Hepatic fatty acid metabolism in pigs and rats - Regulatory, Integrative ...

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ketogenesis in neonatal pigs. J. Nuts. 125: 2541-2549,1995. Pegorier, J. P., P. H. D&e, R.Assan, J. Peret, and J. Girard. Changes in circulating fuels, pancreatic.
Hepatic fatty acid metabolism in pigs and rats: major differences in endproducts, 02 uptake, and P-oxidation SEAN H. ADAMS1 XI LIN,2 XING XIAN YIJ,l JACK ODLE,ly2 AND JAMES lDivision of Nutritional Sciences and 2Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Adams, Sean H., Xi Lin, Xing Xian Yu, Jack Odle, and James K. Drackley. Hepatic fatty acid metabolism in pigs and rats: major differences in end products, O2 uptake, and P-oxidation. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R1641-R1646, 1997. -Models of mammalian hepatic lipid metabolism are based largely on observations made in adult rats, emphasizing ketogenesis as a primary adjunct to mitochondrial p-oxidation. Studies using piglets have illustrated the divergent nature of intermediary metabolism in this model, wherein ketogenesis and p-oxidation are small and acetogenesis is an important route of fuel carbon flux. To clarify potential species differences in hepatic lipid metabolism and its control, we compared 02 consumption and metabolic end products in fasted pig and rat liver homogenates treated with l-[14C]C16:0. Carboxyl carbon accumulation in acid-soluble products (ASP) plus CO2 was threefold greater and 02 consumption was twofold greater in rats (P < 0.05). Unlike rats, pigs showed negligible carboxyl carbon accumulation in ketone bodies (3-7% of ASP), whereas acetate was a carboxyl carbon reservoir in both animals (17-31% of ASP in pigs). Malonate increased (-2-fold) and antimycin/rotenone decreased (40-60%) radiolabel accumulation in acetate. These data concur with the hypotheses that comparatively low hepatic P-oxidative flux in piglets is partially related to a smaller metabolic rate and that substantial acetogenesis occurs intramitochondrially in both pigs and rats. acetate; ketone bodies; metabolic

rate; piglets

HAS BECOME increasingly clear that hepatic lipid metabolism in pigs is not adequately described by traditional models of P-oxidation that emphasize accelerated ketogenesis concurrent with enhanced mitochondrial flux of fatty acids. For instance, the onset of suckling in rats induces upregulation of liver enzymes supporting ketogenesis, including mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3 5) (3 1,35) and carnitine palmitoyltransferase I (CPT-I; EC 23.121) (4, 36). Elevated ketone body production thus results in a neonatal hyperketonemia in some species (see Ref. 12). However, in piglets, low blood ketone bodies are observed (5, 26), and a negligible ketogenic capacity has been reported in vitro (10, 19, 25, 28) and after a medium-chain fatty acid (MCFA) challenge in vivo (1). Furthermore, reported rates of P-oxidation in liver preparations from neonatal rabbits (9, 27) or mature rats (7, 10) are markedly higher than liver from newborn pigs (10, 24, 28). This phenomenon has been ascribed to a propensity for fatty acid esterification vs. oxidation in piglets (28). The etiology of attenuated fatty acid oxidation in piglet liver relative to other species is not clear, but could be related to metabolic enzyme activities (see above) and/or IT

