Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo β-oxidation but are first subjected to fatty acid α-oxidation in which the terminal carboxyl-group is released as CO₂. The mechanism of α-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid α-oxidation, and the complete α-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid α-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Nutrient absorption,storage and remobilization in octopus vulgaris

Average absorption and conversion to ¹⁴CO₂for free leucine included in a meal were 96% and 30% after 24 h. The values for glucose were 98% and 48% and for palmitate 46% and 12.5%. Muscle was the major repository of leucine (38% of the total ingested) and glucose (44%), but the digestive gland contained most of the palmitate (20%).

During normal feeding ¹⁴CO₂ production from octopuses given leucine dropped to low stable levels after only 2 days, those given glucose required 4 to 5, but palmitate apparently did not enter a stable reserve. Fasting increased the release of ¹⁴CO₂ from octopuses given palmitate and leucine, but glucose animals showed little change. A 5‐fold increase in ¹⁴CO₂ production during forced exercise after fasting by octopuses given glucose may indicate increased carbohydrate catabolism. Reduced ¹⁴CO₂production in exercise after other substrates is consistent with this, suggesting muscle carbohydrate reserves may be exclusively for locomotion.

A comparison of nutrient uptake, catabolism and growth suggests that lipids are the limiting nutrient for octopuses on a crab diet.     

INTERACTION OF FATTY ACID AND GLUCOSE OXIDATION BY CULTURED HEART CELLS

Chick embryo heart cells in tissue culture actively oxidize [1-¹⁴C]palmitate to ¹⁴CO₂. Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30–70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in ¹⁴CO₂ production from [1-¹⁴C]palmitate. Conversely, palmitate had a similar sparing effect on [¹⁴C]glucose oxidation to ¹⁴CO₂. Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.     

Fat metabolism in higher plants. LXII. The pathway of ricinoleic acid catabolism in the germinating castor bean (Ricinus communis L.) and pea (Pisum sativum L.)

With cell-free extracts from both germinating peas and castor beans, O-¹⁴Cricinoleate (¹⁴C located at odd-numbered positions in the carbon chain) was β-oxidized at least to the C₁₀ level. With the pea system, formation of unsaturated hydroxy acid intermediates to the C₁₂ level occurred. Acetyl-CoA was the primary product of β-oxidation activity. Although the pathway beyond the C₁₂-intermediate level was not resolved conclusively, two alternative routes may exist in the castor bean system to convert 4-hydroxy-decanoic acid to 2-keto-octanoate, one involving 4-keto-decanoate, the other 2-hydroxy-octanoate. Subsequent degradation of the 2-keto-octanoate tentatively involves an α-oxidation step, releasing CO₂ and heptanoic acid. Further β-oxidation of the latter is envisaged to yield propionyl and acetyl CoA. All necessary enzymes for the catabolism of ricinoleic acid to propionate appear to be associated with the cytosomes.     

Differential effects of acetate on palmitate and octanoate oxidation: Segregation of acetyl CoA pools

In isolated hepatic mitochondria, sodium acetate had little effect on the oxidation of octanoate, but conspicuously inhibited the oxidation of palmitate. This differential effect of acetate on long-chain and short-chain fatty acid oxidation is not due to inhibition of the activation or transfer of long-chain fatty acids into the mitochondria. Both palmitate and octanoate reduced CO₂ production from acetate. Palmitate and octanoate mutually inhibited CO₂ production from each other to the same extent. Acetate stimulated ketogenesis from palmitoyl-1-carnitine to the same extent as it inhibited oxygen uptake and CO₂ production from palmitate. This suggests that acetate causes a redistribution of the end products of palmitate oxidation toward ketogenesis rather than toward total oxidation to CO₂ and H₂O. Acetyl CoA derived from acetate or palmitate may share a common pool or pathway, thus each is mutally inhibitory toward the oxidation of the other. Either because of the existence of separate pools, or because octanoate is the preferred substrate, acetate metabolism does not inhibit O₂ uptake or CO₂ production from octanoate, whereas the oxidation of octanoate dilutes the CO₂ produced from labeled acetate. This may be explained by compartmentation or preferred pathways for the disposition of acetyl CoA derived from different sources.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

Effect of acetaldehyde on fatty acid oxidation and ketogenesis by hepatic mitochondria.

Acetaldehyde inhibited the oxidation of fatty acids by rat liver mitochondria as assayed by oxygen consumption and CO₂ production. ADP-stimulated oxygen uptake was more sensitive to inhibition by acetaldehyde than was uncoupler-stimulated oxygen uptake, suggesting an effect of acetaldehyde on the electron transport-phosphorylation system. This conclusion is supported by the decrease in the respiratory control ratio, associated with fatty acid oxidation. Acetaldehyde depressed ketone body production as well as the content of acetyl CoA during palmitoyl-1-carnitine oxidation. Acetaldehyde was considerably more inhibitory toward fatty acid oxidation than was acetate. Therefore, the inhibition by acetaldehyde is not mediated by acetate, the direct product of acetaldehyde oxidation by the mitochondria. Oxygen uptake was depressed by acetaldehyde to a slightly, but consistently, greater extent in the absence of fluorocitrate, than in its presence. This suggests inhibition of oxygen consumption from β-oxidation to acetyl CoA and that which arises from citric acid cycle activity. The inhibition of fatty acid oxidation is not due to any effect on the activation or translocation of fatty acids into the mitochondria.The depression of the end products of fatty acid oxidation (CO₂, ketones, acetyl CoA) as well as the greater sensitivity of palmitate oxidation compared to acetate oxidation, suggests inhibition by acetaldehyde of β-oxidation, citric acid cycle activity, and the respiratory-phosphorylation chain. Neither the activities of palmitoyl CoA synthetase nor carnitine palmitoyltransferase appear to be rate limiting for fatty acid oxidation.     

