Wet-type grinder-treated okara modulates gut microbiota composition and attenuates obesity in high-fat-fed mice.

Wet-type grinder (WG) is a nanofiber technology used to atomize dietary fiber-rich materials. WG-treated okara (WGO) exhibits high dispersion and viscosity similar to those of viscous soluble dietary fibers. Here, we studied the effect of WGO supplementation on obesity and gut microbiota composition in high-fat diet (HFD)-fed mice. WGO intake suppressed body weight gain and fat accumulation, improved glucose tolerance, lowered cholesterol levels, and prevented HFD-induced decrease in muscle mass. WGO supplementation also led to cecum enlargement, lower pH, and higher butyrate production. The bacterial 16S ribosomal RNA genes (16S rDNA) were sequenced to determine the gut microbiota composition of the fecal samples. Sequencing of bacterial 16S rDNA revealed that WGO treatment increased the abundance of butyrate producer Ruminococcus and reduced the abundances of Rikenellaceae, Streptococcaceae, and Prevotellaceae, which are related to metabolic diseases. Metabolomics analysis of the plasma of WGO- and cellulose-treated mice were conducted using ultra-high-performance liquid chromatography-mass spectrometry. Metabolic pathway analysis revealed that the primary bile acid biosynthesis pathway was significantly positively regulated by WGO intake instead of cellulose. These results demonstrate that WG is useful for improving functional properties of okara to prevent metabolic syndromes, including obesity, diabetes, and dyslipidemia.

Antidepressant properties of voluntary exercise mediated by gut microbiota.

Although regular exercise has been reported to prevent depression, it has not been clarified whether the gut microbiota is involved in the factors that prevent depression through exercise. We investigated the effects of voluntary exercise on the gut microbiota and the prevention of depression-like behaviors using mice. C57BL/6 J male mice were subjected to 10 weeks of sedentary control or wheel running, then they were subjected to social defeat stress (SDS). Exercise attenuated that sucrose drinking was decreased by SDS treatment. Exercise increased the expression of Bdnf and decreased expression of Zo-1 and Claudin5 in the brain. Fecal Turicibacter, Allobaculum, and Clostridium sensu stricto, and propionate in the cecum were decreased by the exercise. Voluntary exercise-induced antidepressant properties might be partially caused by suppression of serotonin uptake into gut microbiota and increase the permeability of the blood-brain barrier via reduced propionate production.

Icing after skeletal muscle injury with necrosis in a small fraction of myofibers limits inducible nitric oxide synthase-expressing macrophage invasion and facilitates muscle regeneration.

Growing evidence from animal experiments suggests that icing after skeletal muscle injury is harmful to muscle regeneration. However, these previous experimental models yielded massive necrotic myofibers, whereas muscle injury with necrosis in a small myofiber fraction (<10%) frequently occurs in human sports activities. Although macrophages play a proreparative role during muscle regeneration, they exert a cytotoxic effect on muscle cells through an inducible nitric oxide synthase (iNOS)-mediated mechanism. In this study, we established an animal injury model with necrosis limited to a small myofiber fraction and investigated the effect of icing on muscle regeneration with a focus on macrophage-related events. Icing after muscle injury of this model resulted in an enlarged size of regenerating myofibers compared with those in untreated animals. During the regenerative process, icing attenuated the accumulation of iNOS-expressing macrophages, suppressed iNOS expression in the whole damaged muscle, and limited the expansion of the injured myofiber area. In addition, icing increased the ratio of M2 macrophages within the injured site at an earlier time point than that in untreated animals. Following these phenomena in icing-treated muscle regeneration, an early accumulation of activated satellite cells within the damaged/regenerating area occurred. The expression level of myogenic regulatory factors, such as MyoD and myogenin, was not affected by icing. Taken together, our results suggest that icing after muscle injury with necrosis limited to a small fraction of myofibers facilitates muscle regeneration by attenuating iNOS-expressing macrophage invasion, limiting muscle damage expansion, and accelerating the accumulation of myogenic cells which form regenerating myofibers.

