Overnutrition and physical inactivity are precipitating factors that cannot be ignored in the occurrence of metabolic complications such as insulin resistance, type2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and MAFLD. MAFLD is a progressive disease, and excessive consumption of fat and/or sugar, particularly fructose, can cause hepatic steatosis and dyslipidemia. A diet constituting 40% fat, 20% fructose, and 2% cholesterol is widely known as the AMLN diet (Research diet, #D09100301) and is the preferred diet to induce many clinically relevant characteristics of NASH. Different periods of AMLN diet will result in different levels of lipid deposition in liver tissue, with consequent changes in oxidative and ER stress levels, as well as mitochondrial function [28]. Interestingly, a recent study revealed that mitochondrial FAO capacity in liver was decreased after 4weeks high fat diet (HFD) feeding, however, this effect was restored at 8 weeks [29], indicating that mitochondria function in liver tissue are highly plastic in diet-induced metabolic alteration. Exercise has also been confirmed to have significant effects on improving hepatic lipid metabolism, mitochondrial function, and the oxidative stress level. Zou et al. [30] recently showed that moderate-intensity exercise induced a greater improvement in antioxidant ability and reduced stress status via regulation of ER stress pathway-related proteins. Therefore, this study first to compare the changes concerning lipid deposition, ER stress signaling pathway, UPRmt, and mitokine secretion after 14 and 18 weeks of AMLN diet intervention, which are two time points during the progression of MAFLD. On this basis, this study further explored the role of an 8-week voluntary wheel running intervention in regulating MAFLD occurrence and progression, focusing mainly on the molecular mechanism involved in the PERK-eIF2α-ATF4 aix and UPRmt. The relationship between UPRmt levels and FGF21 secretion was also investigated.
The AMLN diet is a common dietary formula that induces liver damage in a manner similar to that observed in the human liver diseases. NAFLD development in three stages, including steatosis, steatohepatitis with fibrosis, and cirrhosis [31]. Trevaskis et al. reported that animals had AMLN diet for 12 weeks demonstrated increased body weight and liver fat content without fibrosis, but showed progression toward fibrosis when fed for a chronic 30-week period [32]. Here, mice were fed an AMLN diet for 14 and 18 weeks to establish a model of hepatic steatosis and prefibrosis and found that hepatic lipid deposition occurred significantly in both time periods. Moreover, these changes were associated with abnormal blood lipid levels in the 18-week AMLN diet animals, but not in the AMLN14 group. As the circulating fatty acids uptake is the major way for liver to acquire lipids [33], the absence of dyslipidemia in the 14-week AMLN diet mice may be a result of the increased fatty acid intake and FA storage as TGs in the liver, which normalize serum lipid levels. However, this regulatory mechanisms were disappeared in the 18-week AMLN diet animals. These data suggest that prolonged administration of an AMLN diet not only leads to an abnormal increase in blood lipids but also induces massive macro- and microsteatosis. This may even aggravate oxidative stress and damage mitochondrial function, which contribute to cellular damage and disease progression. In addition, numerous evidence supports that exercise exerts many of its metabolic benefits in liver, adipose tissue, and pancreas [33]. Although the mechanisms by which exercise reduces liver fat are still largely unknown, the inner mechanism involves β-oxidation [34], lipogenesis, and lipid export. In addition, an improvement in insulin resistance with exercise intervention is thought to reduce the uptake of circulating FFAs to the liver. Therefore, exercises can be helpful in orchestrating the lipid synthesis, export [35], and their use as energy substrates. The results showed that liver lipid deposition in AMLN-fed animals at two stages improved significantly after an 8-week voluntary wheel running exercise intervention. However, this study failed to observe significantly decreased blood lipid levels in the early stages mediated by exercise, which may be due to the relatively lower blood lipid levels in the early stages than in the late.
