Use of control analysis to study the regulation of plant lipid biosynthesis.

Control analysis is a powerful method to quantify the regulation of metabolic pathways. We have applied it to lipid biosynthesis for the first time by using model tissue culture systems from the important oil crops, olive ( Olea europaea L.) and oil palm ( Elaeis guineensis Jacq.). By the use of top-down control analysis, fatty acid biosynthesis has been shown to exert more control than lipid assembly under different experimental conditions. However, both parts of the lipid biosynthetic pathway are important, so that attempts to alter oil yield by manipulating the activity of a single enzyme step are very unlikely to produce significant increases.

Carnitine palmitoyl transferase I and the control of beta-oxidation in heart mitochondria.

Mitochondrial beta-oxidation provides much of the fuel requirements of heart and skeletal muscle despite the malonyl-CoA concentration greatly exceeding the IC(50) of carnitine palmitoyl transferase for malonyl-CoA. To try to explore the relationship between inhibition of carnitine palmitoyl transferase I activity and beta-oxidation flux, we measured the flux control coefficient of carnitine palmitoyl transferase I over beta-oxidation carbon flux in suckling rat heart mitochondria. The flux control coefficient was found to be 0.08 +/- 0.05 and 50% of carnitine palmitoyl transferase I activity could be inhibited before beta-oxidation flux was affected. These observations may help to explain the presence of high rates of beta-oxidation despite the high concentration of malonyl-CoA in rat heart; we hypothesize that although not rate-limiting in vitro, carnitine palmitoyl transferase is rate-limiting in vivo because of the high malonyl-CoA concentration in heart and muscle.

Carnitine palmitoyl transferase I and the control of myocardial beta-oxidation flux.

Carnitine palmitoyltransferase I is assumed to be rate limiting for beta-oxidation in all tissues. However, the concentration of malonyl-CoA in heart and muscle is high and is enough to completely inhibit beta-oxidation if this assumption is correct. In this review, we consider whether: (i) there is a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity; (ii) the measured malonyl-CoA concentration in the heart is physiologically meaningful; and (iii) carnitine palmitoyltransferase I is rate-limiting for beta-oxidation in the heart.

Effect of lipopolysaccharide and cytokines on oxidative metabolism in neonatal rat hepatocytes.

BACKGROUND/PURPOSE: Lipopolysaccharide (LPS) and cytokines produced during neonatal sepsis trigger free radical production, which eventually results in inhibition of liver metabolism. Studies in adults have indicated a hypermetabolic response to sepsis; however, evidence for a hypermetabolic response in neonates is equivocal. This study was carried out to determine whether LPS and cytokines can cause liver hypermetabolism in neonates. METHODS: The initial bacterial insult and cytokine cascade were mimicked by the addition of lipopolysaccharide (Escherichia coli 055:B5), tumour necrosis factor (TNF-alpha), and interleukin-6 (IL6) during the isolation of hepatocytes by collagenase digestion from 11- to 13-day-old Wistar rats. Hepatocyte oxygen consumption was measured polarographically with cells respiring on palmitate (0.5 mmol/L). Myxothiazol, a specific inhibitor of mitochondrial respiration, was used to distinguish extra- and intramitochondrial oxygen consumption. Morphologic changes were assessed by electron microscopy. RESULTS: The addition of LPS, TNF-alpha and IL6 during hepatocyte isolation resulted in a 10% decrease in cell yield (P <.05) compared with untreated controls; however, cell viability was unchanged (n = 31). Both total and extramitochondrial oxygen consumption were significantly greater in treated cells compared with untreated controls (P <.05, Student's t test). Electron microscopy indicated that LPS, TNF-alpha, and IL6 did not cause ultrastructural changes to hepatocytes. CONCLUSIONS: The increase in oxygen consumption was predominantly extramitochondrial and likely to be caused by increased oxygen requirement for cytosolic detoxification and repair purposes. This study shows that liver hypermetabolism metabolism can occur in response to LPS and cytokines. However, during in vivo neonatal sepsis, additional free radical damage may blunt this hypermetabolic response.

Neonatal oxidative liver metabolism: effects of hydrogen peroxide, a putative mediator of septic damage.