Acetyl-L-carnitine attenuates Poly I:C-induced sickness behavior in mice.

Fatigue is accompanied by a decrease in physical activity or malaise, and might be reduced by acetyl-L-carnitine (ALC) administration. The purpose of this study was to investigate the preventive effects of ALC on Poly I:C-induced sickness behavior in mice. For the experiment, male C3H/HeN mice were used and treated with ALC for 5 days before Poly I:C administration. ALC administration attenuated the decrease in wheel behavior activity of mice at 24 h after Poly I:C administration and ALC-treated mice quickly recovered from the sickness behavior. The gene expression of brain-derived neurotrophic factor (BDNF) in the cerebrum and hippocampus, which is associated with physical activity, was higher in the ALC-treated group. Translocator protein 18kDa (TSPO), which has cytoprotective effects, was up-regulated in the cerebrum and hippocampus, suggesting that ALC suppressed the decrease in activity induced by Poly I:C treatment through enhancement of cytoprotective effects in the brain.

LPS-induced TNF-α production is attenuated by intake with PHGG via gut microbial fermentation in mice.

OBJECTIVES: Intake of dietary fibers promotes the production of short-chain fatty acids (SCFAs), which can affect host inflammation via gut microbial fermentation. Although partially hydrolyzed guar-gum (PHGG) is a water-soluble dietary fiber with lower viscosity, its benefits in acute inflammation are yet to be determined. The aim of this study was to investigate the effect of PHGG intake on the lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production. METHODS: Nine-wk-old male C3 H/HeN mice were used in this study, and they were randomly divided into control diet (CD) and CD + 5% PHGG (GGCD) groups. After a dietary intervention of 6 wk, LPS (1 mg/kg) was injected into the orbital vein. Plasma TNF-α concentration and SCFAs in cecum contents were then measured. Also, the effect of gut microbiota on LPS-induced TNF-α production was evaluated in PHGG-fed mice before and after antibiotic treatment. RESULTS: PHGG intake accelerated a dramatic suppression of LPS-induced TNF-α production (P < 0.01). PHGG-induced low pH in feces (P < 0.05) indicates that the gut microbiota induced high fermentation. Indeed, SCFAs in cecum contents of GGCD mice were significantly higher than in the CD group (P < 0.05). Furthermore, PHGG intake after antibiotic treatment did not induce the suppression of TNF-α. CONCLUSION: These results demonstrated that inflammation was inhibited by habitual PHGG ingestion, suggesting that this phenomenon might be associated with changes in gut microbiota-induced SCFAs production.

The Effect of Voluntary Exercise on Gut Microbiota in Partially Hydrolyzed Guar Gum Intake Mice under High-Fat Diet Feeding.

Although dietary fiber treatment alters the gut microbiota and its metabolite production, it is unclear whether or not exercise habits can have a supplemental effect on changes in gut microbiota in dietary fiber-treated mice. To clarify the supplemental effect of voluntary exercise on gut microbiota in partially hydrolyzed guar gum (PHGG), which is a soluble dietary fiber, treated mice under high-fat diet (HFD) feeding, 4-week-old male C57BL/6J mice (n = 80) were randomly divided into two dietary groups: the control-diet (CD) and HFD. Then, each dietary group was treated with or without PHGG, and with or without wheel running. After the experimental period, measurement of maximal oxygen consumption, a glucose tolerance test and fecal materials collection for analysis of gut microbiota were carried out. Voluntary exercise load in PHGG treatment under HFD feeding showed the supplemental effect of exercise on obesity (p < 0.01) and glucose tolerance (p < 0.01). Additionally, in both CD and HFD groups, voluntary exercise accelerated the decrease in the Firmicutes/Bacteroidetes ratio in mice fed with PHGG (p < 0.01). These findings suggest that voluntary exercise might activate the prevention of obesity and insulin resistance more via change in gut microbiota in mice administrated with PHGG.

Sparassis crispa Intake Improves the Reduced Lipopolysaccharide-Induced TNF-α Production That Occurs upon Exhaustive Exercise in Mice.