To further understand the alleviated AMLN diet-induced lipid deposition displayed by exercise intervention and with a focus on mitochondria-related metabolic mechanisms, the PERK-eIF2α-ATF4 signaling pathway was first examined, which is ER stress branch pathway and has been shown to be closely related to hepatic lipogenesis and steatosis regulation [36]. The activation of this ER stress or UPR mainly relays on the luminal chaperone GRP78 activation. In terms of its downstream genes, a study showed that decreased hepatic lipogenesis and steatosis in HFD fed mice is accompanied by long-term dephosphorylation of PERK downstream: the elongation initiation factor eIF2α [37]. In this study, the PERK-eIF2α-ATF4 signaling pathway was significantly awakened in both 14- and 18-week AMLN diet mice compared to the ND diet mice, which indicated that abnormal lipid accumulation often coincides with perturbed ER proteostasis in hepatocytes. The more severe the hepatic lipid deposition, the more obvious the PERK-eIF2α-ATF4 signaling pathway. Importantly, after 8 weeks of exercise intervention, the significant elevation of GRP78 and its downstream target gene expression was apparently improved in AMLN diet mice. These results indicant that this improvement induced by exercise in the ER stress signaling pathway is a beneficial adaptive mechanism. However, although ER stress responses were markedly activated by AMLN diets fed for different durations in this experiment and attenuated after exercise intervention, the underlying mechanisms may not be consistent. One clinical research showed that there is a variable degress of ER stress activation in the patients with NAFLD [38]. Prolonged phosphorylation of eIF-2α via activated PERK could specifically increase the downstream effectors ATF4 and CHOP, which enable the cell restore proteostasis. Conversely, there are other potential mechanisms induced by eIF-2α phosphorylation, several studies have shown that ER eIF2a phosphorylated downstream elements, which lead to the severeg oxidative stress by downregulating NFE2-related factor 2 (Nrf2) and depleting glutathione (GSH), eventually leading to apoptosis [39–41]. It has also been shown eIF2α phosphorylation is a key molecular event in mammalian UPRmt activation and is closely connection of the ISR [42]. Under stress conditions, the phosphorylation of eIF2 α triggers ATF4 activation, induces CHOP expression, and promotes UPRmt activation. Elevated transcript levels of ATF4 and CHOP in AMLN diet mice may be related to mitochondrial stress. Thus, next step was to investigate whether mitochondrial function and the UPRmt molecular pathways were altered under different durations of AMLN diet, and the influence mechanism of exercise on it.
The UPRmt intimately associated with mitochondrial quality control system and plays an important role on protein homeostasis by stabilizing mitochondrial function against several pathologies. Although the UPRmt mechanism remains unclear, it promotes development during mild mitochondrial dysfunction. A recent study showed that the hepatic mitochondrial is closely related to the pathogenesis of NAFLD [43]. Results in this study demonstrated that mitochondrial membrane potential was significantly reduced in both 14- and 18-week AMLN diet mice, whereas mitochondrial respiration function, detected by the RCR, and OXPHOS gene expression were markedly decreased only in the 18-week AMLN diet group. These data suggest that mitochondria in excessive lipid deposition hepatocytes display an uncoupling of respiration from ATP production, indicating a reduced ability of ATP generation of mitochondria. However, this alteration did not occur at the early stages of hepatic lipid deposition. It is perhaps reasonable that mitochondrial function was disrupted during MAFLD development, but the changes in UPRmt-associated gene expression were unexpected. In terms of UPRmt, the expression of its downstream effectors (LONP1, mtHSP70, and mtHSP60) were elevated in 14 weeks AMLN diet mice in the presence of UPRmt compared to those fed an AMLN diet for 18 weeks in the absence of UPRmt. This suggests that UPRmt activation was triggered at the early stages of MAFLD progression and was impaired during higher-grade hepatic lipid deposition. It is speculated that the impairment in UPRmt associated with excessive lipid deposition could be due to imbalanced redox homeostasis or the heavily activated PERK-eIF2α-ATF4 pathway, which causes mitochondria to lose their ability to repair themselves. Indeed, a previous study reported that ATF4 negatively regulates TFAM expression and disrupts mitochondrial biogenesis and respiratory function in hepatocytes [44]. Another possible explanation could be that dysfunctional mitochondrial protein import due to the prolonged AMLN diet induced the overall decline in mitochondrial function, which further impeded the process of transporting nuclear genome-encoded mitochondrial chaperones or proteases to the mitochondrial matrix. Taken together, these data point out the possibility that UPRmt may have dual effects: the 14-week AMLN diet associated with slight stress can initiate UPRmt and restore mitochondrial proteostasis, conversely, chronic UPRmt activation may be harmful to cell survival. It is worth noting that, the decreased expression of some UPRmt components in 18-week AMLN diet mice, such as Hsp60, Hsp70, ClpP and LONP1 were restored with exercise intervention, as shown in AMLN18 + E8 group animals. These results highlighted the role of exercise in relieving mitochondrial stress and improving the UPRmt process, which coordinated to ensure mitochondrial function and viability. Future study is needed to probe the deep regulation mechanisms of UPRmt induction after exercise intervention.
