Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 μg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate.     

Isolation and partial characterization of alkaline feruloyl esterases from Aspergillus niger CFR 1105 grown on wheat bran

Feruloyl esterases (FAEs) of a strain of Aspergillus niger (CFR 1105) grown in solid state (ssf) and submerged fermentations (smf) using wheat bran both as carbon source and inducer of the enzyme were studied. The feruloyl esterase activity was maximum after 4 days in solid state as well as in submerged fermentations (32.5 and 31.5 U/g dry weight of wheat bran respectively) and the enzyme titers were comparable. The specific activity was maximum on day 2 in ssf (12.8 U/mg protein) and it decreased thereafter, whereas specific activity was maximum on day 3 (11.7 U/mg protein) in smf and it remained constant up to 5 days. Two isoenzymes of feruloyl esterases were isolated and purified to homogeneity by conventional protein purification methods from the day 5 culture filtrate of A. niger grown in smf. On a DEAE-cellulose column, two enzyme activity peaks designated as FAE-1 and FAE-2 were eluted with 0.3 and 0.35 M NaCl, respectively. They were monomeric glycoproteins with approximate molecular weights of 50 kDa (FAE-1) and 55 kDa (FAE-2), respectively. FAE-1 showed a temperature optimum of 40°C whereas FAE-2 showed a wider temperature optimum of 40–50°C. FAE-1 and FAE-2 exhibited pH optima of 9 and 6, respectively, and both were stable over a pH range of 6–9. The ability of the enzyme to be active in alkaline pH may be advantageous in biotechnological applications.     

Metagenomic mining of feruloyl esterases from termite enteric flora

A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes. The amino acid sequence lengths of the seven FAE encoding open reading frames (ORFs) ranged from 260 to 274 aa and encoded polypeptides of between 28.9 and 31.4 kDa. The highest sequence identity scores for the seven ORFs against the GenBank database were between 45 and 59 % to a number of carboxyl ester hydrolyses. The seven FAE primary structures contained sequence motifs that correspond well with a classical pentapeptide (G-x-S-x-G) serine hydrolyse signature motif which harbours the catalytic serine residue in other FAE families. Six of the seven fae genes were successfully expressed heterologously in Escherichia coli, and the purified enzymes exhibited temperature optima range of 40–70 °C and the pH optima of between 6.5 and 8.0. The k _cat/K _M ratios for the six characterised FAEs showed the following order of substrate preference: methyl sinapate?>?methyl ferulate?>?ethyl ferulate. All six FAEs showed poor conversion rates against methyl p-coumarate and methyl caffeate, both of which lacked the methoxy (O–CH₃) group substituent on the aromatic ring of the ester substrates, emphasising the requirement for at least one methoxy group on the aromatic ring of the hydroxycinnamic acid ester substrate for optimal FAE activity.     

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran

A putative α/β hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75°C, the enzyme retained at least 95% of its original activity for over 80 min; at 80°C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.     

Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts.

We have examined the metabolism of three radiolabeled 1,2-diacylglycerols (DGs) in NIH 3T3 fibroblasts. Since the lipids used are not appreciably taken up by the cells, we used a phosphatidylserine (PS)-based liposome fusion system to rapidly associate the lipid species with the plasma membrane. When 1,2-[1-14C]dioleoyl-sn-3-glycerol ([14C]DOG) is delivered in this way, it is rapidly converted predominantly to phosphatidylcholine (PC) and triacylglycerol (TG) and to a lesser extent, to monoacylglycerol (MG) and fatty acids (FA), as well as phosphatidic acid (PA) and phosphatidylinositol (PI). We present evidence that [14C] DOG is largely utilized as an intact molecule rather than being broken down to FA and then incorporated to cell lipids. Examination of the metabolism of 1-stearoyl-2-[1-14C]myristoyl-sn-3-glycerol ([14C]SMG) and 1-stearoyl-2-arachidonoyl-sn-3-glycerol ([14C]SAG) reveal important differences. Both produce substantial labeling of PC but [14C]SMG gives rise to the highest proportion of TG and the lowest of PA and PI, whereas [14C]SAG yields the opposite pattern. When phosphatidic acid labeled on its glycerol backbone (1,2-dioleoyl-sn-[U-14C] glycero-3-phosphate) was supplied to the cells via the liposomes, rapid appearance of labeled DG was found which then decreased with concomitant labeling of cellular PC and TG. Only small amounts of the glycerol backbone were recovered in PI. Our experiments identify three types of processes involved in the metabolism of plasma membrane DGs: (i) transferase-catalyzed conversions to PC and TG, (ii) lipolytic breakdown to MG and FA, and (iii) phosphorylation to PA and then conversion to PI. The relative proportions of each DG species converted to these different products are strongly dependent on the fatty acyl composition of the particular DG molecular species, even though formation of PC is the major event in all cases. Since DGs are important second messengers, our study supports the view that conversion to PC and TG can play a key role in DG signal attenuation.     

