Nonalcoholic fatty liver disease (NAFLD) is diagnosed when simple intrahepatic fat deposition (steatosis) exceeds 5% of liver weight. When steatosis is combined with severe inflammation, the accumulation of reactive oxygen species (ROS) and fibrosis development trigger nonalcoholic steatohepatitis (NASH) and cirrhosis, ultimately leading to hepatocellular carcinoma1. Therefore, hepatocellular lipid metabolism is tightly regulated and largely leads to two outcomes: lipid acquisition or elimination. When the uptake of free fatty acid (FFA) via FFA transporters, such as CD36 or de novo lipogenesis (DNL) in hepatic cells occur more than export of FFA via very low density lipoprotein (VLDL) packaging or usage of FFA by mitochondrial β-oxidation, hepatocellular fat gradually accumulates, which leads to NAFLD. Many factors are involved in regulating and maintaining hepatic lipid homeostasis2. Aging, mitochondrial dysfunction, and malnutrition, are risk factors that induce an imbalance in hepatic lipid homeostasis and NAFLD development3,4; however, the mechanisms involved in lipid homeostasis in hepatic cells and the causes of its disruption largely remain to be characterized. Physiological changes due to aging are usually accompanied by metabolic syndromes, such as obesity, dyslipidemia, and insulin resistance, which are closely associated with NAFLD4,5. The elevation of visceral fat caused by aging increases FFA levels in blood and induces FFA absorption by the liver6. In addition, the age-dependent reduction in growth hormone/insulin-like growth factor-1 reactions leads to DNL5,7. In contrast, aging decreases the expression of carnitine palmitoyl transferase-1 (CTP-1), the key enzyme regulating mitochondrial β-oxidation8. TG-VLDL is complexed with apoprotein B (apoB) and exported into the blood under tight regulation by insulin9. An increase in insulin resistance along with aging disrupts TG export. All these aging-related dysfunctions together accelerate the accumulation of TG in the liver, which is a typical phenomenon of NAFLD.
As vitamin D prevents various diseases, such as immune diseases and diabetes, many studies on vitamin D have been performed in recent years. The major stable circulating form of vitamin D in blood is 25-hydroxyvitamin D3 (25(OH)D3; that is, vitamin D3). It is generated via cytochrome P450 family 2 subfamily R member 1 (Cyp2R1) in the liver via hydroxylation at the C-25 position of 7-dehydrocholesterol, a nonenzymic product, in the skin mediated by sunlight. The active form of vitamin D in vivo is 1,25(OH)2-VitD3 (1,25VitD3), which is produced in the kidney. 1,25VitD3 binds a nuclear receptor, vitamin D receptor (VDR), thereby regulating many physiological processes, including immune responses and calcium homeostasis maintenance. 1,25VitD3 is ultimately catabolized in the kidney by cytochrome P450 family 24 subfamily A member 1 (Cyp24A1)10. Since 1,25VitD3 shows hormonal activity, cellular mechanisms can maintain appropriate an amount of 1,25VitD3 by balancing metabolic anabolism and catabolism. The production of 7-dehydrocholesterol declines with age, and the amount of vitamin D in the blood gradually decreases with age11,12. Therefore, most elderly people are vitamin D deficient, with the degree of deficiency differing by race and gender13. Vitamin D deficiency is considered a risk factor for NAFLD. Although many studies have shown inverse correlations between serum vitamin D3 levels and NAFLD14,15, the therapeutic effects of vitamin D on NAFLD remain controversial16.
