic pregnancy

Quantitative estimation of rates of urea synthesis, urea and nitrogen excretion and glucose production were done in ten normal, seven insulin-dependent diabetic and three gestationally diabetic subjects, after an overnight fast during the third trimester of pregnancy. Studies were repeated on three normal, three insulin-dependent diabetic, and two gestationally diabetic subjects during postpartum. [15N2]urea and [6,6-2H2]glucose tracers were infused simultaneously at constant rate for five hours from 12–17 hr of fasting. Plasma [15N2]urea and [2H2] glucose enrichments were measured by gas chromatograph-mass spectrometry. Rates of urea synthesis and glucose production were estimated during steady state by tracer dilution. Overnight fast resulted in a decline in plasma glucose. Extension of the fast by another 5 hr resulted in a further decrease in the levels of circulating glucose and rise in the level of betahydroxybutyrate in both normal and diabetic pregnant subjects. There was no significant difference in the plasma concentration of amino acids after 12 hr of overnight fast, between the normal and diabetic subjects during antepartum. However, a significant increase in the plasma concentration of amino acids occurred during the postpartum period. The plasma urea nitrogen concentration was reduced by 60% during antepartum period (8.22 ± 2.24 and 11.49 ± 3.16 mg/dl; mean ± SD). There was a corresponding decrease in the rate of urea nitrogen synthesis (4.17 ± 1.28 and 6.84 ± 2.52 mg/kg hr). In addition, the rates of urinary urea nitrogen and total nitrogen excretion were attenuated during pregnancy. In contrast, the ammonia excretion rate remained unchanged (0.24 ± 0.09 versus 0.23 ± 0.12 mg/kg hr) in the antepartum and postpartum period. Rigorous regulation of diabetes in pregnancy resulted in normalization of urea and glucose metabolism after an overnight fast. The plasma glucose concentration was reduced during pregnancy, while the rate of glucose production expressed per unit body weight was unchanged. The decreased urea synthesis rate during pregnancy resulted in an altered relationship between urea and glucose production rates. These data suggest conservation of amino nitrogen during pregnancy in humans, apparently for tissue protein synthesis. As ureogenesis is a concomitant of gluconeogenesis from amino acids, these data further suggest an attenuated hepatic gluconeogenesis following a brief 17 hr fast. The mechanism of these observations appears to be via decreased delivery of substrate to the liver for ureogenesis possibly as a result of diversion to the fetus.

Dissociation between gene and protein expression of metabolic enzymes in a rodent model of heart failure

Studies in advanced heart failure show down‐regulation of fatty acid oxidation genes, possibly due to decreased expression of the nuclear transcription factors peroxisome proliferator activated receptor α (PPARα) and retinoid X receptor α (RXRα). We assessed mRNA and protein expression of PPARα and RXRα, and for several PPAR/RXR regulated metabolic proteins at 8 and 20 weeks following myocardial infarction induced by coronary artery ligation. Infarction resulted in heart failure, as indicated by reduced LV fractional shortening and increased end diastolic area compared to sham. There was a progressive increase in LV end systolic area, myocardial ceramide content and atrial natriuretic peptide mRNA, and a deterioration in LV fractional area of shortening from 8 to 20 weeks. Protein and mRNA expression of PPARα and RXRα were not different among groups. The mRNA for PPAR/RXR regulated genes (e.g. medium chain acyl‐CoA dehydrogenase (MCAD)) was down‐regulated at 8 and 20 weeks post‐infarction; however, neither the protein expression nor activity of MCAD was reduced compared to sham. In conclusion, reduced mRNA expression of PPAR/RXR regulated genes is not dependent on reduced PPAR/RXR protein expression.

Glucose-alanine relationship in normal human pregnancy.

In order to quantify the glucose-alanine relationship in normal human pregnancy, the turnover rates of alanine and the incorporation of alanine carbon into glucose were quantified in 15 pregnant women during the last 4 weeks of gestation following a ten-hour fast. Eight nonpregnant women of similar age group were studied as controls. L-[2,3-13C2]Alanine and D[6,6-2H2]glucose were infused as tracers. The 13C enrichment of alanine, lactate, and glucose and the deuterium enrichment of glucose were measured by gas chromatography-mass spectrometry. In five pregnant and five nonpregnant women, the contribution of alanine carbon to expired CO2 directly and via glucose was estimated by combining indirect respiratory calorimetry with the tracer infusions. The alanine turnover rates in the pregnant and nonpregnant women were similar (pregnant, 4.43 +/- 0.82 mumol/kg x min; nonpregnant, 4.11 +/- 1.08 mumol/kg x min, mean +/- SD). However, the fraction of alanine incorporated into glucose was significantly lower during pregnancy (23.5 +/- 8.3% v 30.8 +/- 8.2%, P less than .04). In pregnant women, 20% of lactate pool was derived from alanine as compared with 28% in nonpregnant subjects (P less than .02). Twenty-four percent of alanine turnover was converted to CO2 in both pregnant and nonpregnant women. The plasma insulin concentration was increased significantly during pregnancy (P less than .05). These data suggest that gluconeogenesis from alanine is attenuated during pregnancy. This decrease in gluconeogenesis is not the result of decreased alanine flux, but due to intrinsic intrahepatic mechanism such as decreased deamination of alanine mediated by the predominant insulin effect or a decreased hepatic uptake of alanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for isolation of non-esterified fatty acids and several other classes of plasma lipids by column chromatography on silica gel

A method is described for isolation from human plasma of non-esterified fatty acids, cholesteryl esters, triglycerides, cholesterol and diglycerides, monoglycerides, and some phospholipids by extraction and silica gel column chromatography. All of these lipid classes except diglycerides and cholesterol were separated cleanly in seven elution steps. Diglycerides and cholesterol were isolated together. Recovery of model compounds which represent the most significant classes of plasma lipids during the column chromatographic step was nearly complete. The overall recovery of added heptadecanoic acid from plasma specimens was 81% after both sample isolation steps. The overall recovery of added synthetic pentadecanoic acid and heptadecanoic acid ester lipid homologues from plasma was 80-91% after both sample preparation steps. About 6 h are required for extraction and isolation in duplicate of these lipid classes from twenty plasma specimens. Alternatively, non-esterified fatty acids can be isolated from twenty plasma specimens in duplicate within 4 h by a variation of the full procedure.

