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The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outermembrane 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 CO 2 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, CO 2 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 ratelimiting. 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 contol 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.Keywords: flux control coefficients; hepatocytes; mitochondria; carnitine palmitoyltransferase; ketogenesis.During the transient hypoglycaemia immediately after birth [1], when feeding is being established, ketone bodies derived from fat stores provide an alternative perinatal fuel to glucose. Subsequently, throughout the suckling period, hepatic ketogenesis generates the main circulating oxidative fuels for the brain and peripheral tissues and carbon precursors for essential myelination of the neonatal brain [2±5]. However, despite the central importance of this biochemical pathway to metabolic adaptation to extrauterine life, we do not currently understand precisely how its onset and development are regulated and controlled in healthy neonates [6]. Furthermore, it is clear that these mechanisms of regulation and control are impaired in certain clinical infants [7,8]. Without rigorous analyses we shall remain unable to understand and describe quantitatively how hepatic ketogenesis is regulated and controlled in both`normal' healthy infants ...

This study extends our earlier work [ I ] investigating the potential of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) to control ketogenic flux in hepatocytes isolated from suckling rats. Previously we reported ilux control coefficients for CPT I with respect to ketogenic flux from the long-chain fatty acid, palmitate, alone. Here we investigate the flux from a medium-chain fatty acid, octanoate, and develop a more 'physiological model' where 40% of the fatty acid substrates supplied are of medium-chain length. This reflects the situation found in maternal rat milk, where approximately this percentage of the milk triacylglycerols are medium-chain [2].We prepared hepatocytes from Wistar rat pups (1 1-13d), which had been with their mothers and allowed to suckle acl libitum [I]. Cells were incubated in 25ml conical flasks at a density of approximately 3 x lo6 cells in total vdume of 3ml KRB Flasks were gassed with 95%O2:5%CO2 and incubated for 30min at 3 7 T in a reciprocating water bath. Appropriate etomoxir concentrations were added to each flask (OpM-100pM) AAer IOmin. either Iml (2mM, 0.4pCi ['4C]palmitate in KRB, 2% BSA), Iml (8mM; 0.8pCi [''C]octanoate in KRB, 2% BSA) or Iml (ZmM, 0 4pCi [''Clpalmitate and 1.32mM octanoate in KRB, 2% BSA) were added to the flasks. The flasks were regassed, resealed and the incubation continued for 60min Reactions were terminated by the addition of 400p1 of 20% perchloric acid to the incubation medium, via a syringe needle through the subaseal. Where [14C]palmitate, or [ ''C]palmitate:octanoate was used as substrate, the acidified incubation medium was centrihged 0. 41111 supernatant was used directly for assaying ketone body production (I4C-KB). Where [''C]octanoate alone was used as substrate, I .5ml supernatant was added to 4.51111 heptane, shaken and whirlimixed, and centrihged to separate the layers. The upper heptane layer was discarded and a hrther 4ml hepatane added to the tubes. After repeating this washing procedure 3 times, Iml of the aqueous layer was used for assaying ketone body production. Duplicate sets of incubations containing no radiolabel were performed to measure the effects of increasing etomoxir concentration on CPT I activity in each cell preparation. These cells were permeabilised by the addition of Iml of digitonin in KRP and after exactly 10s the suspension was diluted with ice cold KCI buffer and centrihged The permeabilized cells were collected as a pellet and resuspended in KCI medium. This ghost suspension was used to measure CPT I activity using radiolabelled palmitoyl-CoA as substrate, in the presence of 1% BSA, and the amount of butanol-extractable 'H-palmitoyl carnitine measured [3] We calculated flux control coefficients for CPT I with respectAbbreviations used: BSA bovine serum albumin; CPT I outer mitochondrial membrane camitine palmitoyltransferase; KB ketone bodies; KRB Krebs ringer bicarbonate; KRP Krebs ringer phosphate Figure 1 Effwt of etomoxir on CPT I activity and ketogenic flux 0-4 0 2 4 6 8 1054 IW 0-4 0 2...

Why is there interest in neonatal hepatic ketogenesis?Hypoglycaemia characterizes the immediate post-partum period, as the newborn infant adapts to a transient period of starvation and then to the fast-feed cycle of milk feeds [l-31. During this fetal/neonatal transition period, the onset of P-oxidation and ketogenesis provides ketone bodies (acetoacetate and P-hydroxybutyrate, derived from endogenous fat stores accumulated pre-partum) as an alternative perinatal 'energy fuel' to maternally derived glucose, the supply of which is continuous in utem, but ceases abruptly at birth [4,5]. Subsequently, throughout suckling, hepatic ketogenesis generates (from dietary triglycerides) ketone bodies which are the main circulating oxidative fuels for the brain, heart and peripheral tissues, and carbon precursors for essential myelination of the neonatal brain [6-91. At this time, ketone bodies also have a key role as effectors that regulate: whole-body fuel selection/ utilization, neonatal glucose homoeostasis and, Abbreviations used: FFA, free fatty acids; CPT, carnitine palmitoyltransferase; (m)HMG-CoA, (mitochondrial) 3-hydroxy-3-methylglutaryl-CoA; MCA, metabolic control analysis; TPN, total parenteral nutrition; IUGR, intra-uterine growth-retardation; ROS, reactive oxygen species; EM, electron microscopy; CJstep, *CLImk, flux control coefficient describing control exerted by step or block of reactions over flux J.