BACKGROUND/PURPOSE: Surgical neonates are at risk for sepsis and liver dysfunction. These complications are more common in preterm neonates and in those who receive total parenteral nutrition. Elevated levels of reactive oxygen species (eg, hydrogen peroxide) have been reported in these "at-risk" patients and may be the mediators of liver impairment via their effect on oxidative energy metabolism. The aim of this study was to test the hypothesis that elevated levels of hydrogen peroxide (H2O2) impair neonatal liver oxidative energy metabolism. METHODS: An in vitro model to test this hypothesis was developed in hepatocytes isolated from neonatal (11-day to 15-day) rats. The cells, respiring on palmitate (0.5 mmol/L in 2% bovine serum albumin), were exposed to H2O2. Oxygen consumption was measured polarographically. In experiment A, H2O2 was added to the cell preparation at different concentrations (0.5 mmol/L, 1 mmol/L, 1.5 mmol/L, 2 mmol/L) to assess the effect on oxygen consumption. In experiment B, H2O2 (2 mmol/L) was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to define the site of action of H2O2. In experiment C, electron microscopy was performed on hepatocytes after incubation with 1 mmol/L and 2 mmol/L of H2O2. RESULTS: In experiment A, H2O2 significantly reduced hepatocyte oxygen consumption at 1.5 and 2 mmol/L. In experiment B, in the presence of inhibitors of mitochondrial respiration, myxothiazol (inhibitor of substrate oxidation), and oligomycin (inhibitor of adenosine triphosphate (ATP) synthase), no further inhibition by H2O2 occurred, indicating that the effect of H2O2 was intramitochondrial and affecting the synthesis of ATP. In experiment C, microscopic alterations of mitochondria were noticed exclusively in hepatocytes incubated with 2 mmol/L H2O2. CONCLUSIONS: Results of this study demonstrate that H2O2 impairs neonatal liver oxidative metabolism. H2O2 probably directly inhibits ATP synthase. The authors hypothesize that H2O2 may play a role in the biochemical pathogenesis of liver dysfunction associated with sepsis. Identification of the precise target site of H2O2 may be valuable in directing therapy in septic neonates.

Comparisons of flux control exerted by mitochondrial outer-membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats.

The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific pathway studied and (d) the model system.

Analgesic doses of fentanyl impair oxidative metabolism of neonatal hepatocytes.

BACKGROUND/PURPOSE: Studies in human surgical neonates have shown that intraoperative fentanyl analgesia results in greater fall in perioperative body core temperature compared with morphine analgesia. The aim of the study was to compare in a neonatal animal model the biochemical effect of fentanyl and morphine on hepatocyte oxidative metabolism. METHODS: Hepatocytes were isolated from suckling rats and the oxygen consumption from palmitate was measured polarographically. In experiment A, fentanyl and morphine within the respective analgesic serum ranges were added to hepatocytes to assess the effect on oxygen consumption. In experiment B, fentanyl was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to investigate its site of action. In experiment C, hepatocytes were incubated with either fentanyl or morphine, centrifuged, and then examined ultrastructurally by electron microscopy. RESULTS: In experiment A, fentanyl inhibited oxygen consumption by up to 40% (P < .01). Morphine inhibited oxygen consumption to a maximum of 25% (P < .01). In experiment B, in the presence of oligomycin, fentanyl increased the inhibition of oxygen consumption; however, in the presence of myxothiazol, no further inhibition by fentanyl occurred. In experiment C, mild ultrastructural alterations to hepatocytes were observed after incubation with fentanyl but not with morphine. CONCLUSIONS: This study demonstrates that therapeutic doses of two commonly used analgesic drugs impair neonatal hepatic oxidative metabolism. Fentanyl exerts a greater effect than morphine by diminishing liver oxygen consumption by up to 40%. The inhibitory effect of fentanyl occurs directly on the mitochondrial respiratory chain, either on substrate oxidation or on the thermogenic proton leak. The findings of this study are relevant to the perioperative management of surgical neonates.

Hepatic mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme a synthase deficiency.

There are at least two isoenzymes of 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (EC 4.1.3.5) located in the mitochondrial matrix and the cytoplasm of hepatocytes, respectively. The mitochondrial enzyme is necessary for the synthesis of ketone bodies, which are important fuels during fasting. We report a child with a deficiency of this isoenzyme. He presented at 16 mo with hypoglycemia. There was no rise in ketone bodies during fasting or after a long chain fat load but there was a small rise after a leucine load. Measurement of beta-oxidation flux in fibroblasts was normal. Using antibodies specific for mitochondrial HMG-CoA synthase, no immunoreactive material could be detected on Western blotting. Total HMG-CoA synthase activity in liver homogenate was only slightly lower than in control samples. Presumably, as there was no mitochondrial HMG-CoA synthase enzyme protein, this activity arose from the cytoplasmic or other (e.g. peroxisomal) isoenzymes. With avoidance of fasting, our patient has had no problems since presentation and is developing normally at 4 y of age.