Our previous study showed that lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production is inhibited by acute exhaustive exercise in mice, leading to transient immunodepression after exercise. Sparassis crispa (SC), an edible mushroom, has immunopotentiative properties. This study aimed to clarify the effects of SC intake on reduced LPS-induced TNF-α production upon exhaustive exercise in mice. Male C3H/HeN mice were randomly divided into three groups: normal chow intake + resting sedentary, normal chow intake + acute exhaustive treadmill running exercise, and SC intake (chow containing 5% SC powder for 8 weeks) + the exhaustive exercise groups. Each group was injected with LPS immediately after the exhaustive exercise or rest. Plasma and tissue TNF-α levels were significantly decreased by exhaustive exercise. However, this reduction of the TNF-α level was partially attenuated in the plasma and small intestine by SC intake. Although levels of TLR4 and MyD88 protein expression were significantly decreased in tissues by exhaustive exercise, the reduction of TLR4 and MyD88 levels in the small intestine was partially attenuated by SC intake. These results suggest that SC intake attenuates exhaustive exercise-induced reduction of TNF-α production via the retention of TLR4 and MyD88 expression in the small intestine.

Gene Expression Profiles for Macrophage in Tissues in Response to Different Exercise Training Protocols in Senescence Mice.

Age-induced chronic inflammation is prevented by aerobic and resistance exercise training. However, the effects of the mechanism of exercise on chronic inflammation in each tissue remains unclear. The aim of this study was to investigate the effects of resistance and aerobic training on gene expression profiles for macrophage infiltration and polarization (M1/M2 ratio) with chronic inflammation in various tissues of aged model mice. Male 38-week-old SAMP1 (senescence-accelerated prone mouse 1) mice were randomly divided into three groups-sedentary (Aged-Sed-SAMP1), aerobic training (Aged-AT-SAMP1; voluntary running), and resistance training-for 12 weeks (Aged-RT-SAMP1; climbing ladder). Resistance and aerobic exercise training prevented an increase in circulating TNF-α levels (a marker of systemic inflammation) in aged SAMP1 mice, along with decreases in tissue inflammatory cytokine (TNF-α and IL-1ß) mRNA expression in the heart, liver, small intestine, brain, aorta, adipose, and skeletal muscle, but it did not change the levels in the lung, spleen, and large intestine. Moreover, resistance and aerobic exercise training attenuated increases in F4/80 mRNA expression (macrophage infiltration), the ratio of CD11c/CD163 mRNA expression (M1/M2 macrophage polarization), and MCP-1 mRNA expression (chemokine: a regulator of chronic inflammation) in the chronic inflamed tissues of aged SAMP1 mice. These results suggested that resistance and aerobic exercise training-induced changes in gene expression for macrophage infiltration and polarization in various tissues might be involved in the prevention of age-related tissue chronic inflammation, and lead to a reduction of the increase in circulating TNF-α levels, as a marker of systemic inflammation, in aged SAMP1 mice.

Reduction of Real-Time Imaging of M1 Macrophage Chemotaxis toward Damaged Muscle Cells is PI3K-Dependent.

Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the "killed-C2C12" cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.

Resistance training prevents muscle fibrosis and atrophy via down-regulation of C1q-induced Wnt signaling in senescent mice.

