Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Interaction between leucine and palmitate catabolism in 3T3-L1 adipocytes and primary adipocytes from control and obese rats

Metabolic profiling studies have highlighted increases in the plasma free fatty acid (FFA) and branched-chain amino acid (BCAA) concentrations, which are hallmarks of the obese and insulin-resistant phenotype. However, little is known about how the increase of the BCAA concentration modifies the metabolic fate of FFA, and vice versa, in adipocytes. Therefore, we incubated differentiated 3T3-L1 adipocytes or primary adipocytes from rats fed a control or a high-fat diet with: (1) 0, 250, 500 and 1000 μM of leucine and determined the oxidation and incorporation of [1-¹⁴C]-palmitate into lipids or proteins or (2) 0, 250, 500 or 1000 μM of palmitate and evaluated the oxidation and incorporation of [U-¹⁴C]-leucine into lipids or proteins. Leucine decreased palmitate oxidation and increased its incorporation into the lipid fraction in adipocytes; the latter was reduced in adipocytes from obese rats. However, palmitate increased leucine oxidation in adipocytes as well as reduced leucine incorporation into the protein and lipid fractions in adipocytes from obese rats. These results demonstrate that leucine modifies the metabolic fate of palmitate, and vice versa, in adipocytes and that the metabolic interaction between leucine and palmitate catabolism is altered in adipocytes from obese rats.     

Inhibition of Brain Energy Metabolism by the Branched-chain Amino Acids Accumulating in Maple Syrup Urine Disease

In the present work we investigated the in vitro effect of the branched-chain amino acids (BCAA) accumulating in maple syrup urine disease (MSUD) on some parameters of energy metabolism in cerebral cortex of rats. ¹⁴CO₂ production from [1-¹⁴C]acetate, [1-5-¹⁴C]citrate and [U-¹⁴C]glucose, as well as glucose uptake by the brain were evaluated by incubating cortical prisms from 30-day-old rats in the absence (controls) or presence of leucine (Leu), valine (Val) or isoleucine (Ile). All amino acids significantly reduced ¹⁴CO₂ production by around 20–55%, in contrast to glucose utilization, which was significantly increased by up to 90%. Furthermore, Leu significantly inhibited the activity of the respiratory chain complex IV, whereas Val and Ile markedly inhibited complexes II–III, III and IV by up to 40%. We also observed that trolox (α-tocopherol) and creatine totally prevented the inhibitory effects provoked by the BCAA on the respiratory chain complex activities, suggesting that free radicals were involved in these effects. The results indicate that the major metabolites accumulating in MSUD disturb brain aerobic metabolism by compromising the citric acid cycle and the electron flow through the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.     

Glial Metabolism of Isoleucine

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO₂, they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of ¹³C-labelled metabolites generated in the catabolism of [U-¹³C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3-¹³C]glutamine, [4,5-¹³C]glutamine and ¹³C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine. Special issue article in honor of Dr. George DeVries.     

Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-¹³C₂]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [¹³C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-¹³C₂]hexadecane-derived fatty acids contained either two ¹³C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.     

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs–Ringer bicarbonate buffer only, 0–1 mM [3,4-¹³C₂]-4-hydroxynonanoate, and 0–2 mM [5,6,7-¹³C₃]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-¹³C₃]heptanoate perfusates.     

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.     

Biosynthesis of Acyl Moieties of Capsaicin and its Analogues from Valine and Leucine in Capsicum Fruits