Sporotrichum thermophile type C feruloyl esterase (StFaeC): purification,characterization, and its use for phenolic acid (sugar) ester synthesis

A feruloyl esterase (StFaeC) produced by Sporotrichum thermophile was purified to homogeneity. The native StFaeC was homodimer with a subunit of M_r 23,000 and pI 3.1. The enzyme activity was optimal at pH 6.0 and 55 °C. The esterase displayed remarkable stability at pH 10.0 and retained 50% of its activity after 133 and 55 min at 55 and 60 °C, respectively. Determination of k_cat/K_m revealed that the enzyme had a broad spectrum of activity against the (hydroxyl) cinnamate esters indicating a type C feruloyl esterase. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and hydrolysed 4-nitrophenyl-5-O-trans-feruloyl-α-l-arabinofuranoside three times more efficiently than 4-nitrophenyl-2-O-trans-feruloyl-α-l-arabinofuranoside. Ferulic acid was efficiently released from wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 41% total ferulic acid released after 1 h incubation). StFaeC by itself could release FA but at a level almost 10-fold lower than that obtained in the presence of xylanase. The potential of StFaeC for the synthesis of various phenolic acid esters was examined using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. Also StFaeC catalyzed the transfer of the feruloyl group to l-arabinose in a similar system using t-butanol, with about a 40% conversion of l-arabinose to feruloylated derivative was achieved. This work is the first example of enzymatic feruloylation of a carbohydrate.     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Effects of folic acid on growth performance,ruminal fermentation,nutrient digestibility and urinary excretion of purine derivatives in post-weaned dairy calves

This study evaluated the effects of folic acid (FA) supplementation on growth performance, ruminal fermentation, nutrient digestibility and urinary purine derivatives (PD) excretion in dairy calves. Forty-eight Chinese Holstein male dairy calves at 60 ± 3.2 d of age and 89 ± 5.9 kg body weight (mean ± standard error) were assigned to one of four groups in a randomised block design. Calves in control group were fed basal diet, calves in low FA, medium FA and high FA groups with 3.6, 7.2 and 10.8 mg FA per kg basal diet, respectively. The dietary corn silage to concentrate ratio was 50:50 (dry matter [DM] basis). DM intake and average daily gain (ADG) quadratically increased, and feed conversion ratio quadratically decreased with increasing FA supplementation. Ruminal pH linearly decreased, whereas total volatile fatty acids quadratically increased. The unchanged acetate-to-propionate ratio was due to the similar change in acetate and propionate concentration. Ammonia N content quadratically decreased. Digestibility of DM, organic matter, crude protein, ether extract, neutral detergent fibre and acid detergent fibre linearly increased. Activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase linearly increased, but α-amylase and protease quadratically increased. Abundance of Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes linearly increased, but Butyrivibrio fibrisolvens and Prevotella ruminicola quadratically increased. Urinary total PD excretion quadratically increased. The results indicated that FA supplementation increased ADG, ruminal fermentation and nutrient digestibility with promoted ruminal microbial growth and enzyme activity, and the optimum dose was 7.2 mg FA per kg basal diet for calves.     

Enzymatic synthesis of hydroxycinnamic acid glycerol esters using type A feruloyl esterase from Aspergillus niger

We found that hydroxycinnamic acid (HA) glycerol esters such as 1-sinapoyl glycerol and 1-p-coumaroyl glycerol can be synthesized through a direct esterification reaction using a type A feruloyl esterase from Aspergillus niger. The water solubilities of HA glycerol esters were higher than those of the original chemicals. HA glycerol esters absorbed ultraviolet light and scavenged 1,1-diphenyl-2-picrylhydrazyl radicals.     

Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 l) produced activity at 82 nkat g⁻¹ dry substrate (corn cobs) which compared favorably to those reported for the other microorganisms. Use of de-esterified corn cobs as carbon source decreased FAE production by 5.5-fold compared to untreated corn cobs even though ferulic acid (FA) was added to the concentration found in alkali-extracts of corn cobs. Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 °C with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5–8 and up to 40 °C and retained 72 and 40% of its activity after 6 h at pH 9 and pH 10, respectively. After separation by isoelectric focusing electrophoresis, a zymogram indicated one major FAE activity exhibiting pI value of 10.5. This revised version was published online in August 2006 with corrections to the Cover Date.     