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other phenomena such as metabolic rate. Low rates of P-oxidation and a limited capacity for ketogenesis bring up the possibility that alternative, nonketogenic routes of carbon flow may predominate in swine liver (24), an idea supported by recent observations. First, application of radio-high-performance liquid chromatography (HPLC) methodology to characterize organic acid end products of radiolabeled MCFA and long-chain fatty acid (LCFA) metabolism in piglet liver has revealed a substantial accumulation of fatty acid carbon in acetate and not ketone bodies (19, 25). Second, on the basis of residual P-oxidation in the presence of respiratory chain inhibitors (7) or O2 consumption of isolated organelles administered LCFA substrate (37), the relative capacity of peroxisomes to carry at least the first cycle of LCFA P-oxidation might be elevated in pigs compared with rats. Currently accepted principles of P-oxidation and its control have been derived primarily from studies of mature rats. The piglet’s apparent departure from these well-established paradigms raises some intriguing questions addressed in the present study. What is the basis for relatively low P-oxidation in swine? VVhat impact would limited ketogenesis and inhibition of P-oxidation have on cellular carbon trafficking? What is the organellar origin of acetogenesis in liver? The possible contribution of tissue metabolic rate in modulating P-oxidative flux was explored through measurement of O2 consumption in liver homogenates oxidizing fatty acids. Differences in C16:O carboxyl carbon accumulation in metabolic end products were examined after mitochondrial P-oxidation inhibition by antimycin/ rotenone (AR), and mitochondrial acetogenesis/ketogenesis in the presence of the Krebs cycle inhibitor malonate was determined. Because traditional formulations of mammalian fat metabolism are fashioned primarily from the exhaustively studied adult rat system and because direct comparisons of P-oxidation between pigs and other species are rare (1, 7, lo), metabolism in piglet liver was compared with that in ketogenic adult fasted rats under identical conditions. The data support prior reports of relatively low P-oxidation (7, 10) and predominance of acetogenesis rather than ketogenesis (19, 25) in piglet liver. These observations are extended further by results consistent with the hypothesesthat the relatively low rate of P-oxidation in piglets is partially explained by a lower overall metabolism and that mitochondria contribute significantly to acetogenesis. MATERIALS

AND METHODS

Animals and liver sampling. All procedures were approved by the University of Illinois Laboratory Animal Care Advisory the American

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Committee. Commercial cross-bred piglets collected within 12 h of birth (mass, 1,690 t 33 g; n = 3) and mature SpragueDawley rats (284 k 8 g, n = 3) were fasted for 24 h before experimentation. After pentobarbital sodium (20 mg/kg) administration (pigs) or ether anesthesia and cervical dislocation (rats), livers were removed and placed in ice-cold homogenization buffer [(in mM) 220 mannitol, 70 sucrose, 2 N-2hydroxyethylpiperazine-N ‘-2-ethanesulfonic acid, and 0.1 EDTA, pH 7.21, and portions were homogenized manually in 10 volumes of buffer using a Potter-Elvehjem apparatus. Protein yield (in mg protein/g wet wt by biuret analysis) averaged 420 ? 33 and 238 5 66 for rats and pigs, respectively Liver mass was 2.4% (rats) and 1.8% (pigs) of body weight. Incubations. Homogenate incubations to measure LCFA P-oxidation were performed at 37°C as follows: 450+1 aliquots of homogenate (-40 mg liver) were incubated in medium (pH 7.4) containing the following (in mM): 1.0 L-carnitine (Lonza, Basel, Switzerland), 0.5 Na-Cl6:O ([fatty acid/bovine serum albumin ratio, 5: 1; containing [ 1-14C]C 16:O tracer; (ICN Biochemicals, Irvine, CA) at 2.6-3.7 &i/pmol], 13.1 sucrose, 78.1 tris(hydroxymethyl)aminomethane HCl, 10.5 K2HP04, 31.5 KCl, 5.0 ATP, and 1.0 NAD+, as well as 850 PM EDTA and 100 PM CoA in a final volume of 3 ml. Some flasks contained A/R (50 and 10 PM, respectively) or Nazmalonate (10 mM), which inhibit mitochondrial electron transport and succinate dehydrogenase, respectively. Selection of A/R concentrations was based on previous research (13) documenting maximal inhibition. Incubations were initiated by addition of fatty acid after 5-min preincubation, and terminated 30 min later by addition of 250 ~160% trichloroacetic acid. Unless otherwise stated, chemicals were purchased from Sigma Chemical (St. Louis, MO). Radioactivity in acidsoluble products (ASP) and CO2 (corrected for time 0 acidkilled blanks) and homogenate O2 consumption were determined using methods described previously (24). Organic acid anaZysis. The ASP samples were subjected to reversed-phase ion-pairing HPLC to characterize radioactivity associated with ketone bodies and acetate (19). Separation was achieved using a mobile phase of 0.3% H3P04 (pH 2.1, 0.65 ml/min) with a Beckman Ultrasphere IP column (5 pm; 4.6 X 250 mm), and volumes of eluent with retention times corresponding to radioactive peaks of interest (peaks l-6, Fig. 1) were retrieved by a fraction collector (19). Radioactivity in these samples was quantified using liquid scintillation spectrometry and accounted for 80-90% (rats) to 92-100% (pigs) of ASP radioactivity. Not shown are similar analyses of ASP samples using an ion-exchange HPLC separation method (25), which revealed that Krebs cycle intermediates accounted for a minimal fraction (l-5%) of radiolabeled carboxy1 carbon accumulated in ASP of rats and pigs. Real-time characterization of radioactive metabolites (see Fig. 1) was achieved using a Radiomatics Flo-One p-flowmonitor (Packard Instruments, Meridien, CT). Statistics. Data were subjected to analysis of variance for a split-plot design with species as the main plot and treatments as the subplot (Statistical Analysis System, Cary, NC). Values are means 2 SE and are considered significantly different at

