Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level.

Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level. Joo Hyun Lim, Gyu Hee Kim, Hyeon-Ju Jeong, Yoo Jeong Lee, Soo Kyung Koo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2437531/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease. Despite intensive research, considerable NAFLD development remains to be revealed. In this study, we examined the effects of vitamin D on age-induced NAFLD, especially in connection with mitochondrial abnormalities. We observed effective prevention of liver steatosis in 22-month-old C57BL/6 mice fed a vitamin D 3 -supplemented (20,000 IU/kg) diet, in contrast to mice fed a control (1,000 IU/kg) diet. We evaluated whether vitamin D 3 supplementation enhanced mitochondrial functions and found that the mitochondrial contact site and cristae organizing system (MICOS) 60 (Mic60) level was reduced and was specifically restored by vitamin D 3 supplementation in the aged mice. In addition, depletion of Immt , the human gene encoding the Mic60 protein, induced changes in gene expression that led to fat accumulation in HepG2 human hepatocellular carcinoma cells, which is effectively prevented by vitamin D 3 supplementation. In contrast, silencing of Lonp1 , the main matrix protease involved in mitochondrial quality control system and also expressed at a reduced level in aged mice, did not induce triglyceride (TG) accumulation in HepG2 cells. Moreover, VDR-RXR increased Mic60 levels by directly binding to the Immt 5' promoter region spanning from position − 3157 to -2323. Our study demonstrates, for the first time, that a reduction in Mic60 level due to aging may be one of the underlying mechanisms to development aging-associated NAFLD. In addition, vitamin D 3 could positively regulate Mic60 expression, which may be one of the important molecular mechanisms that vitamin D could ameliorate age induced NAFLD. Health sciences/Diseases/Metabolic disorders Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction 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 carcinoma 1 . 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 homeostasis 2 . Aging, mitochondrial dysfunction, and malnutrition, are risk factors that induce an imbalance in hepatic lipid homeostasis and NAFLD development 3 , 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 NAFLD 4 , 5 . The elevation of visceral fat caused by aging increases FFA levels in blood and induces FFA absorption by the liver 6 . In addition, the age-dependent reduction in growth hormone/insulin-like growth factor-1 reactions leads to DNL 5 , 7 . In contrast, aging decreases the expression of carnitine palmitoyl transferase-1 (CTP-1), the key enzyme regulating mitochondrial β-oxidation 8 . TG-VLDL is complexed with apoprotein B (apoB) and exported into the blood under tight regulation by insulin 9 . 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 D 3 (25(OH)D 3 ; that is, vitamin D 3 ). 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 -VitD 3 (1,25VitD 3 ), which is produced in the kidney. 1,25VitD 3 binds a nuclear receptor, vitamin D receptor (VDR), thereby regulating many physiological processes, including immune responses and calcium homeostasis maintenance. 1,25VitD 3 is ultimately catabolized in the kidney by cytochrome P450 family 24 subfamily A member 1 (Cyp24A1) 10 . Since 1,25VitD 3 shows hormonal activity, cellular mechanisms can maintain appropriate an amount of 1,25VitD 3 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 age 11 , 12 . Therefore, most elderly people are vitamin D deficient, with the degree of deficiency differing by race and gender 13 . Vitamin D deficiency is considered a risk factor for NAFLD. Although many studies have shown inverse correlations between serum vitamin D 3 levels and NAFLD 14 , 15 , the therapeutic effects of vitamin D on NAFLD remain controversial 16 . Mitochondria are very dynamic organelles; mitochondrial fusion and fission occur continuously and are precisely regulated 17 . 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: t ranslocase of the o uter m itochondrial membrane (TOM), and s orting and a ssembly m achinery (SAM) in the mitochondrial outer membrane (OM), and t ranslocase of the i nner mitochondrial m embrane (TIM) in the mitochondrial inner membranes (IM), respectively 18 . To prevent the generation of misfolded or incorrectly targeted proteins, the unique mitochondrial unfolded protein response (mtUPR) or mitochondrial quality control system (MQC) is evoked 19 . 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 NAFLD 20 , 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 NASH 22 . 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 i nner b oundary m embrane (IBM) and winding-shaped c rista m embrane (CM). At the entrance point of cristae, where two membranes meet to create narrow bottle neck-like structures called c ristae 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 mi tochondrial co ntact s ite 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 function 24 , 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 m itochondrial i ntermembrane space- b ridging complex (MIB) 26 – 28 . In addition, Mic60 is associated with PTEN-induced kinase 1 (PINK1), a key protein involved in mitophagy 29 . 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 cristae 30 , 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 cristae 32 , 33 . Oma1, a stress-inducible peptidase and a major regulator of mitochondrial fission, is thought to promote apoptosis 34 . 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 resistance 35 . Although the number of studies on the association of MICOS and diseases have gradually recently increased 36 , 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. Materials And Methods Materials Animal Studies Male C57BL/6 mice (3 and 18 months old, n = 10∼12 per group) were purchased from the Animal Facility of Aging Science, Korea Basic Science Institute (KBSI) Gwangju Center (Gwangju, Korea). After a week of adaptation, 3- or 18-month-old mice were randomly divided into two groups and fed for 4 months as follows: The control group was fed a standard chow diet (AIN-93G, Research Diets, NJ, USA) containing vitamin D 3 (1,000 IU/kg), and the vitamin D 3 -supplemented group was fed a standard chow diet enriched with vitamin D 3 (20,000 IU/kg). All animal experiments were performed according to the guidelines of the Korean National Institutes of Health Animal Care and Use Committee (permit number: KCDC-032-20-2A). Silencing of genes Short interfering RNA (siRNA) against Immt (5’-CACCCAAGCUUUAACCGCAtt-3’, 5’-UGCGGUUAAAGCUUGGGUGaa) and LonP1 (5’-GAUUAUCGAGGUUAAAAAUtt, 5’-AUUUUUAACCUCGAUAAUCtt) were synthesized by Ambion (Life Technologies, CA, USA). Transfection with the siRNAs