Hiromi Yano

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.

Exercise attenuates M1 macrophages and CD8+ T cells in the adipose tissue of obese mice.

PURPOSE Obesity is associated with adipose tissue inflammation, which has been attributed to changes in the number and types of leukocytes in adipose tissue. Exercise training is thought to be important for the reduction of adipose tissue inflammation, but the mechanisms by which this may occur are incompletely understood. Here, we evaluated the effect of exercise training on several inflammation-associated changes in adipose tissue, including infiltration of inflammatory macrophages and T cells. METHODS Four-week-old male C57BL/6J mice were randomly assigned to four groups that received a normal diet (ND) plus sedentary (n = 8), an ND plus exercise training (n = 8), a high-fat diet (HFD) plus sedentary (n = 12), and an HFD plus exercise training (n = 12). Mice were fed the ND or the HFD from 4 to 20 wk of age. Mice in the exercise groups ran on a treadmill for 60 min·d, 5 d·wk over the same points. RESULTS Mice fed the HFD had increased numbers of macrophage clusters in adipose tissue, which were reduced by exercise training. Similarly, adipose tissue from the HFD sedentary mice contained higher levels of tumor necrosis factor α mRNA and increased numbers of CD11c inflammatory macrophages and CD8 T cells than adipose tissue from the ND mice, and those were also lowered by exercise training. The mRNA levels of monocyte chemoattractant proteins 1 and 2 and macrophage inflammatory proteins 1α and 1β in adipose tissue were lower in the HFD exercise mice than those in the HFD sedentary mice. CONCLUSIONS The results suggest that exercise training reduces adipose tissue inflammation by suppressing infiltration of inflammatory macrophages and CD8 T cells.

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 β-oxidation. Consequently, the cells may be protected against apoptosis by L-carnitine through inhibition of the fatty acid-induced cytochrome c release.

Curcumin attenuates oxidative stress following downhill running-induced muscle damage

Downhill running causes muscle damage, and induces oxidative stress and inflammatory reaction. Recently, it is shown that curcumin possesses anti-oxidant and anti-inflammatory potentials. Interestingly, curcumin reduces inflammatory cytokine concentrations in skeletal muscle after downhill running of mice. However, it is not known whether curcumin affects oxidative stress after downhill running-induced muscle damage. Therefore, the purpose of this study was to investigate the effects of curcumin on oxidative stress following downhill running induced-muscle damage. We also investigated whether curcumin affects macrophage infiltration via chemokines such as MCP-1 and CXCL14. Male C57BL/6 mice were divided into four groups; rest, rest plus curcumin, downhill running, or downhill running plus curcumin. Downhill running mice ran at 22 m/min, -15% grade on the treadmill for 150 min. Curcumin (3mg) was administered in oral administration immediately after downhill running. Hydrogen peroxide concentration and NADPH-oxidase mRNA expression in the downhill running mice were significantly higher than those in the rest mice, but these variables were significantly attenuated by curcumin administration in downhill running mice. In addition, mRNA expression levels of MCP-1, CXCL14 and F4/80 reflecting presence of macrophages in the downhill running mice were significantly higher than those in the rest mice. However, MCP-1 and F4/80 mRNA expression levels were significantly attenuated by curcumin administration in downhill running mice. Curcumin may attenuate oxidative stress following downhill running-induced muscle damage.

Mechanism of A23187-Induced Apoptosis in HL-60 Cells: Dependency on Mitochondrial Permeability Transition but Not on NADPH Oxidase

Calcium ions (Ca2+) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca2+ in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca2+ induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.

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 Ca2+ 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 β‐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 β‐oxidation. Copyright

The reduction of voluntary physical activity after poly I:C injection is independent of the effect of poly I:C-induced interferon-beta in mice.