Mitochondria are very dynamic organelles; mitochondrial fusion and fission occur continuously and are precisely regulated17. In these processes, most mitochondrial proteins are synthesized in the cytosol and transported into mitochondria through unique translocase complexes located in the outer or inner mitochondrial membranes: translocase of the outer mitochondrial membrane (TOM), and sorting and assembly machinery (SAM) in the mitochondrial outer membrane (OM), and translocase of the inner mitochondrial membrane (TIM) in the mitochondrial inner membranes (IM), respectively18. To prevent the generation of misfolded or incorrectly targeted proteins, the unique mitochondrial unfolded protein response (mtUPR) or mitochondrial quality control system (MQC) is evoked19. Mitochondria are key intracellular organelles that generate energy and are mainly responsible for the β-oxidation of FFAs and ROS production during oxidative phosphorylation (OXPHOS); therefore, in recent years, multiple studies have reported an association between mitochondrial dysfunction with cellular senescence and many chronic diseases, including NAFLD20,21. For instance, Takeochi Y et al. reported that the specific depletion of mitochondrial fission factor (MFF), a mitochondrial fission regulator in the liver, causes high-fat diet-induced NASH22. Although many studies have intensively investigated the link between mitochondrial dysfunction and chronic diseases, the molecular mechanisms have still not been clearly explained.
In contrast to the simple OM structure, the IM is composed of two distinct regions: the linear-shaped inner boundary membrane (IBM) and winding-shaped crista membrane (CM). At the entrance point of cristae, where two membranes meet to create narrow bottle neck-like structures called cristae junctions (CJs)23. In 2011, the Neupert W. group were the first to reported the discovery of a protein complex essential for the maintenance and formation of cristae, namely, the mitochondrial contact site and organizing system (MICOS)24. The MICOS complex comprises two subcomplexes, Mic60-Mic19-Mic25 and Mic10-Mic26-Mic27, with Mic13 (Qil1) being a stabilizer of the Mic60 and Mic10 subcomplexes in humans. TIM complexes are localized in the IBM, on the other hand, MICOS complexes are located in CJs, and OXPHOS complexes are localized in the CM; most notably, ATP synthase (complex Ⅴ) is located at the CM tip. Moreover, depletion of Mic 60 (also known as mitofusin, inner mitochondrial membrane protein (IMMT), and MINOS2), Mic10 (MINOS1) and Atp21, a subunit of ATP synthase, disrupts normal cristae structures and sequentially disturbs mitochondrial function24,25. Whereas Mic10 forms the structural core of MICOS, Mic60 is the main linker between OM and IM, which are connected via TOM, voltage-dependent anion channel (VDAC) and SAM. In particular, Mic60 interacts with Sam50, a SAM component, forming the MICOS-SAM supercomplex, which is also called the mitochondrial intermembrane space-bridging complex (MIB)26–28. In addition, Mic60 is associated with PTEN-induced kinase 1 (PINK1), a key protein involved in mitophagy29. Additionally, several proteins that regulate mitochondrial dynamics interact with the Mic60. For instance, mitochondrial dynamin-like GTPase optic atrophy 1 (Opa1) or SLC25A46, which is involved in mitochondrial fusion, interacts with the Mic60, and these interactions are believed to be involved in the maintenance and formation of cristae30,31. Most papers have concentrated on MICOS structural features, cristae formation and MICOS interactions with proteins. In summary, papers published thus far have indicated that MICOS is thought to be involved in overall systems that maintain proper mitochondrial functions, including membrane potential and ATP formation, due to its participating in cristae formation, mitochondrial biogenesis, and in apoptosis. Via its interaction with Mic60, Opa1 may tighten CJs thereby preventing the release of cytochrome c, which is usually located within cristae32,33. Oma1, a stress-inducible peptidase and a major regulator of mitochondrial fission, is thought to promote apoptosis34. In contrast, Viana MP et al. recently reported that Oma1 stabilized OM–IM supercomplexes by interacting with Mic60 in an Opa1-independent manner and that the depletion of Oma1 resulted in apoptotic resistance35. Although the number of studies on the association of MICOS and diseases have gradually recently increased36, little is known about these relationships.
In this study, we provided the first evidence showing that the Mic60 level declines with age and that its depletion induces TG accumulation in liver cells. In addition, we showed that vitamin D treatment rescued age-associated NAFLD by directly inducing Mic60 expression in a VDR-RXR-binding-dependent manner. Collectively, these findings implied that MICOS 60 participates in the development of NAFLD and that the direct upregulation of Mic60 expression mediated via vitamin D supplementation may be a molecular mechanism underlying the effective prevention of NAFLD development, especially in elderly people.