was performed using Lipofectamine RNAiMAX (Invitrogen, Cambridge, UK) reagent for 24 hr. Measurement of TG concentration Liver tissues or HepG2 cells were homogenized in 1 ml of 5% NP-40 solution using a homogenizer for 30 s. The samples were slowly heated to 99°C in a heat block for 5 min and then cooled to room temperature. The samples were centrifuged for 2 min at top speed with a microcentrifuge to remove insoluble material. For tissue obtained from animals, samples were diluted 10-fold with ddH 2 O, and for cells obtained from the HepG2 cell culture, the samples were diluted 3-fold with ddH 2 O before TG analysis. Prepared samples were measured using a triglyceride assay kit (Ab65336, Abcam, Cambridge, UK). Immunohistochemistry Paraffin sections (5 µm) were deparaffinized and hydrated with xylene and ethanol. The sections were blocked with 2.5% normal horse serum at room temperature for 30 min and then incubated overnight at 4°C with an anti-Mic60 antibody (Abcam, CO, USA). After washing, the sections were incubated with Alexa Fluor-488 secondary antibody (Invitrogen, CA, USA) for 1 hr and washed with PBS. The sections were stained with DAPI (H-1200, Vector Laboratories, CA, USA) and observed under a fluorescence microscope. Images of the whole slide were captured using a confocal laser scanning microscope (FV3000-OSR, Olympus Corp., Japan). The relative fluorescence intensity was measured using CellSens (Olympus Corp., Japan) software. Measurement of electron microscopy HepG2 cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 overnight. The samples were sequentially dehydrated in 50% ethanol, 70% ethanol, 90% ethanol, 95% ethanol and 100% ethanol. After embedding with Epon 812 (Merck, Darmstadt, Germany), 70-nm sections were sliced with an ultrathin microtome. The sections were stained with 1% uranyl acetate for 5 min, incubated with 1% lead citrate for 3 min, and observed with a transmission electron microscope (LIBRA-120, Carl Zeiss, Germany). Chromatin immunoprecipitation (ChIP) ChIP analysis was performed according to the manufacturer's instructions (Millipore, Darmstadt, Germany). Briefly, HepG2 cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine. Cells were resuspended and sonicated in SDS lysis buffer. Lysates were incubated overnight at 4°C with the following antibodies: anti-mouse IgG (Santacruz, Dallas, U.S.A., sc-2025), an anti-VDR (Santacruz, sc-131333X) antibody and an anti-RXRα (Santacruz, sc-515929X) antibody. Protein G agarose was added to form the immunocomplexes, and were washed and subjected to elution. The samples were treated with RNase A and Proteinase K. DNA was subsequently purified using PCR purification spin columns (QIAGEN, Hilden, Germany). The primers used to amplify the Immt promoter regions, designated R1 (-3986~-3203), R2 (-3157~-2323), R3 (-2312~-1724), R4 (-1845~-1159), R5 (-1179~-550) and R6 (-574 ~ 115) from the transcription start site (TSS), respectively. The primers are listed in supplementary Table 1. Statistical analysis All results are expressed as the means ± standard errors of the means (SEMs). Statistical analysis was performed using GraphPad Prism software (GraphPad, CA, USA). Comparisons between two groups were performed with Student’s t test or nonparametric Mann–Whitney U test. For multiple group comparisons, one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons was performed to evaluate significant differences. P values < 0.05 were considered to be statistically significant. Results Vitamin D supplementation restored the age-dependent reduction in liver Mic60 level Vitamin D is thought to have beneficial functions in preventing NAFLD; however, its effects remain controversial 16 , 37 . Moreover, most studies have investigated only vitamin D deficiency in NAFLD and have not explored combination of vitamin D with multiple other risk factors, such as aging or mitochondrial dysfunction. In this study, we examined the precise molecular mechanisms through which vitamin D induces age-induced NAFLD, especially in relation to mitochondria. We first measured the liver mass and degree of lipid accumulation by hematoxylin and eosin (H&E) staining samples from young (7-month-old) and aged (22-month-old) C57BL/6 mice. We observed a significant reduction in the abnormal expansion of the total liver mass and lipid accumulation within the liver only in the aged mice fed a vitamin D 3 -supplemented diet (20,000 IU/kg) (Fig. 1 b and c). In addition, the circulating FFA and hepatic TG levels were inversely correlated with serum 25(OH)D 3 levels (Fig. 1 d, e). Next, we measured gene expression patterns related to lipid homeostasis in the liver in samples extracted from each group of animals. Aging altered lipid metabolism to promote TG accumulation, and vitamin D 3 supplementation led to the opposite results only in aged mice, corresponding with histological changes (Fig. 1 f). In particular, we observed profound alterations in the expression of the following genes: peroxisome proliferator-activated receptor (PPAR)γ and cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) involved in DNL 38 . Moreover, the expression of PPARα and carnitine palmitoyltransferase 1 (CPT-1), which are key regulators of β-oxidation, was reduced 39 . A marked increase in the expression of CD36, very low density of lipoprotein receptor (Vldlr) and monoacylglycerol O -acyltransferase 1 (Mgat-1), a diacylglycerol (DAG) and TG synthesis catalase, was observed 40 . Aging and a decline in mitochondrial functions are closely associated 41 , and mitochondrial dynamics and MQC are important for the maintenance of optimal mitochondrial function 42 . Therefore, we measured changes in the levels of proteins related to mitochondrial dynamics and MQC. In aged mice, the expression of proteins involved in OXPHOS (Supplementary Fig. 1) and mitochondrial fusion was reduced, and this decrease was reversed by vitamin D 3 supplementation (Fig. 2 a). Previously, Ryan Z. C. et al. reported similar effects of vitamin D, which increased the levels of mitochondrial fusion proteins, such as mitofusin1 (Mfn1) and Opa1, and reduced the levels of fission proteins, mitochondrial fission protein 1 (fis1) and Oma1 in skeletal muscle 43 . In agreement with the results shown in Fig. 1 , the marked compensatory effects of vitamin D 3 were observed only in the aged mice. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and lon protease 1 (LonP1) are representative MQC proteins located in the mitochondrial matrix 44 . We observed that only the level of LonP1 was specifically reduced in the aged mice and that the level was restored by vitamin D 3 treatment (Fig. 2 b). MICOS is a relatively newly discovered mitochondrial protein complex; therefore, its role, especially in diseases, is largely unknown. To determine whether the level of MICOS was altered in our animal model system, we performed real-time PCR and Western blot analysis. Interestingly, we found that the expression of Mic60 was specifically reduced by aging, and the level was restored by vitamin D 3 supplementation only in the aged mice (Fig. 2 c). To confirm these results, we performed immunohistochemistry with an anti-Mic60 antibody and determined that the expression of Mic60 was inversely correlated with aging and vitamin D 3 supplementation (Fig. 2 d). Considering these results, we demonstrated that aging, mitochondrial dysfunction, and NAFLD are closely related and that vitamin D 3 supplementation is effective only when the vitamin D 3 concentration in the blood is too low, as it is in aged mice. In particular, we observed a reduction in Mic60 and LonP1 levels due to aging and found that vitamin D 3 supplementation restored these levels in aged mice. Depletion of Mic60 specifically induced TG accumulation in the liver To examine whether the decrease in Mic60 or Lonp1 level with aging is related to fat accumulation in the liver, we first transfected HepG2 human hepatoma cells with siRNAs against Immt and lonp1 . In all cases, transfection of both siRNAs led to specific depletion of individual genes (Fig. 3 a), and the cellular ATP production level and mitochondrial membrane potential were gradually decreased (Supplementary Fig. 2). Next, we examined whether the expression pattern of MICOS subunits is altered by depletion of Mic60 or LonP1. Significant changes were observed in MICOS subunits, except Mic27, after Immt silencing, but no profound reduction in MICOS subunit levels was observed after siLonP1 silencing (Fig. 3 b). Interestingly, we observed opposite changes in the Sam50 and SLC25A46 levels after the depletion of Mic60 or LonP1. We therefore examined the amount of both proteins in our animal models. As shown in Fig. 3 c, the protein expression of Sam50 and SLC25A46 was reduced in aged mice and restored by vitamin D 3 , similar to the protein expression of Mic60. Both Sam50 and SLC25A46 interact with Mic60 28,31 but not with LonP1. Therefore, the reduction in the Sam50 and SLC25A46 levels in aged mice seemed to be related to the reduction in the Mic60 level in aged mice. In addition, we observed the influences of depleting either Mic60 or Lonp1 on mitochondrial dynamics. We found that silencing Mic60 led to upregulated Fis1, Oma1 and dynamin-related protein 1 (Drp1) expression and downregulated Mfn1 and Mfn2 expression; moreover, it led to decreased expression of Opa1, which caused changes in the directions towards to the mitochondrial fragmentation (Fig. 3 d). In contrast, depletion of LonP1 did not lead to any significant alteration in the levels of proteins involved in mitochondrial dynamics. To determine whether these alterations in mitochondria caused by silencing of specific RNAs are related to fat accumulation in the liver, we measured the amount of TG after silencing Immt or lonp1 . Interestingly, we found that only the loss of Mic60 specifically increased TG accumulation in the liver (Fig. 3 e). In addition, we determined that the DNL and lipid uptake rates were greatly increased by the loss of Mic60, similar to the observations in our animal model. These outcomes were obvious only in the absence of Mic60 but not in the absence of Lonp1 (Fig. 3 f, g). From these results, we suggest that there is a specific relationship between the loss of Mic60 and TG accumulation in liver cells. Vitamin D restores mitochondrial function and prevents TG accumulation after the loss of Mic60 In aged mice, vitamin D 3 supplementation effectively prevented the reduction in Mic60 level. To test whether vitamin D 3 affects Mic60 expression in cell models, we treated cells with 1,25VitD 3 after silencing Immt . Even though siImmt transfection completely depleted Mic60 in cells, the 1,25VitD 3 treatment restored some level of the Mic60 protein and other MICOS components (Fig. 4 a, left panel). Notably, mitochondrial fusion proteins, membrane potential and cellular ATP levels were also restored by vitamin D (Fig. 4 a, right panel and 4b). We also observed that 1,25VitD 3 treatment effectively prevented TG accumulation that had been caused by a decrease in the Mic60 level (Fig. 4 c). To verify the fat accumulation specifically induced by the loss of Mic60 and the prevention of this outcome after 1,25VitD 3 treatment, we performed an oil red O staining assay after siImmt transfection in the presence and absence of 1,25VitD 3 . We observed significant fat accumulation after siImmt transfection and a reduction in the number of lipid droplets after 1,25VitD 3 treatment (Fig. 4 d). Recently, Stephan T. et al. showed that after depletion of Immt in HeLa cells, normal cristae formation was largely disrupted, and most cristae formed scattered small ladder-like shapes 45 . We next examined whether vitamin D can restore mitochondrial morphology that had been disrupted by the depletion of Mic60. We observed mitochondrial shapes through an electron microscope 24 hr after siImmt treatment in the presence or absence of 50 nM 1,25VitD 3 . In addition to a decrease in the Mic60 level, we found that the number of normal mitochondria was reduced, and 1,25VitD 3 treatment significantly restored the overall number and normal morphology of these mitochondria (Fig. 4 e). To confirm that aging and a reduction in Mic60 level were related to each other, we established a cell model similar to that of the aging animal model by treating HepG2 cells with doxorubicin (Dox) as indicated 46 . By increasing the concentration of Dox, the levels of representative senescence marker proteins, such as p21 and p53, were greatly increased. In contrast, the level of Mic60 was gradually decreased, as observed in our animal models (Fig. 5 a). We next examined the effects of vitamin D after the induction of cellular senescence. The levels of a senescence marker protein and Mic60 were inversely correlated, and vitamin D specifically upregulated the expression of Mic60, consistent with observations in the animal model (Fig. 5 b). To verify the recovery of Mic60 induced by vitamin D treatment, we measured Immt expression after Dox treatment in the absence or presence of 1,25VitD 3 and confirmed that vitamin D specifically upregulated Immt expression (Fig. 5 c). We also measured the alteration of proteins involved in mitochondrial dynamics after Dox treatment and observed changes similar to those observed after the depletion of Mic60: A decrease in mitochondrial fusion and an increase in fission was observed, and these effects were reversed by 1,25VitD 3 treatment (Fig. 5 d). Next, we examined whether silencing Immt triggered a counteraction to cellular senescence. As shown in Fig. 5 e, the loss of Immt significantly induced several senescence