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P < 0.05. RESULTS

P-Oxidation and oxygen consumption. Accumulation of Cl6:O carboxyl carbon in oxidative end products (ASP and C02) is shown in Table 1. Malonate, an inhibitor of succinate dehydrogenase, did not significantly affect the total P-oxidative flux, but, as expected,

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RETENTIONTIME (min) Fig. 1. Example of reverse-phase high-performance liquid chromatography radiochromatograms of the acid-soluble fraction derived from l-day-old fasted newborn pig and mature fasted rat liver homogenates incubated with [1-14C3C16:0 (see MATERIALS AND METHODS for details). Incubations were carried out in the presence of antimycin/ rotenone (A/R; A) or malonate (B) or in the absence of malonate and A/R (control; C). Radioactivity peaks l-6 correspond to unknown A, acetate, acetoacetate, P-hydroxybutyrate, unknown B, and unknown C, respectively. Note minimal carboxyl carbon radioactivity accumulated in ketone bodies in piglet samples, the lowered accumulation in ketone bodies with a high proportion of P-hydroxybutyrate vs. acetoacetate in A/R-treated rat samples, and increased acetate accumulation with malonate treatment.

depressed the accumulation of carboxyl carbon in CO2 by 60% in rats and fivefold in pigs. Carboxyl carbon accumulation in ASP plus CO2 in piglet homogenates was just 35% (control and malonate) to 61% (A/R) of that in rat preparations. Oxygen consumption in control piglet homogenates was only one-half that observed in rat preparations. Inclusion of A/R slowed mitochondrial metabolism/poxidation in both species (Table 1). Liver O2 consumption decreased significantly in the presence of these electron-transport inhibitors to levels just 45% of controls, whereas carboxyl carbon accumulation in CO2 was lowered by 85 and 95% in pigs and rats, respectively (Table 1). ASP analysis. Example radiochromatographs from HPLC separation of ASP samples derived from C16:O

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Table 1. Oxygen consumption and carboxyl carbon accumulation in ASP and CO2 from incubations of rat or pig liver homogenates with [1 J4C]C1 6:0 t additions Ofmalonate or antimycin / rotenone (see MATERIALS AND METHODS for details)

Treatment

Adult rats Control MaIonate Antimycin/rotenone Piglets Control MaIonate Antimycin/rotenone

ASP

co2

166 + 20** 1612 16** 50 + gb*

115 1” 7+0.!jb* 0.6 + 0.03”

lO+l* 2:0.3b

51+6*

5829 30 t 2b

1+0.2b

02

Consumption

177?21** 168+16”* 51+ sbt

744 + 141$ 6795223 34156

61k7" 60 5 lO*

379224 4165 10 16829

31+2b -

Values are means + SE for n = 3 replicates/treatment in nmol . min-’ l g liver? ASP, acid-soluble product. Different letters within a column denote significant difference within a species (PC 0.05). * Significantly different versus treatment-matched piglet value (P C 0.05); “r P = 0.07. $02 consumption was lower in piglets (P < O.Ol), and an effect of treatment was observed (control = malonate > antimycin/rotenone, P < 0.01).