One characteristic of sickness behavior in mice is demonstrated by a reduction in voluntary wheel-running activity during infection. Among synthetic double-stranded (ds) RNAs, polyriboinosinic: polyribocytidylic acid (poly I:C) activates to produce interferon (IFN) -beta, which plays an important role in anti-viral activity and host-defense. However, how voluntary wheel-running activity is regulated during poly I:C infection is unknown. To determine whether poly I:C-induced IFN-beta production is responsible for reduced spontaneous physical activity, we measured poly I:C-induced changes in voluntary wheel-running activity in mice. In this experiment, the mice were injected with poly I:C (0-5 mg/kg i.v.) and/or anti-IFN-beta neutralizing antibody (1.5x10(5) U/kg i.v.). We also observed the direct effect of injection of recombinant IFN-beta (rIFN-beta: 5.0x10(4) and 2.5x10(5) U/kg) on wheel-running behavior. Poly I:C treatment dose-dependently reduced wheel-running activity, and induced an increase in plasma IFN-beta in mice. However the activity was not attenuated by the neutralizing antibody specific to IFN-beta treatment. Additionally, the wheel-running activity in rIFN-beta treated mice was maintained, although they showed a higher IFN-gamma inducible protein (IP)-10 concentration in plasma compared with that of the vehicle group. Our results suggest that the transient reduction in physical activity after poly I:C injection is induced dose dependently, but that the mediator might not be poly I:C-induced IFN-beta.

Mechanism of 3-nitropropionic acid-induced membrane permeability transition of isolated mitochondria and its suppression by L-carnitine

3‐Nitropropionic acid (3NP) functions as an irreversible inhibitor of succinic acid dehydrogenase (complex II) and induces neuronal disorders in rats similar to those in patients with Huntingtons disease. It is well known that L‐carnitine (LC), a carrier of long chain fatty acid into the mitochondrial matrix, attenuates the neuronal degeneration in 3NP‐treated rats. From these findings it has been suggested that 3NP induces certain neuronal cell death through mitochondrial dysfunction and that LC preserves the neurons against the dysfunction of mitochondria caused by 3NP. However, the detailed mechanism of cell death by 3NP and the protective actions of LC against the mitochondrial dysfunction have not been fully elucidated yet. Thus, we studied the molecular mechanism of the effects of 3NP and LC on isolated rat liver mitochondria. 3NP inhibited succinate respiration and the decreased respiratory control ratio of isolated mitochondria without affecting oxidative phosphorylation. 3NP induced a membrane permeability transition (MPT), which plays an important role in the mechanism of apoptotic cell death. 3NP stimulated Ca2+ release from mitochondria, decreased membrane potential, induced mitochondrial swelling, and stimulated cytochrome c release from mitochondria. 3NP‐induced swelling was suppressed by bovine serum albumin, inhibitors of phospholipase A2 and by an inhibitor of classic MPT, cyclosporin A. Furthermore, LC suppressed the changes brought about by 3NP in mitochondrial functions in the presence of ATP. These results suggest that MPT underlies the mechanism of 3NP‐induced cell death, and that LC attenuates mitochondrial MPT by decreasing long chain fatty acids generated by phospholipase A2. Copyright

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-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 O2 consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A2 (PLA2) 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 PLA2 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 β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA2.

Exercise training attenuates adipose tissue fibrosis in diet-induced obese mice

Tissue fibrosis, such as that which occurs in obesity, is associated with chronic inflammatory diseases. Although regular exercise reduces adipose tissue inflammation, the mechanisms regulating the effects of exercise on adipose tissue fibrosis are unclear. This study aimed to clarify whether exercise training attenuates adipose tissue fibrosis with consequent reduction of extracellular matrix including collagens. Male C57BL/6J (4-week old) mice were randomly assigned to four groups that received a normal diet (ND) plus sedentary (n=8), an ND plus exercise training (n=8), a high-fat diet (HFD) plus sedentary (n=12), and an HFD plus exercise training (n=12). Mice were fed the ND or HFD from 4 to 20 weeks of age. The exercise groups were trained on a motorized treadmill for 60 min/day, 5 times/week over the same period. Histological hepatic fibrosis detected by Sirius red and α-smooth muscle actin staining were attenuated in HFD exercise mice compared with HFD sedentary mice. mRNA levels of transforming growth factor-β and tissue inhibitors of metalloproteinase-1, major regulators of tissue fibrosis, were increased in HFD sedentary mice but were attenuated in HFD exercise mice. Similarly, adipose tissue from the HFD sedentary mice contained higher macrophages than adipose tissue from the ND mice, and this was also lowered by exercise training. These findings suggest that exercise training may be effective for attenuating adipose tissue inflammation in obesity.