markers, including p53, p21 and interleukin 1A (IL-1A). From these experiments, we confirmed that the Mic60 level was dramatically decreased with aging and cellular senescence and that vitamin D specifically restored the senescence-dependent reduction in Mic60 level. In contrast, an absence or reduction in Mic60 level may trigger cellular senescence and aging. Vitamin D regulates Immt expression by the direct binding of VDR-RXR to Immt Vitamin D is an important regulator of Ca 2+ homeostasis 10 , multiple immune responses and antioxidative responses 47 . Binding of vitamin D to VDR, together with its heterodimeric receptor retinoid X receptor (RXR), usually triggers conformational changes, which enables the recognition of vitamin D-responsive elements (VDREs) on vitamin D target genes. VDRE usually does not comprise rather than a conserved linear sequences, it is more three-dimensional and variant VDRE sequences may influence unique complexes carrying VDR-RXR 10 , 48 . To test whether elevation of Mic60 level by vitamin D exerts indirect effects, such as alterations in Ca 2+ homeostasis or oxidative stress, or whether vitamin D directly regulates Mic60 level by VDR binding, we performed chromatin immunoprecipitation (ChIP) using anti-VDR and anti-RXR antibodies. First, we searched for possible retinoid X receptor responsive elements (RXREs) or VDREs throughout the whole Immt gene, from the 5´ intron to 3´ tail sequence, using NCBI blast suite SRA (SRX100497; HepG2_IP RXR) and found five candidate regions (Supplementary Table 1). We assessed the recruitment of VDR and RXRα to all these candidate RXRE or VDRE sites in the regulatory region upstream in the Immt promoter and found one specific high-affinity VDR-RXR binding site located in the region spanning positions − 3157 ~ -2323 from the transcription start site (TSS). The binding of both VDR and RXRα to this VDRE was dramatically reduced by Dox treatment but was very strong in the presence of the VDR-RXR ligand 1,25VitD 3 (Fig. 6 ). These results reveal, for the first time that VDR-RXR directly binds to Immt in a region ranging from position − 3157 to -2323, thereby specifically upregulating Immt expression mediated by vitamin D. Discussion The aging population is rapidly increasing worldwide; therefore, interest in research on aging-associated diseases, such as Parkinson’s disease and metabolic syndrome, has been increasing. The prevalence of NAFLD is also closely related to aging. In this study, we showed, for the first time, that depletion of Mic60 was directly associated with age-induced NAFLD development. In this work, we report the following interesting facts: (ⅰ) Aging triggered a specific reduction in Mic60 level. (ⅱ) Depletion of Mic60 disrupted lipid homeostasis in liver cells, which directly led to TG accumulation. (ⅲ) Vitamin D 3 prevented lipid accumulation in aged mice and in senescence-induced HepG2 cells, which coincided with the recovery of the Mic60 level. (ⅳ) The loss of Mic60 itself disrupted mitochondrial dynamics and triggered cellular senescence. (ⅴ) Vitamin D 3 upregulated Mic60 expression via the recruitment of VDR-RXRα to the promoter of the Immt gene. Although mitochondrial dysfunction and concomitant elevation of oxidative stress are associated with numerous diseases, including chronic diseases and even cancer 49 . Even though MICOS is important for cristae formation and related mitochondrial functions, most studies on MICOS have thus far focused on MICOS structural effects, and studies on the relationship of MICOS and human diseases have recently been reported. To date, representative studies on the association between MICOS and chronic diseases include the following reports: Baseler W.A. et al. reported a reduction in Mic60 level in type 1 diabetic heart 50 , and Thapa D. et al. showed that transgenic cardiac-specific overexpression of Mic60 ameliorated diabetic cardiomyopathy 51 . Additionally, Guarani V. et al . demonstrated that the Mic13-null mutant was critical to early onset fetal mitochondrial encephalopathy with liver diseases 52 and that Mic26 was overexpressed in the human diabetic heart 53 . Our finding clearly showed, for the first time, that age-dependent reduction in the Mic60 level or the depletion of Mic60 specifically induces TG accumulation, a key characteristic of NAFLD (Fig. 4 ). We observed age-dependent reduction in Mfn1, Opa1 and LonP1 levels, consistent with previous reports 44 (Fig. 2 ). Since many studies have been performed on mitochondrial dynamics, we selectively depleted Mic60 and LonP1, which are less known among the proteins reduced in our aged mice. Although deletion of both Mic60 and LonP1 led to a reduction in the cellular ATP level and mitochondrial membrane potential (Supplementary Fig. 2), only depletion of Mic60 specifically led to TG accumulation in HepG2 cells (Fig. 4 ). These findings imply the possibility that a mechanism in addition to the reduction in the overall mitochondrial function is involved with TG accumulation. We observed a specific reduction not only in the level of Mic19, one of the MIC60-Mic19-Mic25 subunits, but also in the levels of sam50, SLC25A46, Opa1 and Mfn1 and an increase in the levels of Oma1 and Fis1 (Fig. 3 b, d). These outcomes were observed only in the absence of Mic60, not in the absence of LonP1 (Fig. 3 ). Recently, Tang J. et al . reported that the Sam50-Mic19-Mic60 interaction is important to the connection between the OM and the IM and is involved in normal cristae shape formation 54 . In addition, they found that Oma1 disrupted this membranous connection by cleaving Mic19. Recently, using liver-specific Sam50-knockout mice, Chen L. et al . reported that depletion of Sam50 induced liver inflammation and liver injury 55 . In addition, the SAMM50 polymorphism has been reported to be associated with NAFLD 56 . Considering these studies, we hypothesize that a reduction in Sam50 level induced by the depletion of Mic60 might be a specific reason for the TG accumulation induced by the loss of Mic60. We also suggest that Mic60 may play a central role not only in mitochondrial cristae formation and the maintenance of mitochondrial dynamics but also in the connection between mitochondrial function and the environment outside organelles via its interactions with multiple proteins; thus, Mic60 may influence the development of NAFLD. Although Lonp1 is important to maintain overall mitochondrial function, we found that loss of Lonp1 did not induce TG accumulation. We suggest that LonP1 did not induce fat accumulation because it did not induce a change in the mitochondrial structure and thus exerted no overall influence on the connection between the OM and IM, even though Lonp1 plays a central role in MQC and is important to maintain mitochondrial function. However, many studies remain to be