Mitochondria have been considered to crosstalk with other distant tissues through serum cytokine non-cell autonomously [44]. This study next focused on FGF21, a mitokine, mediated by the UPRmt-related eIF2α-ATF4-CHOP ER stress response axis, and is also a well-known exercise-induced hepatokine that stimulates lipolysis and fatty acid oxidation and suppresses lipogenesis in the liver [45, 46]. Myung-Shik Lee’s group demonstrated that ATF4-dependent FGF21 induction was accompanied by impaired mitochondrial function, which contribute to suppress the diet-induced hepatic steatosis in animals [47]. The present study found that compared to ND mice, the FGF21 levels in serum and liver tissue were significantly enhanced after 14-week AMLN diet intervention, but the content was markedly reduced in 18-week AMLN diet mice, which is consistent with the previous UPRmt alteration. This observation suggests a relationship between UPRmt and FGF21 secretion. Yu-Wei Cheng recently reported that the ISR/ATF4-dependent induction of FGF21 was triggered by mitochondrial stress, which can mediate beneficial effects systemically [48]. Surprisingly, it found a positive correlation between UPRmt-related gene expression levels, including LONP1, HSP60, HSP70, and ClpP, and hepatic FGF21 levels as well as serum FGF21 content, suggesting that FGF21 is secreted by cells experiencing mitochondrial stress. Different from linear correlation, the relationship between mitochondrial perturbations-mediated UPRmt and hepatic lipid accumulation demonstrated a mitohormetic response. This mitohormesis concept suggests that mild mitochondrial stress initiates a diverse set of retrograde stress responses from mitonuclear interaction, whereas higher doses of stress can have harmful effects on cellular function [49]. Notably, this study also demonstrated that the decrease in circulating and hepatic FGF21 associated with excessive lipid accumulation was improved in response to exercise intervention. Based on the evidence presented above, this result may be due to the effect of exercise on regulating PERK-eIF2α-ATF4 or the UPRmt-related ISR/ATF4 pathway, both of which induce adaptive responses by triggering mitochondrial to nuclear communications, thereby recovering mitochondrial UPR and enabling mitochondrial self-healing. Previous studies have proposed an important role of cytokine factors as regulators of systemic energy metabolism. Secretion of mitokine during a specific period of disease progression has been proved to be a vital adaptive response that occurs [50]. In line with the concept of mitohormesis, exercise-mediated FGF21 secretion may be largely dependent on mitohormesis effect. Under normal conditions, long-term exercise can enhance cellular adaptations to an extent by triggering mitochondrial stress and mitonuclear communication. Moreover, in a state of severe lipid deposition in liver cells, exercise intervention could induce the mitohormetic response by improving the levels of oxidative stress and mitochondrial function, enhancing mitochondrial chaperones and proteases expression, and secreting mitokines.
Study strengths and limitations
This study demonstrated how an additional 4 weeks on the AMLN diet can progress the development of steatohepatitis, as well as the effects it has on the proteins involved in ER stress signaling pathway, and UPRmt. Moreover, this study further explored the effectiveness of exercise to reduce the development of MAFLD and to normalize the protein content and activation of proteins involved in ER signaling and UPRmt. However, several limitations are existed in this study. First, this study is limited to animal experiments. Although serum FGF21 levels are showed closely related to liver UPRmt level here, whether it can be used for clinical diagnosis of MAFLD still needs further confirmation from clinical experiments. Second, owing to the complex of the pathogenesis of MAFLD, this study mainly focus on the mitochondira, and the depth mechanisms related to UPRmt, such as mitonuclear communication and the interaction of mitochondria with the endoplasmic reticulum during MAFLD development deserves further investigation.