Increased complement component 1q (C1q) secretion with aging leads to muscle fibrosis and atrophy whereas resistance training attenuates circulating C1q levels. This study aimed to clarify whether resistance exercise-induced reduction of C1q secretion contributes to the inhibition of fibrosis and atrophy in aged muscles. Young (13-wk-old) and aged (38-wk-old) senescence-accelerated mouse prone 1 mice were randomly assigned to one of 4 groups: a young or aged sedentary control group, or a young or aged resistance training (climbing a ladder 3 d/wk for 12 wk) group. We found that resistance training ameliorated muscle fibrosis and atrophy in aged mice, concomitant with decreased circulating and muscle C1q levels and attenuated activation of muscle Wnt signaling (glycogen synthase kinase ß/ß-catenin), including ß-catenin in satellite (Pax7+/DAPI+) and fibroblast (vimentin+/DAPI+) cells. Furthermore, during muscle regeneration after mice were injured by cardiotoxin injection, we observed a reduction in circulating C1q levels, the inhibition of muscle fibrosis and repair, and decreased in the activation of muscle cytoplasmic and nuclear ß-catenin in aged mice from the resistance training group, but these effects were cancelled by a single preadministration of exogenous recombinant C1q. In addition, resistance training attenuated aging-related muscle loss concomitant with decreased expression of both muscle ring-finger protein 1 and muscle atrophy F-box in the muscle. Thus, resistance training-induced changes in circulating C1q levels may contribute to the prevention of muscle fibrosis and atrophy via muscle Wnt signaling in senescent mice.-Horii, N., Uchida, M., Hasegawa, N., Fujie, S., Oyanagi, E., Yano, H., Hashimoto, T., Iemitsu, M. Resistance training prevents muscle fibrosis and atrophy via down-regulation of C1q-induced Wnt signaling in senescent mice.

Palmitoleic acid induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

Exhaustive exercise increases the TNF-α production in response to flagellin via the upregulation of toll-like receptor 5 in the large intestine in mice.

Although intense exercise may induce temporary immune depression, it is unclear whether exercise stimulates tumor necrosis factor-alpha (TNF-α) production in response to flagella protein flagellin (FG), which binds to toll-like receptor 5 (TLR5) and induces the production of pro-inflammatory cytokines. Male C3H/HeN mice were administered FG (1mg/kg, i.v.) after exhaustive exercise (EX), and the plasma TNF-α concentrations were examined. The production of TNF-α and the TLR5 expression in both RAW264 and Caco2 cells were measured under FG conditions in vitro. Although the plasma TNF-α concentrations were observed to significantly increase in both the EX and non-EX (N-EX) mice (p<0.01, respectively) following FG injection, the TNF-α levels in the EX mice were significantly higher than those observed in the N-EX mice (p<0.01). Epinephrine (Ep) treatment accelerated the FG-induced TNF-α production and TLR5 expression on the Caco2, but not RAW264 cells. Interestingly, a high Ep-induced TLR5 expression was observed on the Caco2 cell surface, which was inhibited by an inhibitor of phosphoinositide3-kinase (PI3K), Ly294002, as well as a ß-adrenergic blocker, propranolol. In addition, the EX-induced TNF-α production observed in response to FG was also attenuated by pretreatment with propranolol. Our findings suggest that exhaustive exercise upregulates the production of TNF-α in response to FG via a high expression of TLR5 on the intestinal cell surface following the stimulation of ß-adrenergic receptors with exercise.

Relationship between macrophage differentiation and the chemotactic activity toward damaged myoblast cells.

We investigated the effect of macrophage differentiation on the chemotactic activity to invade local damaged myoblasts using in vitro models of muscle injury. We found that: 1) the chemotactic activity of macrophages toward areas of damaged myoblasts might be induced more by live myoblasts than dead ones, 2) the chemotactic activity of macrophages is not due to velocity, but depends on the directionality toward damaged myoblast cells, and 3) macrophage differentiation strongly influence the chemotactic activity toward damaged myoblast cells through the expression of CCR2 and/or F-actin.

Exercise training attenuates hepatic inflammation, fibrosis and macrophage infiltration during diet induced-obesity in mice.

