Tomomi Ookawara

Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training.

The effects of endurance training on the enzyme activity, protein content, and mRNA abundance of Mn and CuZn superoxide dismutase (SOD) were studied in various phenotypes of rat skeletal muscle. Female Sprague-Dawley rats were randomly divided into trained (T, n = 8) and untrained (U, n = 8) groups. Training, consisting of treadmill running at 27 m/min and 12% grade for 2 h/day, 5 days/wk for 10 wk, significantly increased citrate synthase activity ( P < 0.01) in the type I (soleus), type IIa (deep vastus lateralis, DVL), and mixed type II (plantaris) muscles but not in type IIb (superficial vastus lateralis, SVL) muscle. Mitochondrial (Mn) SOD activity was elevated by 80% ( P < 0.05) with training in DVL. SVL and plantaris muscle in T rats showed 54 and 42% higher pooled immunoreactive Mn SOD protein content, respectively, than those in U rats. However, no change in Mn SOD mRNA level was found in any of the muscles. CuZn SOD activity, protein content, and mRNA level in general were not affected by training, except for a 160% increase in pooled CuZn SOD protein in SVL. Training also significantly increased glutathione peroxidase and catalase activities ( P < 0.05), but only in DVL muscle. These data indicate that training adaptations of Mn SOD and other antioxidant enzymes occur primarily in type IIa fibers, probably as a result of enhanced free radical generation and modest antioxidant capacity. Differential training responses of mRNA, enzyme protein, and activity suggest that separate cellular signals may control pre- and posttranslational regulation of SOD.

Superoxide dismutase gene expression in skeletal muscle: fiber-specific effect of age

The influence of ageing on the expression of two superoxide dismutase (SOD) isozymes was examined in three different skeletal muscle fiber types of young (Y, 8 mo) and old (O, 25 mo) rats. Total SOD activity was increased with age in the gastrocnemius (Gas, type II(mix)) and superficial vastus lateralis (SVL, type IIb) but unchanged in the soleus (Sol, type I). The increased SOD activity in SVL was due to increased cytosolic SOD (CuZn SOD), whereas both mitochondrial (Mn SOD) and CuZn SOD activities were increased in Gas. In Sol, Mn SOD activity was significantly increased in aged rats. Mn SOD mRNA level was significantly decreased with age in all three muscles examined, while Mn SOD protein content was not altered. Ageing did not affect CuZn SOD mRNA abundance in any of the muscles, but significantly increased CuZn SOD protein content in aged Gas and Sol. Binding of two redox-sensitive transcription factors, nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP-1) was significantly decreased with age in all three muscle types. These results indicate that increased SOD activity in aged skeletal muscle is not associated with higher levels of gene transcription. Increases in Mn SOD activity seen in aged Gas and Sol are the result of post-translational modification of the enzyme, whereas increases in CuZn SOD activity during ageing may be due to both translational and post-translational control.

Effect of aging and late onset dietary restriction on antioxidant enzymes and proteasome activities, and protein carbonylation of rat skeletal muscle and tendon.

Many studies have shown that lifelong dietary restriction (DR) can retard aging processes. Very few reports, however, are found that examined the effect of late onset DR on biochemical parameters in aging animals [Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002b. Dietary restriction initiated in late adulthood can reverse age-related alterations of protein and protein metabolism. Ann. NY Acad. Sci. 959, 50-56]. We studied the effect of every-other-day feeding, initiated at the age of 26.5 months and continued for 3.5 months, on antioxidant enzymes, protein carbonyls, and proteasomes of the gastrocnemius muscle and tendon in rats. Age-related increase in the activity and content of Cu, Zn-SOD and the content of Mn-SOD was attenuated by the DR in both tissues. The same was true for glutathione peroxidase and catalase activities. Significant increase with age in protein reactive carbonyl derivatives (RCD) in the tendon was noted that was partially reversed by the DR. No significant change of RCD, however, was observed in the skeletal muscle. The age-related and DR-induced changes of the RCD in the tendon appeared to be associated with proteasome activity that decreases with age and increases by the DR. It is suggested that the late onset DR can have beneficial effects on the locomotive functions by reducing age-associated potentially detrimental oxidative protein damage in the tendon.