oxidation are shown in Fig. 1. Six distinct radioactive peaks were observed, but only acetate, acetoacetate (AcAc), and P-hydroxybutyrate (P-OHB) could be positively identified. Quantification of the carboxyl carbon accumulated in these metabolites yielded a number of important observations. First, acetate was the predominant identifiable end product of Cl6:O oxidation in the 24-h fasted l-day-old pig liver, constituting 17 t 1,31? 2, and 17 t 2% ofthe total carboxyl carbon accumulation in ASP under control, malonate, and A/R treatments, respectively (see Fig. 2). Second, the addition of malonate doubled C16:O carboxyl carbon accumulation (in nmol min-l g liver-l) l

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in acetate for rats (to 14.2 t 2.3) and piglets (to 18.2 t 3.8) (Fig. 2). Third, piglet liver produced minimal ketone bodies (3-7% of ASP carboxyl carbon), which is in stark contrast to rats (Fig. 2).

l

These data provide direct evidence in support of previous suggestions that P-oxidation is lower in pigs compared with other species (7, 9, 10, 23, 27, 28). The basis for comparatively low fatty acid oxidation in piglet hepatic tissue is not clear, but might be related to a lower tissue-specific metabolic rate (0, consumption per unit mass) and thus a diminished demand for P-oxidation to meet cellular ATP requirements (23). In fact, O2 consumption in control piglet liver preparations was only 50% that determined in rats (Table l), indicating that differences in the metabolic rate can at least partially account for the relatively low P-oxidation observed in piglets. Piglet body mass was sixfold greater than rats (see MATERIALS AND METHODS), and it is well established that, across a wide range of adult mammalian species, the mass-specific basal metabolic rate decreases with increasing body size (15). Furthermore, this whole animal relationship between body mass and O2 consumption holds for liver, such that pig hepatocytes consume O2 at -50% the rate of rats (29,30). The limitation of this explanation when comparing tissues from neonates of one species to adults of another is recognized, and it is likely that factors in addition to metabolic rate (i.e., metabolic enzyme activities; see Perspectives) also contribute to differences in P-oxidation across species. Comparisons of in vitro metabolic rate based on O2 consumption could be confounded by the potential impact of extramitochondrial metabolic pathways that consume O2 but are not directly linked

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ntal Oxidation (ASP + C02)

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Fig. 2. Accumulation of carboxyl carbon in the major identifiable organic acids in acid-soluble products (ASP) derived from from incubation of liver homogenates fasted adult rats (Left) and fasted 1.-dayold piglets (right) -and incubated with [1-14C3C16:0 (see MATERIALS AND METHODS for details). For each species, bars represent control incubations and incubations containing malonate or A/R treatments. Pooled SE are shown in the first set of bars. Treatment had no effect on accumulation in ketone bodies in piglets (P > 0.1). However, in the rat, accumulation in acetoacetate (crosshatched bars) rose in the presence of malonate (*P < 0.05 vs. control) but fell with A/R (**P < 0.0001 vs. control and malonate). Carboxyl carbon in P-hydroxybutyrate (hatched bars) dropped with malonate (*P < 0.0001 vs. control) and with A/R (**P < 0.01 vs. control and malonate). Malonate elicited a rise (*P < 0.01 vs. control) whereas A/R caused a fall (**P < 0.05 vs. control and malonate) in accumulation in acetate (solid bars) in both species.

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to ATP production [i.e., peroxisomal acyl-CoA oxidase (EC 1.3.3.6) and catalase (EC 1.11.1.6) utilize and liberate Og, respectively]. Nevertheless, O2 consumption (Table 1) reflected true differences in mitochondrial ATP production because peroxisomal pathways of O2 utilization could account for only a fraction of measured O2 uptake.l The addition of A/R in liver homogenate incubations inhibited mitochondrial metabolism (Table 1 and Fig. 2), but did not completely abolish O2 consumption (Table 1). Diminished but continued O2 consumption in the presence of respiratory poisons (also see Refs. 5 and 29) may be explained by a drop in mitochondrial metabolism concurrent with continued activities of extramitochondrial 02-consuming enzymes that are A/R insensitive (29). The large drop in carboxyl carbon accumulation in ketone bodies and COB in the rat (Table 1 and Fig. 2) supports this assertion. Incomplete inhibition of mitochondrial respiration could also explain O2 consumption in the presence of A/R. This was signaled by the following observations: 1) C16:O carboxy1 carbon accumulation in ketone bodies (Fig. 2) remained at -30% of control values in rat homogenates exposed to A/R; and 2) in both species, only a portion of observed O2 consumption in the presence ofA/R (Table 1) could be attributed to peroxisomes under the assumption that ASP in the presence of A/R is peroxisomally derived. The lack of total A/R inhibition indicates that common estimates of peroxisomal metabolism (cyanide- or AR-insensitive P-oxidation) could overestimate the true rate. Nevertheless, the results in no manner discount the assertion that significant speciesrelated differences exist with regard to the relative contribution of peroxisomes to total hepatic P-oxidation