黑曲霉阿魏酸酯酶在毕赤酵母中的组成型表达

【目的】实现黑曲霉来源的阿魏酸酯酶在毕赤酵母(Pichia pastoris GS115)中的组成型表达。【方法】以黑曲霉(Aspergillus niger)基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(AnfaeA),将其与载体pGAP9K相连,构建重组表达载体p GAP9KAnfae A,经SalI线性化后电转入毕赤酵母GS115中,得到重组菌株。高效液相色谱法测定发酵液中阿魏酸酯酶活力,并对重组菌进行了发酵优化。【结果】克隆得到783 bp的阿魏酸酯酶编码基因并实现了其在毕赤酵母中的组成型表达。重组菌发酵84 h后,上清液中酶活达5.72±0.10 U/m L。重组酶(reAnfaeA)经分离纯化后比酶活为59.75 U/mg,大小约为40 k D。发酵优化结果为:葡萄糖40.0 g/L,蛋白胨10.0 g/L,酵母膏30.0 g/L,CaCO_3 0.2 g/L,种龄28 h,接种量3%(体积比),装液量50 m L/250 m L。在此条件下发酵培养,酶活达15.60±0.23 U/m L。【结论】阿魏酸酯酶在毕赤酵母中的组成型表达,对研究毕赤酵母组成型表达系统和阿魏酸酯酶的发酵生产具有一定的借鉴意义。     

Chemoenzymatic synthesis of feruloyl d-arabinose as a potential anti-mycobacterial agent

The feruloyl esterase (StFaeC) produced by Sporotrichum thermophile transfered the feruloyl group to d-arabinose using a mixture of n-hexane, t-butanol and water. About 45% conversion of d-arabinose to the feruloylated derivative was achieved. The compound had an MIC value against Mycobacterium bovis BCG of 25 μg/ml.     

In vitro propagation and bioactive compound accumulation in regenerated shoots of <Emphasis Type="Italic">Dioscorea birmanica</Emphasis> Prain &amp; Burkill

Dioscorea birmanica Prain & Burkill is a Thai medicinal plant, which is often used with other medicinal plants for the treatment of cancers, AIDS, and septicemia diseases. Large numbers of this desirable plant can be produced using the plant tissue culture techniques. The objectives of this study were to investigate techniques of in vitro propagation and to examine the bioactive compounds: diosgenin-3-O-α-l-rhamnopyranosyl (1 → 2)–β-d-glucopyranoside (DBS1) content, total phenolic content, and antioxidant activity of the regenerated shoots compared to those of rhizomes growing in the field. For shoot induction, the highest numbers of shoots (2.8 ± 0.5) and nodes per shoot (5.7 ± 0.8) occurred after the single-nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg/l BA (6-benzyladenine) for 4 weeks. Shoot multiplication was achieved on MS medium supplemented with 0.01 % activated charcoal (AC) and 2 mg/l BA in combination with 0.1 mg/l IAA or 0.2 mg/l NAA. The regenerated shoots were rooted on ½ MS medium supplemented with 0.01 % AC, 2 mg/l BA and 4 mg/l NAA for 8 weeks. The survival percentage was 71.88 and small rhizomes developed after transplanting for 4–6 weeks. The quantities of 0.37 ± 0.03 % (w/w) DBS1, 44.24 ± 8.47 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 53.67 ± 4.16 µg/ml were determined from the regenerated shoots, while 3.27 ± 0.04 % (w/w) DBS1, 259.67 ± 7.34 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 11.42 ± 3.28 µg/ml were found in the mother rhizomes.     

Synthesis of fatty acid esters and diacylglycerols at elevated temperatures by alkalithermophilic lipases from Thermosyntropha lipolytica