Prevention of ongoing lipid peroxidation by wound excision and superoxide dismutase treatment in the burned rat

To determine if wound excision and superoxide dismutase (SOD) treatment prevent ongoing lipid peroxidation after burn injury, the plasma, kidney, and lung lipid peroxide (LPO) levels in 25% total body surface area (TBSA) burned rats was studied. The animals were given intraperitoneal bovine copper-, zinc-SOD (Cu/Zn-SOD) (50,000 U/kg dissolved in saline) or saline immediately after burns and were operated by wound debridement and allograft 2 hours after the burn. We measured LPO levels of 6-hour postburn plasma and tissues by the thiobarbituric acid (TBA) method, and measured the manganese SOD (Mn-SOD) by an enzyme-linked immunosorbent assay. Wound excision alone prevented the increase of plasma LPO levels but could not prevent the increase in tissues. The combination of wound excision and Cu/Zn-SOD treatment markedly inhibited the increase in both plasma and tissue LPO levels after the burn, but did not prevent the increase in Mn-SOD. Wound excision in conjunction with SOD-treatment might be therapeutic in the management of severe burns.

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.

Voluntary exercise attenuates obesity-associated inflammation through ghrelin expressed in macrophages

Chronic low-level inflammation is associated with obesity and a sedentary lifestyle, causing metabolic disturbances such as insulin resistance. Exercise training has been shown to decrease chronic low-level systemic inflammation in high-fat diet (HFD)-induced obesity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Ghrelin is a peptide hormone predominantly produced in the stomach that stimulates appetite and induces growth hormone release. In addition to these well-known functions, recent studies suggest that ghrelin localizes to immune cells and exerts an anti-inflammatory effect. The purpose of the current study was to investigate the role of ghrelin expressed in macrophages in the anti-inflammatory effects of voluntary exercise training. Expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1 and F4/80 was increased in adipose tissue from mice fed a HFD (HFD mice) compared with mice fed a standard diet (SD mice), whereas the expression of these inflammatory cytokines was markedly decreased in mice performing voluntary wheel running during the feeding of a HFD (HFEx mice). The expression of TNF-α was also increased in peritoneal macrophages by a HFD and exercise training inhibited the increase of TNF-α expression. Interestingly, expression of ghrelin in peritoneal macrophages was decreased by a HFD and recovered by exercise training. Suppression of ghrelin expression by siRNA increased TNF-α expression and LPS-stimulated NF-κB activation in RAW264 cells, which is a macrophage cell line. TNF-α expression by stimulation with LPS was significantly suppressed in RAW264 cells cultured in the presence of ghrelin. These results suggest that ghrelin exerts potent anti-inflammatory effects in macrophages and functions as a mediator of the beneficial effects of exercise training.

Is insulin-like growth factor I involved in brown adipose tissue enlargement?

During cold exposure, the expression of insulin-like growth factor I (IGF-I) mRNA in rat brown adipose tissue (BAT) increased significantly with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated (CA) rats enhanced the proliferation of brown adipocyte precursor cells in primary culture and the expression of IGF-I mRNA in them compared to those from warm-acclimated rats. The cell proliferation was considerably inhibited by anti-growth hormone (GH) antibody. These results suggest that IGF-I produced by brown adipocytes may play a role in BAT enlargement during cold acclimation. It is probable that some factors (including GH) concerned with the proliferation of brown adipocyte precursor cells are involved in the blood plasma from CA rats.

Immunoreactivity and activity of mitochondrial superoxide dismutase following training and exercise

Superoxide dismutases (SOD) are physiological antioxidant proteins which scavenge superoxides, a reactive oxygen species capable of producing diverse pathophysiological consequences. Mangano-SOD (Mn-SOD) is a mitochondrial enzyme. Our study presents the first evidence that a single bout of exhaustive exercise (EE) is associated with an increase in plasma Mn-SOD immunoreactivity (IR). Compared to untrained controls, the increase was less in endurance-trained rats supplemented with exogenous glutathione. EE-associated changes in the activity and IR of Mn-SOD were studied in the plasma, liver, skeletal muscles, heart, kidney and lung of trained and control rats.

N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells

Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewisx structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase.

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.