(6, 7,13,37). ASP analysis. ASP generated from radiolabeled fatty acids have been primarily associated with ketone bodies in adult fasted rat liver (20-22). However, piglets represent an animal model with minimal ketogenic potential (2, 10, 28), which suggested that fatty acid carbon must flow via alternative pathways in this species (24). Development of radio-HPLC methods that characterize acid-soluble end products of metabolism have confirmed that, in piglets, ketogenesis is negligible (19,25), with acetogenesis and production of other unknown compounds being more predominant (19). The present work illustrates further that, in the liver, major differences in the pattern of carboxyl carbon accumulation in acetate, ketone bodies, and various metabolites exist across species and in the presence of l A hypothetical maximal peroxisomal capacity for 02 consumption under control conditions in the rat (330 nmol min-l g liver-l) can be calculated assuming that 100% of ASP is peroxisomally generated and C16:O undergoes four cycles of peroxisomal P-oxidation at one-half 02 consumed per cycle. This maximal value, which greatly overestimates the peroxisomal contribution to ASP, accounts for just -40% of the observed 02 uptake (Table 1). At more realistic estimates of peroxisomal P-oxidation in fasted rats (520%), theoretical peroxisomal 02 uptake falls to only -2-10% of total 02 consumed, consistent with prior determinations of nonmitochondrialO2 consumption (29). l

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mitochondrial inhibitors (Figs. 1 and 2). Similar results were obtained with fasted piglet hepatocytes administered the substrates C7:O and C&O radiolabeled in the carboxyl carbon position (19). Acetogenesis has long been recognized as an alternative route of fatty acid catabolism (16,32,33), but it operates at a rate (in nmol C2 equivalents min1 mg mitochondrial protein) just 2-6% of ketogenesis in liver mitochondrial preparations from adult rats (33), consistent with our control rat homogenates (i.e., carboxyl carbon accumulation in acetate was 9% of that in ketone bodies). Under control conditions, the absolute accumulation of Cl6:O carboxy1 carbon in acetate was not different across species (Fig. 2). Overall metabolism, P-oxidation, and ketogenesis are less in piglets compared with rats (Table 1 and Fig. 2), and it follows that, in relative terms, acetogenesis has a substantial impact on the fate of fatty acid carbon in the neonatal pig liver. A significant portion of acetate production appears to occur intramitochondrially in both species, as indicated by the following observations. First, addition of malonate doubled carboxyl carbon accumulation in acetate (Fig. 2). Thus a mitochondrial acetyl-CoA pool provided acetate precursors when Krebs cycle disposal was slowed. It is notable that Krebs cycle blockage by malonate has been associated with exclusive flux of C16:0-carnitine to AcAc in newborn piglet mitochondria (10, 11). However, this assumption based on measured vs. theoretical O2 consumption is readily explained if the primary end products include acetate or acetylcarnitine. Second, the mitochondrial inhibitors A/R markedly dampened carboxyl carbon accumulation in acetate in both species (Fig. 2). Third, in the piglet liver, there was a substantial attenuation of in vitro MCFA carboxyl-carbon accumulation in acetate brought about by inclusion of valproate (19), a P-oxidation inhibitor that is a poor peroxisomal substrate (8,38). In addition, MCFAof eight or fewer carbons serve as weak peroxisomal substrates (17), yet contribute to acetogenesis in piglets (19) and adult rats (18, 32, 33). Peroxisomes also have acetogenic capacity (14, 18) and may have contributed to acetate production in our study because inclusion of A/R did not abolish LCFA carboxyl carbon accumulation in acetate (Fig. 2). Similarly, valproate did not completely block acetogenesis from MCFA in piglet liver (19). The data underscoring poor ketogenic potential in piglets (Figs. 1 and 2) are consistent with previous reports (10, 19, 25) and indicate that ketone bodies should not be assumed to compose the majority of acid-soluble end products of liver fatty acid p-oxidation in all mammalian species. It should be noted that, under the conditions used, ketone bodies accounted for only 50% of ASP radiolabel in rats [% of ASP label in AcAc and p-OHB were as follows: 22 t 2 and 33 t 2% (controls), 9 t 0.3 and 41 t 1% (malonate), and 45 2 3 and 8 t 0.2% (AR)]. The remaining radioactivity was largely found in unknowns A and B (see MATERIALS AND METHODS and Fig. 1). Unknown A accumulated little radiolabel in malonate or A/R incubations, which is suggestive of a mitochondrial origin. Under control l

