The present study investigated the influence of diet-induced obesity on liver Mt O·2− production and membrane fluidity in rats that had consumed HFD for 11 weeks. Energy metabolism was higher and O·2− production was lower in HFD-fed rats than in ND-fed rats. No significant changes were observed in Mt membrane fluidity.
The period from 4 to 15 weeks old, during which rats were fed the HFD, accounts for suckling over puberty, which corresponds to adolescence . Body weights (Fig. 3) and abdominal circumferences (Table 1) were markedly higher in the HFD group than in the ND group, and AI was higher in the HFD group (Table 2). Regarding the respiratory activity of liver Mt, RCR was 3 or higher, and the P/O ratio was 2.6 or higher , showing that it was possible to extract Mt in a favorable state under both conditions. The P/O ratio, reflecting the amount of ATP synthesized by Mt, and substrate oxygen consumption were higher in the HFD group than in the ND group (Fig. 4). In rats fed a HFD, fatty acid metabolism was found to be promoted, energy metabolism increased , and citrate synthase activity was enhanced . The amount of ATP synthesized increases in the liver in the compensatory phase , and the ingestion of a HFD increases the total amount of Mt . RCR and the P/O ratio reflect the state and impermeability of the Mt inner membrane, substrate oxidization, and conjugation with oxidative phosphorylation. In HFD-fed rats, Mt functions, such as ATP synthesis and oxidative phosphorylation, were promoted to metabolize the fatty acids ingested.
O·2− production was measured in samples after removing the scavenging activity of Mn-SOD by ultrasonication using the adrenaline method. In the adrenaline method, adrenaline is oxidized by O·2−, and O·2− is quantitated based on one molecule of O·2− producing one molecule of adrenochrome. In Mt, 3–5% of oxygen becomes O·2− during ATP synthesis in the electron transport chain [13, 14] and is scavenged by the SOD, Mn-SOD . O·2− production was lower in the HFD group than in the ND group (Fig. 5). In elderly rats fed a HFD, hydrogen peroxide production in the liver increased, RCR decreased , and hydrogen peroxide production in skeletal muscle increased . In obese individuals, the amount of Mt decreased, thereby reducing Mt functions, such as oxygen consumption [38, 39]. We also previously demonstrated that oxidative stress may lead to cell damage in the skeletal muscle of diet-induced obese rats . Regarding antioxidant capacity, the transcription level of antioxidative enzyme-related genes decreased in rats fed a HFD . The increase in oxygen consumption and decrease in O·2− production accompanying the promotion of energy metabolism indicate a reduced antioxidant capacity, such as Mn-SOD expression . In the present study, the ingestion of the HFD increased ATP synthesis and reduced O·2− production due to the promotion of fatty acid metabolism in the liver, suggesting that a reduction in antioxidant capacity occurred in the body. The genomic DNA of Mt is not protected by histones and is readily impaired by radicals. The respiratory chain, which plays a central role in energy metabolism, is located in close proximity, making it more susceptible to damage by ROS than in other cells. In a state of increased ROS production, such as exercise, exposure to ROS increases due to a reduction in the antioxidant capacity in the liver, which increases the possibility of cell damage by oxidative stress.
Moderate O·2− production is beneficial for up-regulating the infection-protective immune system and signal transmission for apoptosis in the body [9–11, 42]. A previous study reported that ROS production by Mt increases the phagocytic and migration abilities of macrophages . Taking the function of ROS as a signaling molecule into account, the ingestion of a HFD from the juvenile period may impair liver Mt and have a negative influence due to reductions in ROS production in Mt
In the present study, rats were fed lard with a high content of polyunsaturated fatty acids, which are cell membrane components. Many previous studies have employed aging model rats, dietary restrictions, and measurements of membrane fluidity in white blood cells [31, 44–46]. The spin label method used in the present study employed the labeling agent, 5-NS, and outer membrane fluidity was measured based on the nitroxide group binding to the alkyl chain of the phospholipid head on the external membrane surface. The relationship between O·2− generation and membrane fluidity in Mt has not yet been examined. Therefore, we attempted to investigate this relationship in the livers of rats fed a HFD. Lipids account for 25–30% of Mt membrane components, and polyunsaturated fatty acids and cholesterol are abundant . We hypothesized that diet-induced obesity promotes structural changes in membrane phospholipids localized in the electron transport chain and increases O·2− leakage; however, no diet-induced change was noted in the fluidity of the Mt outer membrane or electron transport chain isolated by ultrasonication (Fig. 6). Although previous studies reported that membrane fluidity was altered with changes in the contents of cholesterol and phospholipids [15, 16], these findings were not consistent with the present study. The ingestion of a HFD has been shown to promote fatty acid oxidation due to changes in the fat composition of Mt and impaired oxidative phosphorylation . Therefore, the promotion of lipid metabolism in the liver may have resulted in a decrease in lipid infiltration in the liver and had no influence on the Mt membrane.
Regarding the limitations of the present study, we isolated and analyzed the electron transport chain from Mt, but did not quantify O·2− in Mt after substrate-permeable cell processing. Future studies are needed in order to investigate this and examine the impact of the duration of a HFD on Mt and O·2−. Another aspect that warrants further study is the impact of an exercise protocol on antioxidant capacity and O·2− production.