performed to determine the precise molecular mechanisms through which depletion of Mic60 may promote NAFLD. Many reports, including those of Roth C. L. et al. , suggested therapeutic effects of vitamin D on NAFLD 57 . In this study, we observed that vitamin D effectively prevented TG accumulation only in aged mice. Similarly, we observed the preventive effect of vitamin D 3 on pancreatic dysfunction and hyperinsulinemia defects only in aged mice 58 . Pines A. et al. showed that the beneficial effects of vitamin D 3 supplementation were greater on aged men than on younger men 59 . The optimal vitamin D 3 concentration varies considerably, and the normal concentration of vitamin D 3 in human serum is typically considered to be 25 ~ 80 ng/mL 13 . In our mouse model, the serum vitamin D 3 level was insufficient only in the aged mice fed a normal chow diet, and it was restored in the aged mice fed a diet supplemented with vitamin D 3 (Fig. 1 e). Healthy human hepatocytes contain relatively low levels of VDR, but nonparenchymal cells produce a large amount of VDR 60 . As Keane J. T. et al . clearly described in their review, the amount of VDR in hepatocytes is increased in the disease state 37 . Interestingly, we found that vitamin D 3 restored the expression of Mic60 only in aged mice fed a diet supplemented with vitamin D 3 (Fig. 2 c, d) and in senescence-induced HepG2 cells (Fig. 5 b). In addition, we showed that VDR-RXR bound directly to Immt to regulate its expression; that is, the effect of vitamin D on Mic60 was not indirectly mediated by alterations in the cellular environment. Considering these observations, we suggest that it is important to maintain a sufficient serum vitamin D and VDR level in the hepatocytes in aged mice to preserve a sufficient Mic60 level in the liver and prevent mitochondrial dysfunction and subsequent age-related NAFLD development. In conclusion, in this study, we first demonstrated that depletion of Mic60 is related to the development of age-induced NAFLD and that vitamin D can prevent NAFLD by upregulating Mic60 expression in a VDR-RXR-dependent manner. Further studies are needed to determine whether the loss of Mic60 is related to the development of age-dependent NAFLD in humans Declarations Acknowledgments This study was supported by an intramural research grant from the Korea National Institute of Health (2020-NG-014-02). We are appreciated to Jee Woong Kim for the technical assistance of electron microscope measurement Author Contributions J.H.L designed this study and wrote the manuscript. G.H.K. mainly performed experiments and analyzed data. H.J. wrote some part of this manuscript and performed experiments. Y.J.L performed animal study. S.K.K. commented on the manuscript. Conflict of Interest The authors declare that they have no conflict of interest. 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Additional Declarations (Not answered) Supplementary Files SIKimetalEMM.docx Cite Share Download PDF Status: Published Journal Publication published 04 Jan, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 24 May, 2023 Review # 2 received at journal 23 May, 2023 Reviewer # 2 agreed at journal 01 May, 2023 Review # 1 received at journal 10 Feb, 2023 Reviewer # 1 agreed at journal 26 Jan, 2023 Reviewers invited by journal 26 Jan, 2023 Submission checks completed at journal 03 Jan, 2023 First submitted to journal 03 Jan, 2023 Editor assigned by journal 03 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2437531","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":170803134,"identity":"623bd421-67ab-4c60-a75b-778d3a490ae1","order_by":0,"name":"Joo Hyun Lim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACZjYgYcAgJwHhJhCvxZgELQxsYDJxBtFadNvZEh9XFNikz2zvffjgB0NaPkEtZofZDhueMUjLnc1z3NiwhyHHsoGwFvY2yQaDw7nzJNLYJHgYKgyIsAWiJV1OIo395x/itLAdA2lJkAbawszDkEOUlmTDBoM0w5k9x5ilZQzSiNBy/pjhw4Y/NvISx9sYP76pSCasBQ2QrGEUjIJRMApGAVYAAJLpMzR9lCmLAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5107-3774","institution":"Korea National Institute of Health","correspondingAuthor":true,"prefix":"","firstName":"Joo","middleName":"Hyun","lastName":"Lim","suffix":""},{"id":170803135,"identity":"273ab946-42e0-4f7e-96b6-874c9a1fd234","order_by":1,"name":"Gyu Hee Kim","email":"","orcid":"","institution":"Korea National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Gyu","middleName":"Hee","lastName":"Kim","suffix":""},{"id":170803136,"identity":"bd490b30-bf88-40ef-9611-26898f81559f","order_by":2,"name":"Hyeon-Ju Jeong","email":"","orcid":"https://orcid.org/0000-0001-9025-2559","institution":"Korea National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Hyeon-Ju","middleName":"","lastName":"Jeong","suffix":""},{"id":170803137,"identity":"38f43845-fbf4-422c-84b2-1678ffbaa9bc","order_by":3,"name":"Yoo Jeong Lee","email":"","orcid":"","institution":"Korea National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Yoo","middleName":"Jeong","lastName":"Lee","suffix":""},{"id":170803138,"identity":"a3411452-5d3e-4ab7-8f33-3a020d8321c4","order_by":4,"name":"Soo Kyung Koo","email":"","orcid":"","institution":"KNIH","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Kyung","lastName":"Koo","suffix":""}],"badges":[],"createdAt":"2023-01-03 05:35:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2437531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2437531/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-023-01125-7","type":"published","date":"2024-01-04T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32226299,"identity":"70b49895-0bfd-44cb-aa29-2f2fd27e1d1c","added_by":"auto","created_at":"2023-01-30 18:27:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":824928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin D\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e specifically prevents the development of NAFLD in aged mice.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Body weight changes in 3 month- or 18-month-old mice in an 18-week study (n=10~12). \u003cstrong\u003eb\u003c/strong\u003e The weight of livers extracted from mice as indicated. \u003cstrong\u003ec\u003c/strong\u003e Representative images of H\u0026amp;E stained liver tissue sections of 7- or 22-month-old mice. Scale bar = 30 μm. \u003cstrong\u003ed\u003c/strong\u003e Tissue TG and serum free fatty acid (FFA) levels were measured using ELISA kits according to the manufacturer’s instructions. Each value was normalized to that of the protein concentration (n=5). \u003cstrong\u003ee\u003c/strong\u003e Serum vitamin D\u003csub\u003e3 \u003c/sub\u003elevel (n=5). \u003cstrong\u003ef\u003c/strong\u003e Quantitative real-time PCR of genes related to lipid metabolism (n=5). Statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test for multiple comparisons; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/6231d2937c6c3d403dab5d06.png"},{"id":32226304,"identity":"14348e83-dd80-4504-a4e0-d96bea646e3d","added_by":"auto","created_at":"2023-01-30 18:27:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":987718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration in MICOS and mitochondrial dynamics-associated protein levels in aged mice.\u003c/strong\u003e Changes in the levels of proteins involved in either mitochondrial dynamics \u003cstrong\u003ea\u003c/strong\u003e or MQC \u003cstrong\u003eb\u003c/strong\u003e (n=3). Relative values compared to value of young mice fed a chow diet are presented as the means ± SEMs (n=3). \u003cstrong\u003ec\u003c/strong\u003e Protein levels of MICOS subunits (left panel) and quantitative real-time PCR analysis of MICOS subunits (right panel, n=5). \u003cstrong\u003ed\u003c/strong\u003e Immunofluorescence images of mouse liver tissue obtained using an anti-Mic60 antibody as indicated. Scale bars = 50 μm. Statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test for multiple comparisons; *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/872114a113d111b2192ca0cf.png"},{"id":32226300,"identity":"aeea6085-6d31-41e9-82e4-3d8fd5da0f0c","added_by":"auto","created_at":"2023-01-30 18:27:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":461050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of Mic60 specifically led to TG accumulation in HepG2 cells.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Immunoblot analysis after transfection of individual siRNAs as indicated. \u003cstrong\u003eb\u003c/strong\u003e Changes in the protein level of MICOS. \u003cstrong\u003ec\u003c/strong\u003e Immunoblots showing Sam50 and SLC25A46 levels in the liver tissue obtained from the animals as indicated (n=3). \u003cstrong\u003ed\u003c/strong\u003e Alterations in the levels of proteins involved in mitochondrial dynamics after transfection of \u003cem\u003esiImmt\u003c/em\u003eor \u003cem\u003esiLonP1\u003c/em\u003e. \u003cstrong\u003ee\u003c/strong\u003e The TG level was measured with a TG assay kit following the manufacturer’s instruction (n=3). \u003cstrong\u003ef, g\u003c/strong\u003eQuantitative real-time PCR of genes related to lipid metabolism after transfection of either \u003cem\u003esiImmt\u003c/em\u003e or \u003cem\u003esiLonP1\u003c/em\u003e. Statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test for multiple comparisons; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 (n=3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/37f6d340fe7a2ca83682d945.png"},{"id":32226777,"identity":"295ee75c-6300-4f7a-a499-0f0e29e4af7f","added_by":"auto","created_at":"2023-01-30 18:35:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1354841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin D\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e supplementation prevents TG accumulation by restoring the Mic60 level.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Cells were treated with 1,25VitD\u003csub\u003e3\u003c/sub\u003e 24 h after \u003cem\u003esiImmt\u003c/em\u003e transfection. Immunoblot analyses were performed using antibodies as indicated. \u003cstrong\u003eb\u003c/strong\u003e Mitochondrial membrane potential was measured with a JC-1 assay kit, and cellular ATP levels were measured with an ATP assay kit (n=3). \u003cstrong\u003ec\u003c/strong\u003e TG levels were determined using a TG assay kit following the manufacturer’s instructions (n=3). \u003cstrong\u003ed\u003c/strong\u003e Cellular lipid droplets were visualized by oil red O staining (n=3). \u003cstrong\u003ee\u003c/strong\u003e Electron microscopy images of HepG2 cells after the treatment indicated. Scale bars = 1μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/d0f84fbb69d41781ddafa325.png"},{"id":32226297,"identity":"d490411a-a058-48dc-a2b1-8558a39246f6","added_by":"auto","created_at":"2023-01-30 18:27:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":382827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of cellular senescence triggered a reduction in the Mic60 level.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e MICOS protein levels were altered by increasing the concentration of doxorubicin for 24 h. \u003cstrong\u003eb\u003c/strong\u003e Western blot analysis after induction of senescence in the presence or absence of 20 nM 1,25VitD\u003csub\u003e3\u003c/sub\u003e. \u003cstrong\u003ec\u003c/strong\u003e Measurement of \u003cem\u003eImmt\u003c/em\u003e expression by qRT‒PCR (n=5). \u003cstrong\u003ed\u003c/strong\u003e Analysis of the effects on the mitochondrial dynamics, as determined by immunoblotting, as indicated. \u003cstrong\u003ee\u003c/strong\u003e Silencing of \u003cem\u003eImmt\u003c/em\u003e triggered the expression of cell senescence markers (n=3).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/bde873e525e1b33abc1d64ca.png"},{"id":32226298,"identity":"1fdb8b44-1d70-486b-8243-3c2ee4e1ed14","added_by":"auto","created_at":"2023-01-30 18:27:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":62250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVDR-RXR upregulated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eImmt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression by direct binding the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eImmt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene in HepG2 cells. \u003c/strong\u003eChromatin immunoprecipitation (ChIP) assay with control vehicle or 500 µM doxorubicin treatment of HepG2 cells in combination with 20 nM or 50 nM 1,25VitD\u003csub\u003e3 \u003c/sub\u003eusing putative VDREs or RXREs. The recruitment of VDR or RXRa to the position ranging from -3157 to -2323 was the representative value for comparisons to the IgG control (three sets per group). Statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test for multiple comparisons; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/bea3bddcf087147ca6727f77.png"},{"id":49166061,"identity":"55fc467f-5e59-4d68-bd64-1f343418af89","added_by":"auto","created_at":"2024-01-04 08:05:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2547574,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/28065cdf-494e-4caa-bea3-71ab3a25e3cc.pdf"},{"id":32226776,"identity":"a567919c-b8ec-4a3c-8ae0-58f2cb5b6204","added_by":"auto","created_at":"2023-01-30 18:35:35","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":272235,"visible":true,"origin":"","legend":"","description":"","filename":"SIKimetalEMM.docx","url":"https://assets-eu.researchsquare.com/files/rs-2437531/v1/17a43d8b6a23e117eff8ab1e.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNonalcoholic 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 carcinoma\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. 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 \u003cem\u003ede novo\u003c/em\u003e 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 homeostasis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Aging, mitochondrial dysfunction, and malnutrition, are risk factors that induce an imbalance in hepatic lipid homeostasis and NAFLD development\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e; 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 NAFLD\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The elevation of visceral fat caused by aging increases FFA levels in blood and induces FFA absorption by the liver\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition, the age-dependent reduction in growth hormone/insulin-like growth factor-1 reactions leads to DNL\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In contrast, aging decreases the expression of carnitine palmitoyl transferase-1 (CTP-1), the key enzyme regulating mitochondrial β-oxidation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. TG-VLDL is complexed with apoprotein B (apoB) and exported into the blood under tight regulation by insulin\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eAs 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 D\u003csub\u003e3\u003c/sub\u003e (25(OH)D\u003csub\u003e3\u003c/sub\u003e; that is, vitamin D\u003csub\u003e3\u003c/sub\u003e). 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 \u003cem\u003ein vivo\u003c/em\u003e is 1,25(OH)\u003csub\u003e2\u003c/sub\u003e-VitD\u003csub\u003e3\u003c/sub\u003e (1,25VitD\u003csub\u003e3\u003c/sub\u003e), which is produced in the kidney. 1,25VitD\u003csub\u003e3\u003c/sub\u003e binds a nuclear receptor, vitamin D receptor (VDR), thereby regulating many physiological processes, including immune responses and calcium homeostasis maintenance. 1,25VitD\u003csub\u003e3\u003c/sub\u003e is ultimately catabolized in the kidney by cytochrome P450 family 24 subfamily A member 1 (Cyp24A1)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Since 1,25VitD\u003csub\u003e3\u003c/sub\u003e shows hormonal activity, cellular mechanisms can maintain appropriate an amount of 1,25VitD\u003csub\u003e3\u003c/sub\u003e 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 age\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Therefore, most elderly people are vitamin D deficient, with the degree of deficiency differing by race and gender\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Vitamin D deficiency is considered a risk factor for NAFLD. Although many studies have shown inverse correlations between serum vitamin D\u003csub\u003e3\u003c/sub\u003e levels and NAFLD\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, the therapeutic effects of vitamin D on NAFLD remain controversial\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondria are very dynamic organelles; mitochondrial fusion and fission occur continuously and are precisely regulated\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. 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: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et\u003c/span\u003eranslocase of the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eo\u003c/span\u003euter \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eitochondrial membrane (TOM), and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003es\u003c/span\u003eorting and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ea\u003c/span\u003essembly \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eachinery (SAM) in the mitochondrial outer membrane (OM), and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et\u003c/span\u003eranslocase of the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ei\u003c/span\u003enner mitochondrial \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eembrane (TIM) in the mitochondrial inner membranes (IM), respectively\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To prevent the generation of misfolded or incorrectly targeted proteins, the unique mitochondrial unfolded protein response (mtUPR) or mitochondrial quality control system (MQC) is evoked\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. 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 NAFLD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For instance, Takeochi Y \u003cem\u003eet al.\u003c/em\u003e reported that the specific depletion of mitochondrial fission factor (MFF), a mitochondrial fission regulator in the liver, causes high-fat diet-induced NASH\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Although many studies have intensively investigated the link between mitochondrial dysfunction and chronic diseases, the molecular mechanisms have still not been clearly explained.\u003c/p\u003e \u003cp\u003eIn contrast to the simple OM structure, the IM is composed of two distinct regions: the linear-shaped \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ei\u003c/span\u003enner \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003eoundary \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eembrane (IBM) and winding-shaped \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003erista \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eembrane (CM). At the entrance point of cristae, where two membranes meet to create narrow bottle neck-like structures called \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003eristae junctions (CJs)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. 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 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emi\u003c/span\u003etochondrial \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eco\u003c/span\u003entact \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003es\u003c/span\u003eite and organizing system (MICOS)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. 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 function\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. 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 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eitochondrial \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ei\u003c/span\u003entermembrane space-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003eridging complex (MIB)\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In addition, Mic60 is associated with PTEN-induced kinase 1 (PINK1), a key protein involved in mitophagy\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. 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 cristae\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. 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 cristae\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Oma1, a stress-inducible peptidase and a major regulator of mitochondrial fission, is thought to promote apoptosis\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In contrast, Viana MP \u003cem\u003eet al.