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conditions, unknown B contained between 16 (rats) and 44% (pigs) of the ASP radioactivity (data not shown). This peak might represent acetylcarnitine, an end product observed in adult rat liver preparations oxidizing LCFA in the presence of exogenous carnitine (20, 21) F’inally, LCFA carboxyl carbon accumulation in ketone bodies in the presence of respiratory inhibitors in rat liver has been observed (Figs. 1 and 2) (5a, 20). Although accumulation of C16:O carboxyl in AcAc and p-OHB in rats fell to 7 and 64% of controls (controls: 55 2 6 and 35 5 2 nmolmin-l ‘g liver-l, respectively) (Fig. 2) and radiolabeled CO2 production dropped 95% in the presence of A/R (Table l), ketone bodies accounted for 53% of ASP under this condition. As discussed previously, this result is likely due to incomplete mitochondrial inhibition by AIR. Nevertheless, AJR successfully slowed mitochondrial metabolism (Table 1 and Fig. 2) and decreased matrix NADH disposal; the latter was reflected in the dramatic shift of the P-OHBI AcAc ratio toward P-OHB in rats (Figs. 1 and 2) (also see Ref. 5a). Perspectives The physiological ramifications of species differences in hepatic fatty acid metabolism are currently being clarified. Ketogenic capacity is trivial in neonatal piglets (2,10,19), and oxidation of P-OHB under physiological conditions meets less than 3% of the metabolic requirements of a typical piglet (34). In contrast, whole animal turnover of endogenously produced acetate in piglets indicates that up to 20% of their energy budget could potentially be derived from this fuel source (3). The etiologic basis for attenuated ketogenesis and comparatively low hepatic P-oxidation in piglets remains an active area of research. Modulation of CPT-I by malonyl-CoA inhibition (22) is generally accepted as the predominant control site for ketogenesis and fatty acid oxidation in liver. This system may be important in neonatal swine because pig CPT-I is particularly sensitive to malonyl-CoA inhibition (lo), and oxidation of [ 1-14C]C8:0 to ASP and CO2 surpasses that of [1-14C]C16: 0 by fourfold in piglet hepatocytes (25). An intramitochondrial site of ketogenic control has also been suggested (1), which is an idea strongly supported by reports of negligible activity of the ketogenic enzyme mitochondrial HMG-CoA synthase in pig liver (l,lO). Comparatively low activity of this enzyme results from suppressed gene expression in neonatal pigs and may be related to posttranscriptional modification and/or specific differences in enzyme kinetics (1). It remains plausible that low mitochondrial HMG-CoA synthase activity in pigs exerts some degree of control over hepatic P-oxidation in this species. Clearly, comparative analyses of intermediary metabolism and its regulation promise to yield new insight into common or unique mechanisms controlling P-oxidation across taxonomic boundaries. This material is based on work of Illinois Agricultural Experiment

supported Station

in part by the University and by the Cooperative

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State Research, Education, and Extension Service, U.S. Department ofAgriculture, under Agreement 92-37203-7993 (Project 350525). Present addresses: S. H. Adams, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, Dallas, TX 75235-9135; X. Lin and J. Odle, Dept. of Animal Sciences, North Carolina State University, Raleigh, NC 27695-7621; and X. X. Yu, Dept. of Biochemistry, Emory University, Atlanta, GA 30322. Address for reprint requests: J. Odle, PO Box 7621, Raleigh, NC 27695-7621. Received

19 August

1996;

accepted

in final

form

10 December

1996.