Urinary 3-hydroxydicarboxylic acids in pathophysiology of metabolic disorders with dicarboxylic aciduria

Dicarboxylic aciduria occurs during increased mobilization or inhibited beta-oxidation of fatty acids. In these conditions, a number of 3-hydroxydicarboxylic acids are excreted in the urine. These 3-hydroxydicarboxylic acids include 3-hydroxyadipic (3OHDC6), 3-hydroxyoctanedioic (3OHDC8), 3-hydroxydecanedioic (3OHDC10), 3-hydroxydodecanedioic (3OHDC12), and a number of unsaturated homologues. The metabolic origin of these 3-hydroxydicarboxylic acids is from the omega-oxidation of 3-hydroxy fatty acids. Subsequent beta-oxidation of the dicarboxylates yields lower-chain 3-hydroxydicarboxylic acids. A new defect in fatty acid oxidation characterized by increased urinary ratios of 3OHDC6, 3OHDC12, and unsaturated 3OHDC14s relative to 3OHDC10 is described. This pattern is consistent with a defect in long-chain 3-hydroxyacyl-CoA dehydrogenase (LHAD), which was confirmed by enzyme assay in fibroblasts. In contrast, patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency had lower ratios of 3OHDC6 and 3OHDC8 to 3OHDC10, consistent with a decreased activity of MCAD. Nonketotic dicarboxylic aciduria, other than MCAD and LHAD deficiencies, is shown to have a normal 3-hydroxydicarboxylic acid profile when compared with fasting normal controls. Since increased excretion of 3-hydroxydicarboxylic acids was observed in all patients with dicarboxylic aciduria, an increased excretion of these compounds is not an adequate criterion to suspect a defect in 3-hydroxyacyl-CoA dehydrogenases. The analysis of the metabolite ratios (3OHDC6 and 3OHDC12 relative to 3OHDC10) is a more useful indicator for defects in LHAD.

Distinction of dicarboxylic aciduria due to medium-chain triglyceride feeding from that due to abnormal fatty acid oxidation and fasting in children

Increased amounts of dicarboxylic acids are excreted in human urine under conditions of medium-chain triglyceride (MCT) feeding, abnormal fatty acid oxidation (FAO) and fasting. Criteria to distinguish dicarboxylic aciduria originating from MCT feeding and other conditions are needed in urinary organic acid profiling for detecting inborn errors of metabolism. Patterns of dicarboxylic aciduria in children under various conditions were compared. The relative amounts of medium-chain saturated dicarboxylic acids in urine are not reliable for identifying MCT-induced dicarboxylic aciduria. On the other hand, low ratios of unsaturated to saturated dicarboxylic acids (<0.1) and 3- hydroxydecenedioic to 3-hydroxydecanedioic acids were found to be useful in identifying dicarboxylic aciduria due to MCT ingestion. Additional unique features of dicarboxylic aciduria from MCT are low ratios of 3-hydroxydodecanedioic to 3-hydroxydecanedioic acid (<0.14) and 3-hydroxyadipic to adipic acid (<0.02).

Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6-benzylguanine.

O6‐Benzylguanine and its metabolite, 8‐oxo‐O6‐benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6‐alkylguanine‐DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6‐benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60‐min constant infusion). A two‐compartment model fits the plasma concentration versus time profile of O6‐benzylguanine. O6‐Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2α and t1/2β are 2 ± 2 min and 26 ± 15 min [mean ± SD, n = 7], respectively), and its plasma clearance (513 ± 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6‐benzylguanine and 8‐oxo‐O6‐benzylguanine and classical metabolite kinetics methods. With increasing doses of O6‐benzylguanine, the plasma clearance of 8‐oxo‐O6‐benzylguanine decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.

Urinary 3-hydroxyadipic acid 3,6-lactone: structural identification and effect of fasting in adults and children

Increased urinary excretion of medium-chain dicarboxylic acids is a general feature of disordered fatty acid metabolism. The physiological role of the metabolic pathways involved in dicarboxylic acid production has been a subject of controversy. In the present investigation, the existence of 3-hydroxyadipic acid 3,6-lactone, possibly representing a metabolic intermediate in the beta-oxidation of adipic acid to succinic acid, has been demonstrated. The identity of this compound was established by electron-impact mass spectrometry of its trimethylsilyl derivative and by comparison with synthetic authentic samples. 3-Hydroxyadipic acid 3,6-lactone is present in almost every urine sample we have examined. In the nonfasting state, urinary concentrations of 0.9 +/- 0.5 micrograms/mg creatinine were observed in the adults. During fasting, the urinary excretion of this compound increased with time. It reached 19.1 +/- 8.5 micrograms/mg creatinine by the end of the third day. The responses in children were even higher; urinary concentrations of 82 +/- 50 micrograms/mg creatinine were observed by the end of 36 hours. The urinary excretion of the lactone is closely correlated (r2 = 0.8) with that of adipic acid, an indicator of fatty acid omega-oxidation activity. Non-ketotic dicarboxylic aciduria appears to vary in different defects based on the ratio of urinary lactone to adipic acid.