Biosynthetic pathways of acyl moieties of capsaicinoid in intactCapsicum fruits and spheroplasts prepared from placentas ofCapsicum fruits were examined using a radioisotopic technique.In intact Capsicum fruits, L-[U-¹⁴C] valine was incorporatedinto capsaicin and dihydrocapsaicin, the acyl constituents ofwhich are even-number branched chain fatty acids, while L-[U-¹⁴C]leucine was incorporated into nordihydrocapsaicin and homodihydrocapsaicin,which have odd-number branched chain facty acids as the acylmoieties. The intermediates of the odd- and even-number branchedchain fatty acids were identified with GLC/GPC after the spheroplastshad been incubated with L-[U-¹⁴C] valine or L-[U-¹⁴C] leucine.After incubation with L-[U-¹⁴C] valine, isobutyric acid and8-methyl nonanoic acid were detected, while isopentanoic acidand 9-methyl decanoic acid were found after incubation withL-[U-¹⁴C] leucine. The involvement of a-ketoisovalerate or a-ketoisocaproatein the biosynthesis of acyl moieties of capsaicinoid was alsodemonstrated in vitro using cell-free extracts of the placentasof Capsicum fruits. These findings suggest that the acyl moietiesof individual capsaicinoids in Capsicum fruits are synthesizedby pathways similar to those proposed for adipose tissue andbacteria. ¹Formation and Metabolism of Pungent Principle of Capsicum Fruits.Part IX. (Received September 2, 1980; Accepted November 17, 1980)     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Catabolism of nutritionally essential amino acids in developing porcine enterocytes

This study was conducted using the piglet model to test the hypothesis that mucosal cells of the neonatal small intestine can degrade nutritionally essential amino acids (EAA). Enterocytes were isolated from the jejunum of 0-, 7-, 14-, and 21-day-old pigs, and incubated for 45 min in Krebs buffer containing plasma concentrations of amino acids and one of the following L-[1-¹⁴C]- or L-[U-¹⁴C]-amino acids plus unlabeled tracees at 0.5, 2, or 5 mM: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In these cells, branched-chain amino acids (BCAA) were extensively transaminated and 15–50% of decarboxylated branched-chain α-ketoacids (BCKA) were oxidized to CO₂ depending on the age of piglets. BCAA transamination increased but their decarboxylation decreased between 0 and 14 days of age. Addition of 1 and 2 mM α-ketoglutarate to incubation medium dose-dependently stimulated BCAA transamination without affecting their decarboxylation. Western blot analysis revealed that the abundance of mitochondrial BCAA aminotransferase declined but cytosolic BCAA aminotransferase increased between 0 and 14 days of age, with the cytosolic protein being the major isoform in 7- to 21-day-old pigs. BCKA dehydrogenase protein existed primarily as the phosphorylated (inactive) form in enterocytes of newborn pigs and its levels were markedly reduced in older pigs. All measured parameters of BCAA metabolism did not differ between 14- and 21-day-old pigs. In contrast to BCAA, catabolism of methionine and phenylalanine was negligible and that of other EAA was absent in enterocytes from all ages of piglets due to the lack of key enzymes. These results indicate that enterocytes are an important site for substantial degradation of BCAA but not other EAA in the neonatal gut.     

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of ¹⁴C from ¹⁴C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene.     

Studies on the Bacterial Formation of Peptide Antibiotic,Colistin

The biosynthetic mechanism of 6-methyloctanoic and isooctanoic acids, which are present in the amide linkage with the α-amino group of the terminal α, γ-diaminobutyric acid residue of colistin A and B, respectively was investigated. From the isotopic experiments using isoleucine-U-¹⁴C, valine-U-¹⁴C and acetic acid-2-¹⁴C, it was concluded that 6-methyloctanoic and isooctanoic acids were derived from isoleucine and valine, respectively.

Amino acids pooled in colistin-producing cells grown in the synthetic medium were abundant in isoleucine, valine and leucine, which were probable precursors of the abovedescribed fatty acid components of colistin and cellular fatty acids. On the other hand, 6-methyloctanoic and isooctanoic acids were not found in the cellular fatty acids, while C-15 and C-16 branched chain fatty acids usually found in Bacillus sp. were abundantly contained in the cells, indifferently of an improved capacity of colistin formation.     

The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis

1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-¹⁴C]stearate, [U-¹⁴C]oleate, [U-¹⁴C]palmitate and [1-¹⁴C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4·2mg./min./kg. body wt. for acetate, and 0·18, 0·28 and 0·42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18–21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [¹⁴C]acetate infusion, milk triglyceride fatty acids of chain length C₄–C₁₄ showed specific radioactivities that were 75–90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C₄–C₁₄ arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.     

Transfer of fatty acids and proteins between cytoplasmic membranes and mesosomes of Bacillus subtilis var. niger

A transfer of labelled branched-chain fatty acids or proteins between mesosomes, periplasmic space and protoplasts, is suggested in vivo and demonstrated in vitro.In the sole mesosomal fraction, [¹⁴C]valine or [¹⁴C]isoleucine are, always, more incorporated in fatty acids than in proteins.Mesosomal fatty acids can be transferred to the protoplasts, but protoplasts seems to give essentially amino acids to mesosomes.     

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H⁺ gradient, but not on a Na⁺ or K⁺ gradient at the cytoplasmic membrane level.     

Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly 14C-labeled BCAA, together with [methyl-3H]methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of 14C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Sequential ordered fatty acid alpha oxidation and Delta9 desaturation are major determinants of lipid storage and utilization in differentiating adipocytes

Herein, we exploit the power of global lipidomics to identify the critical role of peroxisomal processing of fatty acids in adipocyte lipid storage and metabolism. Remarkably, 3T3-L1 differentiating adipocytes rapidly acquired the ability to alpha oxidize unbranched fatty acids, which is manifested in the accumulation of odd chain length unbranched fatty acids in all major lipid classes. Moreover, in differentiating adipocytes, unsaturated odd chain length fatty acids in TAG molecular species contained exclusively Delta9 olefinic linkages. Unsaturated fatty acids (e.g., oleic and palmitoleic acids) were not subject to alpha oxidation, resulting in the absence of Delta8 unsaturated odd chain length fatty acids. This highly selective substrate utilization resulted in the obligatory sequential ordering of alpha oxidation prior to Delta9 desaturation. On the basis of these results, a putative type 2 peroxisomal localization sequence was identified at the N-terminus of mouse stearoyl-CoA desaturase I (SCD I) comprised of (30)KVKTVPLHL(38). Kinetic analysis demonstrated that the rate of alpha oxidation of exogenously administered [9,10-(3)H]palmitic acid increased 4-fold during differentiation. Similarly, quantitative PCR demonstrated a 4-fold increase in phytanoyl-CoA alpha hydroxylase (PAHX) and fatty acyl-CoA oxidase (FACO) mRNA levels during differentiation. Collectively, these results underscore the role of peroxisomal fatty acid processing as an important determinant of the metabolic fate of fatty acids in the differentiating adipocyte.     

Metabolic Compartmentation in Cortical Synaptosomes: Influence of Glucose and Preferential Incorporation of Endogenous Glutamate into GABA

Metabolism of glutamine was determined under a variety of conditions to study compartmentation in cortical synaptosomes. The combined intracellular and extracellular amounts of [U-¹³C]GABA, [U-¹³C]glutamate and [U-¹³C]glutamine were the same in synaptosomes incubated with [U-¹³C]glutamine in the presence and absence of glucose. However, the concentration of these amino acids was decreased in the latter group, demonstrating the requirement for glucose to maintain the size of neurotransmitter pools. In hypoglycemic synaptosomes more [U-¹³C]glutamine was converted to [U-¹³C]aspartate, and less glutamate was re-synthesized from the tricarboxylic acid (TCA) cycle, suggesting use of the partial TCA cycle from -ketoglutarate to oxaloacetate for energy. Compartmentation was studied in synaptosomes incubated with glucose plus labeled and unlabeled glutamine and glutamate. Incubation with [U-¹³C]glutamine plus unlabeled glutamate gave rise to [U-¹³C]GABA but not labeled aspartate; however, incubation with [U-¹³C]glutamate plus unlabeled glutamine gave rise to [U-¹³C]aspartate, but not labeled GABA. Thus the endogenous glutamate formed via glutaminase in synaptic terminals is preferentially used for GABA synthesis, and is metabolized differently than glutamate taken up from the extracellular milieu.