REFERENCES S. H., C. S. Alho, G. Asins, F. G. Hegardt, and P. F. 1. Adams, Marrero. Gene expression of mitochondrial3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal. Effect of starvation during the neonatal period in the piglet. B&hem. J. In press. 2. Adams, S. H., and J. Odle. Plasma P-hydroxybutyrate after octanoate challenge: attenuated ketogenic capacity in swine. Am. J. PhysioZ. 265 (Endocrinol. Metab. 28): E761-E765,1993. 3. Adams, S. H., and J. Odle. Pickled pigs: acetogenesis and its potential physiological relevance to metabolism in newborns from a species with low ketogenic potential (Abstract). PhysioZ. ZooZ. 68: 105,1995. 4. Asins, G., D. Serra, G. Arias, and F. G. Hegardt. Developmental changes in carnitine palmitoyltransferases I and II gene expression in intestine and liver of suckling rats. Biochem. J. 306: 379-384,1995. 5. Bengtsson, G., 3. Gentz, J. Hakkarainen, R. Hellstrom, and B. Persson. Plasma levels of FFA, glycerol, P-hydroxybutyrate and blood glucose during the postnatal development of the pig. J. Nutr. 97: 311-315,1967. r-oa.Berry, M. N., R. B. Gregory, A. R. Grivell, and P. G. Wallace. Compartmentation of fatty -acid oxidation in liver cells. Eur. J. Biochem. 131: 215-222,1983. 6. Crockett, E. L., and B. D. Sidell. Peroxisomal P-oxidation is a significant pathway for catabolism of fatty acids in a marine teleost. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R1004-R1009,1993. 7. Drackley, J. K., X. X. Yu, X. Lin, and J. Odle. Effect of palmitate concentration on mitochondrial and peroxisomal p-oxidation in piglet and adult rat liver (Abstract). FASEB J. 9: A468, 1995. 8. Draye, J. P., and J. Vamecq. The inhibition by valproic acid of the mitochondrial oxidation of monocarboxylic acids and o-hydroxymonocarboxylic acids: possible implications for the metabolism of gamma-aminobutyric acid. J. Biochem. 102: 235-242, 1987. 9 Duee, P. H., J. P. Pegorier, L. El Manoubi, C. Herbin, C. Kohl, and J. Girard. Hepatic triglyceride hydrolysis and development of ketogenesis in rabbits. Am. J. Physiol. 249 (Endocrinol. Metab. 12): E478-E484,1985. 10. Duee, P. H., J. P. Pegorier, P. A. Quant, C. Herbin, C. Kohl, and J. Girard. Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Biochem. J. 298: 207-212,1994. 11. Duke, P. H., J. P. Pegorier, R. Robin, C. Herbin, and J. Girard. Some characteristics of mitochondrial fatty acid oxidation in the liver of the newborn pig: preliminary results. Reprod. Nutr. Dev. 26: 633-634,1986. 12. Girard, J., P. Ferre, J. P. Pegorier, and P. H. Duee. Adaptations of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition. Physiol. Rev. 72: 507562,1992. 13. Grum, D. E., L. R. Hansen, and J. K. Drackley. Peroxisomal P-oxidation of fatty acids in bovine and rat liver. Comp. Biochem. PhysioZ. 109B: 281-292,1994. 14. Hovik, R., B. Brodal, K. Bartlett, and H. Osmundsen. Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of acetate and acetoacetyl-CoA. J. Lipid Res. 32: 993-999,199l. 15. Kleiber, M. The Fire oflife. Huntington, NY: Krieger, 1975. 16. Knowles, S. E., I. G. Jarrett, 0. H. Filsell, and F. J. Ballard. Production and utilization of acetate in mammals. Biochem. J. 142: 401-411,1974.

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Lazarow, fatty

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acids.

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I? B. Rat liver peroxisomes catalyze J. BioZ. Chem. 253: 1522-1528,1978.

Leighton, F., S. Bergseth, T. Rortveit, and J. Bremer. Free acetate production

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the P-oxidation

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