Fatty Acid Chain Elongation in Palmitate-perfused Working Rat Heart: MITOCHONDRIAL ACETYL-CoA IS THE SOURCE OF TWO-CARBON UNITS FOR CHAIN ELONGATION*

Rat hearts were perfused with [1,2,3,4-¹³C₄]palmitic acid (M+4), and the isotopic patterns of myocardial acylcarnitines and acyl-CoAs were analyzed using ultra-HPLC-MS/MS. The 91.2% ¹³C enrichment in palmitoylcarnitine shows that little endogenous (M+0) palmitate contributed to its formation. The presence of M+2 myristoylcarnitine (95.7%) and M+2 acetylcarnitine (19.4%) is evidence for β-oxidation of perfused M+4 palmitic acid. Identical enrichment data were obtained in the respective acyl-CoAs. The relative ¹³C enrichment in M+4 (84.7%, 69.9%) and M+6 (16.2%, 17.8%) stearoyl- and arachidylcarnitine, respectively, clearly shows that the perfused palmitate is chain-elongated. The observed enrichment of ¹³C in acetylcarnitine (19%), M+6 stearoylcarnitine (16.2%), and M+6 arachidylcarnitine (17.8%) suggests that the majority of two-carbon units for chain elongation are derived from β-oxidation of [1,2,3,4-¹³C₄]palmitic acid. These data are explained by conversion of the M+2 acetyl-CoA to M+2 malonyl-CoA, which serves as the acceptor for M+4 palmitoyl-CoA in chain elongation. Indeed, the ¹³C enrichment in mitochondrial acetyl-CoA (18.9%) and malonyl-CoA (19.9%) are identical. No ¹³C enrichment was found in acylcarnitine species with carbon chain lengths between 4 and 12, arguing against the simple reversal of fatty acid β-oxidation. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl-CoA with simultaneous inhibition of carnitine palmitoyltransferase 1b and 2) catalyze the palmitoyl-CoA-dependent incorporation of ¹⁴C from [2-¹⁴C]malonyl-CoA into lipid-soluble products. In conclusion, rat heart has the capability to chain-elongate fatty acids using mitochondria-derived two-carbon chain extenders. The data suggest that the chain elongation process is localized on the outer surface of the mitochondrial outer membrane.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Characterization of the Glycolipid Associated with Alzheimer Paired Helical Filaments

Abstract: In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)-assisted carbohydrate linkage-analysis, one- and two-dimensional NMR, and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The ¹H NMR spectrum of acid-hydrolyzed protein-resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two-dimensional homonuclear correlated spectroscopy, homonuclear Hartmann-Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the ¹H and ¹³C NMR spectrum of native PHF were assigned to a nonreducing terminal α-1,6-glycosidically linked glucose, an internal α-1,6-linked glucose, and an α-1,2,6-linked glucose. The narrow line-widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF-associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal α-1,6-linked glucose and α-1,2,6-linked glucose in a molar ratio of 2:1:1. Three components of the PHF-associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI-MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C₂₀ fatty acid (Glc₁₃ + C₂₀ or Glc₁₃ + C_20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C₁₄, C₁₆, or C₁₈ esterified fatty acids {D[(Glc₅ + C₁₈) + (Glc₆ + C₁₆)] or D[(Glc₅ + C₁₄) + (Glc₆ + C₁₄)]}. The ratio of glucose to fatty acid calculated from these best-fit structures of the more abundant mass components (5.5 ± 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid-hydrolyzed prcPHF (6.2 ± 1.6). Structural similarities between PHF-associated glycolipid and other glycolipid amphiphiles known to form PHF-like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.     

Recovery of cells in vitro from the effects hypoxia and hyperoxia

The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO₂ environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O₂ exposure for at least 20 days while continuous exposure to high O₂ atmosphere results in degeneration and death after 7–10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O₂ tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO₂ atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.     

Interactions between substrate,fermentation end-products,buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro

Summary In animal nutrition, incubation of feed samples with CO₂/HCO₃-buffered rumen fluid is used to predict the nutritional values of the feed. During fermentation, volatile fatty acids (VFAs) are produced, which release CO₂ from the buffer through their H⁺ ions. This indirect gas production amounted to 20.8 ml gas per mmol VFA. By incubating glucose, rice starch and cellulose, the relationship between direct and indirect gas production in relation to fermentation kinetics was studied. The total amount of gas formed was found to be dependent on the composition of the fermentation end-products formed. This could be described by: ml gas = M_v·mmol HAc + 2M_v·mmol HB + 0.87M_v·mmol Tot. VFA where HAc = acetic acid; HB = butyric acid; and M_v = molar gas volume. No clear relationship was found between the rate of fermentation and total gas production. From rice starch more total gas was produced than from glucose and cellulose, which were fermented faster and slower, respectively. Correspondence to: S. F. Spoelstra     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

Studies on the role of β-cell metabolism in the insulinotropic effect of α-ketoisocaproic acid

α-Ketoisocaproic acid has been shown to be apotent insulin secretagogue but the mechanism has not been elucidated. To define the role of β-cell metabolism in the insulinotropic activity of α-ketoisocaproic acid the utilization of glucose and the oxidation of α-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured.Glucose metabolism was never enhanced by α-ketoisocaproic acid. The same ¹⁴CO₂ amounts were released from the non-secretagogue [1-¹⁴C]isovaleric acid (10 mM) or from α-keto [2-¹⁴C]isocaproic acid (5–20 mM). Pyruvate (20 mM) did not inhibit α-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of α-ketoisocaproic acid by more than 40%.The results indicate that stimulated insulin release in response to α-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in α-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.