Previous studies have shown that butyric acid or its pre-derivatives has beneficial effects on glucose homeostasis and body weight maintenance[3, 12]. However, few of them consider the effect of sodium butyrate on skeletal muscle mitochondria and different fat phenotypes. In the present study, we confirmed that NaB has the similar effect as previous studies did, NaB intervention could effectively reduce the body weight of rats in the OP and OR group, and reduce insulin resistance, plasma lipid. Moreover, our study showed that the regulation of NaB significantly increases the activity of mitochondrial antioxidant enzymes MnSOD and GSH-Px, mitochondrial membrane potential, NADH/NAD+ ratio, acetyl-CoA and ATP production, TFAM and PGC-1α expression, and mitochondrial DNA copy number. 6% NaB intervention in OP rats, 4%NaB intervention in OR rats was more significant, indicating that there is dose effect of NaB intervention in different obesity phenotypes.
Oxidative stress in HFD-fed rats was presented as higher plasma ROS and lower plasma antioxidant levels in accordance with previous studies[13, 14]. In the present study, the results indicated that OP and OR rats showed different degrees of oxidative damage. NaB intervention mitigated HFD-induced oxidative damages (ROS, MDA) and increased antioxidant capacity biomarkers (SOD, GSH/GSSG and T-AOC) in gastrocnemius muscle and pancreatic. 4% and 6% NaB intervention showed better effect in OR and OP groups respectively. Nrf2-ARE signaling is the main antioxidant pathway in vivo. Nrf2, as a key antioxidant factor, can regulate downstream NQO-1, HO-1 and other antioxidant enzymes transcription and expression[15]. PI3K and GSK-3β could regulate the proportion of Nrf2 into the nucleus, activate PI3K or down-regulated the expression of GSK-3β in order to enhance Nrf2 entry [16]. Oxidative stress induced by high-fat diet is closely related to Nrf2-ARE pathway, in this study, the results showed that NaB significantly up-regulated PI3K and down-regulated GSK-3β expression, indicating that the mechanism NaB protects against oxidative stress may relate to the regulation of Nrf2-ARE signaling pathway. Meanwhile, these data showed that 6% NaB and 4% NaB had significant effects on the expression of Nrf2-ARE pathway-related genes in OP and OR rats, indicating that there is a dose-effect NaB antioxidant protection.
Mitochondrial function shows an crucial impact on whole-body metabolism. As important antioxidant enzymes in mitochondria, Mn-SOD and GSH-Px are essential for the maintenance of mitochondrial redox homeostasis. Acetyl-CoA and NADH/NAD+ ratios are the major indicators of mitochondrial energy metabolism. In our study, NaB intervention could restore the above indicators in obesity rats. Our previous study found that H2O2 induced oxidative damage in HepG2 cells, and reduced mitochondrial acetyl CoA content, membrane potential and ATP production. Resveratrol can restore mitochondrial metabolism by increasing the levels of Mn-SOD and GSH-Px in AF mice[17]. Our data also show that NaB can improve mitochondrial function, increase mitochondrial membrane potential, increase ATP production, and reduce lactic acid accumulation in gastrocnemius muscle by increasing the levels of Mn-SOD and GSH-Px. In addition to mitochondrial dysfunction, we also found that mitochondrial mRNA copy number decreased in OP and OR rats. It has been reported that PGC1α and TFAM regulate mitochondrial copy number[18, 19]. Three doses of NaB intervention could promote PGC1α and TFAM expression that contribute to mitochondrial copy number in OP rats. Recent study showed that accumulation of TFAM protein could increase the expressions of mtDNA in skeletal muscle, indicating that NaB may enhance mitochondrial biogenesis by enhancing the PGC1α and TFAM expression in rats.
Mechanistic Target of Rapamycin (mTOR) signaling pathway plays an important role in protein synthesis and cell growth. mTOR participates in cell differentiation, proliferation and protein synthesis by activating the downstream protein S6K1[20]. Previous studies have shown that skeletal muscle mTOR levels and downstream S6K1 phosphorylation levels increase after exercise, and protein synthesis rate increases. Myostain, a member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors, plays an important role in muscle development [21]. In mice knocking out the myostain gene, skeletal muscle weight was significantly increased, and excessive muscle growth was also observed in mice with decreased myostain expression. The myogenin gene is a myogenic determinant and necessary factor for skeletal muscle differentiation [22]. Previous studies have shown that the risk of obesity in C57BL/6J mice may be related to the over-activation of mTOR-S6K1 signaling pathway and decreased AKT levels[23], which is consistent with the results of this study. In our study, we found that OP + 6% and OR + 4% NaB significantly increased the expression of muscle and related genes, which may be an important factor in NaB intervention to reduce body weight.
SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase. In mice lacking SIRT1 deacetylase activity, skeletal muscle insulin signaling is diminished and insulin resistance occurs [24]. The AMPK signaling pathway is the central link regulating cell energy status. It activates and promotes phosphorylation of downstream signaling molecules [25]. Peng [26] found that NaB treatment of colon increased AMPK activity, consistent with the results of this study. SIRT1 and AMPK are intracellular energy receptors that regulate the activity of PGC-1α by phosphorylation and deacetylation.
Previous studies found that Glut 4 mRNA levels in skeletal muscle of type 2 diabetic mice was significantly decreased, spironolactone can significantly increase the expression of Glut 4 and improve glucose utilization[27]. Compared with the control group, the expression of IRS-1 mRNA in skeletal muscle of diabetic rats was significantly decreased, and chromium supplementation up-regulated the expression of IRS-1 mRNA and increased insulin resistance. Our study found that NaB intervention for 12 weeks can significantly increase the expression of IRS-1 and Glut 4 mRNA, and OP + 6%NaB, OR + 4%NaB has a significant effect on the improvement of insulin sensitivity and promote glucose intake.
Pancreatic redox homeostasis is closely related to insulin secretion. High-fat diet induced imbalanced redox homeostasis in obese mice, reduced pancreatic dysfunction and insulin secretion. The expression of Pdx1 and MafA in obese mice induced by high fat decreased accomponied with dysfunction of pancreas [28]. This study found that NaB intervention could increase the expression of Pdx1 and MafA mRNA and insulin secretion in the pancreas.
Consistent with previous publications, our results showed that the expression of Bcl-2 were significantly down-regulated after a HFD with the increase of Bax and Caspase 3 mRNA levels. Qi et al [29] showed that radiation significantly reduced the expression of Bcl-2 mRNA, increased the expression of Bax and Caspase 3, and excessive oxidative stress altered the expression level of apoptosis-related genes. The study found that Schisandra oil can significantly reduce the content of MDA in pancreatic tissue of diabetic rats, up-regulate the levels of SOD and CAT and increase the level of Bcl-2 protein, indicating that the improvement of oxidative stress can inhibit apoptosis[30]. In this study, NaB intervention significantly improved pancreatic cell function, improve oxidative stress, and inhibit pancreatic cell apoptosis, and OP + 6% NaB and OR + 4% NaB had significant effects. Based on the above findings, we found that NaB regulates the main pathways of blood glucose homeostasis in OP and OR rats: on the one hand, down-regulates the expression of Bax and Caspase 3, up-regulates Bcl-2 expression, inhibits pancreatic cell apoptosis; On the one hand, the expression levels of Pdx1 and Mafa are up-regulated and insulin secretion is promoted; in addition, the expression of IRS-1 and GLUT4 mRNA is increased, muscle synthesis is promoted, gastrocnemius insulin sensitivity is increased, and blood glucose and insulin resistance are controlled.