Nonalcoholic steatohepatitis, which is considered the hepatic event in metabolic syndrome, was recently associated with the innate immune system. Although regular exercise reduces hepatic injury markers like serum alanine aminotransferase (ALT) levels, the mechanisms regulating the effects of exercise on steatohepatitis are unclear. This study aimed to clarify whether exercise training suppresses hepatic injury, inflammation, and fibrosis by suppressing macrophage infiltration. Male C57BL/6J (4-week old) mice were randomly divided into four groups: normal diet (ND) control (n=7), ND exercise (n=5), high-fat diet and high-fructose water (HFF) control (n=11), and HFF exercise (n=11) groups. Mice were fed the ND or HFF from 4 to 20 weeks of age. The exercise groups were trained on a motorized treadmill for 60 min/day, five times/week. The nonalcoholic fatty liver disease (NAFLD) activity score and plasma ALT activity, indicators of liver injury, were increased in HFF control mice but were attenuated in HFF exercise mice. Hepatic inflammation, indicated by hepatic tumor necrosis factor (TNF)-α levels and hepatic resident macrophage infiltration, was significantly lower in HFF exercise mice than in HFF control mice. Hepatic fibrosis markers (histological hepatic fibrosis detected by Sirius red and α-smooth muscle actin staining and tissue inhibitor of matrix metalloproteinase-1 mRNA) were attenuated in HFF exercise mice compared with HFF control mice. These results suggest that exercise training reduces hepatic inflammation, injury, and fibrosis by suppressing macrophage infiltration.

Interferon-beta, but not tumor necrosis factor-alpha, production in response to poly I:C is maintained despite exhaustive exercise in mice.

It remains unclear whether immune response to viral infection is inhibited by severe exercise. We determined whether exhaustive exercise inhibits interferon (IFN)-ß and tumor necrosis factor (TNF)-α production after injection of synthetic double-stranded (ds) RNAs, a polyriboinosinic polyribocytidylic acid (poly I:C), as viral infection model. Male C3H/HeN mice, which were divided into exhaustive-exercised and non-exercised groups, were injected with poly I:C (5 mg/kg). Although TNF-α in response to poly I:C was significantly inhibited by exhaustive exercise, IFN-ß was no different in both groups. In in-vitro experiments, catecholamines inhibited poly I:C-induced TNF-α, but not IFN-ß, production in macrophages. These results suggest that anti-virus cytokine IFN-ß in response to poly I:C might be maintained despite severe stressful exercise.

Protective action of L-carnitine on cardiac mitochondrial function and structure against fatty acid stress.

Cardiovascular risks are frequently accompanied by high serum fatty acid levels. Although recent studies have shown that fatty acids affect mitochondrial function and induce cell apoptosis, L-carnitine is essential for the uptake of fatty acids by mitochondria, and may attenuate the mitochondrial dysfunction and apoptosis of cardiocytes. This study aimed to elucidate the activity of L-carnitine in the prevention on fatty acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoyl-CoA-induced mitochondrial respiration that was observed with L-carnitine was inhibited with oligomycin. The palmitoyl-CoA-induced mitochondrial membrane depolarization and swelling were greatly inhibited by the presence of L-carnitine. In ultrastructural observations, terminally swollen and ruptured mitochondria with little or no distinguishable cristae structures were induced by treatment with palmitoyl-CoA. However, the severe morphological damage in cardiac mitochondria was dramatically inhibited by pretreatment with L-carnitine. Treatment with L-carnitine also attenuated 4-hydroxy-L-phenylglycine- and rotenone-induced mitochondrial swelling even when the L-carnitine could not protect against the decrease in oxygen consumption associated with these inhibitors. Furthermore, L-carnitine completely inhibited palmitoyl-CoA-induced cytochrome c release. We concluded that L-carnitine is essential for cardiac mitochondria to attenuate the membrane permeability transition, and to maintain the ultrastructure and membrane stabilization, in the presence of high fatty acid ß-oxidation. Consequently, the cells may be protected against apoptosis by L-carnitine through inhibition of the fatty acid-induced cytochrome c release.

L-carnitine is essential to beta-oxidation of quarried fatty acid from mitochondrial membrane by PLA(2).

Mitochondrial beta-oxidation is an important system involved in the energy production of various cells. In this system, the function of L-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O(2) consumption without substrates, is caused by L-carnitine treatment. In this study, we investigated whether L-carnitine is essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A(2) (PLA(2)) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and L-carnitine. The effect of L-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with L-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of L-carnitine. Moreover, the L-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA(2) inhibitors were treated before ADP treatment. The L-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that L-carnitine might be essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by PLA(2).

Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937.

Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.

L-Carnitine suppresses oleic acid-induced membrane permeability transition of mitochondria.

Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation.