Peroxisomal beta-oxidation of branched chain fatty acids in rat liver. Evidence that carnitine palmitoyltransferase I prevents transport of branched chain fatty acids into mitochondria

H Singh,K Beckman,A Poulos

doi:10.1016/s0021-9258(17)36911-9

H Singh, K Beckman + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(17)36911-9

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Abstract

Fatty acid beta-oxidation was investigated in highly purified mitochondrial and peroxisomal preparations from rat liver. Under isotonic conditions, pristanic and homophytanic acid beta-oxidation in purified peroxisomes was severalfold greater compared to the oxidation in purified mitochondria. Branched chain fatty acid beta-oxidation in purified mitochondria was very low, and the oxidation was not stimulated by exogenous L-carnitine or L-malate. In contrast, stearic acid beta-oxidation by purified mitochondria depended upon exogenous L-carnitine, and the oxidation was stimulated by L-malate. Both mitochondrial and peroxisomal beta-oxidation of branched chain fatty acids was strongly inhibited by fatty acid-free bovine serum albumin, whereas stearic acid oxidation was either unaffected or slightly inhibited by bovine serum albumin. The results presented clearly indicate that branched chain fatty acids are mainly degraded in peroxisomes in rat liver. Branched chain fatty acids were efficiently converted to coenzyme A thioesters by purified mitochondria, peroxisomes, and microsomes. Although pristanic and phytanic acids were rapidly converted to pristanoyl-CoA and phytanoyl-CoA, respectively, they were not converted to carnitine esters by mitochondrial outer membranes. The results indicate that acyl-CoA synthetase and carnitine acyltransferase located at the mitochondrial outer membranes regulate entry of branched chain fatty acids into mitochondria. Mitochondrial carnitine acyltransferase I appears to be highly specific for straight chain fatty acids and restricts entry of branched chain fatty acids into mitochondria. Thus, branched chain fatty acids which cannot be transported across the mitochondrial membranes via the carnitine acyltransferase system are directed to peroxisomes for beta-oxidation. The results reported indicate that phytanic acid, the fatty acid which can be initially degraded by alpha-oxidation due to the presence of a beta-methyl group in the molecule, cannot be transported across the mitochondrial membranes. The data presented strongly suggest that phytanic acid alpha-oxidation occurs in organelles other than mitochondria and possibly in peroxisomes.

Highlights

Fatty acid P-oxidation was investigatedin highly pu- fatty acid (>22 carbons, VLCFA) oxidation is confined to perrified mitochondrial and peroxisomal preparations oxisomes
Branchedchain fatty acid P-oxi- Poulos, 1988).In addition tVo LCFA,branched chain fattyacids dation in purified mitochondria was verylow, and the such as phytaniaccid (3,7,11,15-tetramethylhexadecanoiaccid) oxidation was not stimulated by exogenous L-carnitine and pristanic acid (2,6,10,14-tetramethylpentadecanoicacid) mcortiihrnfiiatLeeoi,d-ncamhnfmaoadlntiatttdtyoherce.iahIaocnolixadncinsddodarnwitptaiaroeasdnrsoestxt,pwriseosatnnoesdsgmatelriyadimcl iuuanPlpcha-oiotdinebxdiPitedbe-xayodotxigboLiyden-onamfftoiabaoutrltnsaaytbneLyca.-hcpcBieauddor--tnhi-TfainalesvtnotoyBlavraceicncdikudimsne. utPtlhahaelyt.eo,taxi1nnid9ica9pt2eairo)co,nixdsoiuswfgoiigmstoehasplatriedPnni-gsomeitdahe-satdhetesyrpl(ievPgreorodouxulbiosprsoatmenhtceehosaemrl.ed,atiy1cc9ahl8abl8yien; free bovine serum albumin, whereasstearic acid oxida- cannot be degraded by P-oxidation, anditis believed to tion was either unaffected or slightly inhibited by bo- be degraded by a-oxidation, i.e. l-carbon cleavage
The results indicate that acyl-CoA synthetase Stokke, 1987; Watkins et al, 1990; Wanders et al, 19911, indiand carnitine acyltransferase located at the mitochon- cating that initial a-oxidation and possibly subsequent P-oxidrial outer membranesregulate entry of branched chain dation of branched chain fatty acids takes place in mitochonfatty acids into mitochondria

Summary

EXPERIMENTAL PROCEDURES Materials

Sodium [14C]cyanide(47-55 mCi/mmol),used for radiolabeling of fatty acids, and [l-14Clstearicacid (59 mCi/mmol) were purchased from DuPont NEN.Nycodenz, used for gradient centrifugationw, as obtained synthase, alkyl dihydroxyacetone phosphate synthase. Crude mitochondrial fractions (2 ml) were layeroedn t o 1.0-1.5) with 40 p1of 10 M HCI, and the free fatty acid produced by the top of each of the discontinuous Percoll gradients, and the tubes alkaline hydrolysis was removed by washing the aqueous phasetwice were centrifugedat 16,500 xg for 60 min. Thecell pellet major microsomal protein band in 25% Percoll was collected, diluted (free from trypsin) was dispersed inDulbecco's phosphate-buffered sawith a n equal volume of buffered sucrose, and centrifugedat 7,300 x g line and usedfor fatty acid oxidation assays (totavlolume 0.2 ml). 0.6 l-14C-Fatty acids ( a 1 4 PM) were incubated with purified rat liver mitochondria (15 pgof protein) a t 37 “C for 10 min using mitochondrial. Me, lated fatty acid oxidation (Table 11).Peroxisomal oxidation of fatty acids depended upon ATP, coenzyme A, and NAD’

RESULTS

Peroxi1s8o4mes 336

DISCUSSION

Findings

Carnitine acyltransferase