LipA and LipB of Thermosyntropha lipolytica DSM 11003 as previously published are the most alkalithermophilic (pH _opt ^25°C  = 9.4–9.6, T _opt = 96°C) and thermostable (T _1/2 ^24 h  = 74–76°C) lipases currently known. The purified enzymes were analyzed in organic solvents for their ability to catalyze synthesis of diacylglycerols and various alcohol fatty acids. To obtain 100% recovery and avoid a 40% and 50% loss of catalytic activity during lyophilization of purified LipA and LipB, respectively, addition of 1 mg/ml bovine serum albumin (BSA) and 25% polyethylene glycol (PEG400) was required. LipA and LipB catalyzed esterification of fatty acids and alcohols with the highest yields for octyl oleate (LipA) and lauryl oleate (LipB) and also catalyzed synthesis of 1,3-dioleoyl glycerol, 1-oleoyl-3-lauroyl glycerol, and 1-oleoyl-3-octoyl glycerol. Isooctane was the most efficient solvent for esterification reactions at 85°C. Similar to the positional specificity for the hydrolytic reaction in aqueous solutions, LipA and LipB catalyzed in organic solvents the synthesis of diacylglycerol with esterification of position 1 and 3 with a yield of 62% for di-oleoyl glycerol. The reported conversion rates do not represent the full potential of these enzymes, since only 1/100th–1/1,000th of the protein concentrations usually used in commercial processes were available. However, use of slightly increased protein concentrations confirmed the trend to higher yields with higher protein concentrations. The obtained specificity and variety of the reactions catalyzed by LipA and LipB, and their high thermostability allowing synthesis to occur at 90°C, demonstrate their great potentials for industrial applications, particularly in structured lipid biosynthesis for substrates that are less soluble at mesobiotic temperatures.     

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.     

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC₅₀ 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC₅₀ 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

     

ACE-Inhibitory and Antioxidant Activity of Temporin-Ra Peptide: Biochemical Characterization and Molecular Modeling Study

In this study, the inhibitory effect of Temporin-Ra (FP-14 peptide) on angiotensin converting enzyme (ACE) was evaluated. Inhibition mechanism was investigated by kinetic studies and molecular docking simulation. Lineweaver–Burk plot revealed that Temporin-Ra behaved as a non-competitive ACE inhibitor supported by the docking simulation. The IC₅₀ and K_i values were determined to be 22.19 μM and 36 µg/ml, respectively. Molecular docking simulation showed that Temporin-Ra bound to both of N- and C-domains of ACE by forming hydrogen bonds and electrostatic interactions; Temporin-Ra displayed higher affinity to C-domain than N-domain. Antioxidant activity of Temporin-Ra was examined using different methods. The antioxidant activity of Temporin-Ra (0.2 mg/ml) in the inhibition of linoleic acid autoxidation was evaluated to be 57 %. 1,1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt radicals scavenging activities were 60 % at 0.5 mg/ml and 37 % at 0.3 mg/ml, respectively. The hydroxyl radical scavenging of FP-14 peptide at 0.33 mg/ml was 55 %. The results suggest that Temporin-Ra is a multifunctional peptide that could be exploited to develop new anti-hypertension drugs and bio-compatible natural antioxidants.     

Linkage of p-coumaroyl and feruloyl groups to cell-wall polysaccharides of barley straw

Treatment of cell walls of barley straw with Oxyporus “cellulase” (a mixture of polysaccharide hydrolases) released compounds containing p-coumaroyl and feruloyl groups bound to carbohydrates, two of which were identified as O-[5-O-(trans-p-coumaroyl)-α-l-arabinofuranosyl]-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (PAXX) and O-[5-O-(trans-feruloyl)-α-l-arabinofuranosyl]-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (FAXX).     

Insulin increases FA uptake and esterification but reduces lipid utilization in isolated contracting muscle

We examined the effect of insulin on the synthesis and degradation of muscle lipid pools [phospholipid (PL), diacylglycerol (DG), triacylglycerol (TG)] and palmitate oxidation in isolated resting and contracting (20 tetani/min) soleus muscles. Lipid metabolism was monitored using the previously defined pulse-chase procedure. At rest, insulin significantly increased total palmitate uptake into soleus muscle (+49%, P < 0.05), corresponding to enhanced DG (+60%, P < 0.05) and TG (+61%, P < 0.05) esterification, but blunted palmitate oxidation (-38%, P < 0.05) and TG hydrolysis (-34%, P < 0.05). During muscle contraction, when total palmitate uptake was increased, insulin further enhanced uptake (+21%, P < 0.05) and esterification of fatty acids (FA) to PL (+73%, P < 0.05), DG (+19%, P < 0.05), and TG (+161%, P < 0.01). Despite a profound shift in the relative partitioning of FA away from esterification and toward oxidation during contraction, the increase in palmitate oxidation and TG hydrolysis was significantly blunted by insulin [oxidation, -24% (P = 0.05); hydrolysis, -83% (P < 0.01)]. The effects of insulin on FA esterification (stimulation) and oxidation (inhibition) during contraction were reduced in the presence of the phosphatidylinositol 3-kinase inhibitor LY-294002. In summary, the effects of insulin and contraction on palmitate uptake and esterification are additive, while insulin opposes the stimulatory effect of contraction on FA oxidation and TG hydrolysis. Insulin's modulatory effects on muscle FA metabolism during contraction are mediated at least in part through phosphatidylinositol 3-kinase.