\u003c/em\u003e recently reported that Oma1 stabilized OM\u0026ndash;IM supercomplexes by interacting with Mic60 in an Opa1-independent manner and that the depletion of Oma1 resulted in apoptotic resistance\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Although the number of studies on the association of MICOS and diseases have gradually recently increased\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, little is known about these relationships.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eAnimal Studies\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (3 and 18 months old, n\u0026thinsp;=\u0026thinsp;10\u0026sim;12 per group) were purchased from the Animal Facility of Aging Science, Korea Basic Science Institute (KBSI) Gwangju Center (Gwangju, Korea). After a week of adaptation, 3- or 18-month-old mice were randomly divided into two groups and fed for 4 months as follows: The control group was fed a standard chow diet (AIN-93G, Research Diets, NJ, USA) containing vitamin D\u003csub\u003e3\u003c/sub\u003e (1,000 IU/kg), and the vitamin D\u003csub\u003e3\u003c/sub\u003e-supplemented group was fed a standard chow diet enriched with vitamin D\u003csub\u003e3\u003c/sub\u003e (20,000 IU/kg). All animal experiments were performed according to the guidelines of the Korean National Institutes of Health Animal Care and Use Committee (permit number: KCDC-032-20-2A).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSilencing of genes\u003c/h2\u003e \u003cp\u003eShort interfering RNA (siRNA) against \u003cem\u003eImmt\u003c/em\u003e (5\u0026rsquo;-CACCCAAGCUUUAACCGCAtt-3\u0026rsquo;, 5\u0026rsquo;-UGCGGUUAAAGCUUGGGUGaa) and \u003cem\u003eLonP1\u003c/em\u003e (5\u0026rsquo;-GAUUAUCGAGGUUAAAAAUtt, 5\u0026rsquo;-AUUUUUAACCUCGAUAAUCtt) were synthesized by Ambion (Life Technologies, CA, USA). Transfection with the siRNAs was performed using Lipofectamine RNAiMAX (Invitrogen, Cambridge, UK) reagent for 24 hr.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of TG concentration\u003c/h2\u003e \u003cp\u003eLiver tissues or HepG2 cells were homogenized in 1 ml of 5% NP-40 solution using a homogenizer for 30 s. The samples were slowly heated to 99\u0026deg;C in a heat block for 5 min and then cooled to room temperature. The samples were centrifuged for 2 min at top speed with a microcentrifuge to remove insoluble material. For tissue obtained from animals, samples were diluted 10-fold with ddH\u003csub\u003e2\u003c/sub\u003eO, and for cells obtained from the HepG2 cell culture, the samples were diluted 3-fold with ddH\u003csub\u003e2\u003c/sub\u003eO before TG analysis. Prepared samples were measured using a triglyceride assay kit (Ab65336, Abcam, Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin sections (5 \u0026micro;m) were deparaffinized and hydrated with xylene and ethanol. The sections were blocked with 2.5% normal horse serum at room temperature for 30 min and then incubated overnight at 4\u0026deg;C with an anti-Mic60 antibody (Abcam, CO, USA). After washing, the sections were incubated with Alexa Fluor-488 secondary antibody (Invitrogen, CA, USA) for 1 hr and washed with PBS. The sections were stained with DAPI (H-1200, Vector Laboratories, CA, USA) and observed under a fluorescence microscope. Images of the whole slide were captured using a confocal laser scanning microscope (FV3000-OSR, Olympus Corp., Japan). The relative fluorescence intensity was measured using CellSens (Olympus Corp., Japan) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of electron microscopy\u003c/h2\u003e \u003cp\u003eHepG2 cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 overnight. The samples were sequentially dehydrated in 50% ethanol, 70% ethanol, 90% ethanol, 95% ethanol and 100% ethanol. After embedding with Epon 812 (Merck, Darmstadt, Germany), 70-nm sections were sliced with an ultrathin microtome. The sections were stained with 1% uranyl acetate for 5 min, incubated with 1% lead citrate for 3 min, and observed with a transmission electron microscope (LIBRA-120, Carl Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eChIP analysis was performed according to the manufacturer's instructions (Millipore, Darmstadt, Germany). Briefly, HepG2 cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine. Cells were resuspended and sonicated in SDS lysis buffer. Lysates were incubated overnight at 4\u0026deg;C with the following antibodies: anti-mouse IgG (Santacruz, Dallas, U.S.A., sc-2025), an anti-VDR (Santacruz, sc-131333X) antibody and an anti-RXRα (Santacruz, sc-515929X) antibody. Protein G agarose was added to form the immunocomplexes, and were washed and subjected to elution. The samples were treated with RNase A and Proteinase K. DNA was subsequently purified using PCR purification spin columns (QIAGEN, Hilden, Germany). The primers used to amplify the \u003cem\u003eImmt\u003c/em\u003e promoter regions, designated R1 (-3986~-3203), R2 (-3157~-2323), R3 (-2312~-1724), R4 (-1845~-1159), R5 (-1179~-550) and R6 (-574\u0026thinsp;~\u0026thinsp;115) from the transcription start site (TSS), respectively. The primers are listed in supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (SEMs). Statistical analysis was performed using GraphPad Prism software (GraphPad, CA, USA). Comparisons between two groups were performed with Student\u0026rsquo;s t test or nonparametric Mann\u0026ndash;Whitney U test. For multiple group comparisons, one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test for multiple comparisons was performed to evaluate significant differences. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eVitamin D supplementation restored the age-dependent reduction in liver Mic60 level\u003c/h2\u003e \u003cp\u003eVitamin D is thought to have beneficial functions in preventing NAFLD; however, its effects remain controversial\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Moreover, most studies have investigated only vitamin D deficiency in NAFLD and have not explored combination of vitamin D with multiple other risk factors, such as aging or mitochondrial dysfunction. In this study, we examined the precise molecular mechanisms through which vitamin D induces age-induced NAFLD, especially in relation to mitochondria. We first measured the liver mass and degree of lipid accumulation by hematoxylin and eosin (H\u0026amp;E) staining samples from young (7-month-old) and aged (22-month-old) C57BL/6 mice. We observed a significant reduction in the abnormal expansion of the total liver mass and lipid accumulation within the liver only in the aged mice fed a vitamin D\u003csub\u003e3\u003c/sub\u003e-supplemented diet (20,000 IU/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c). In addition, the circulating FFA and hepatic TG levels were inversely correlated with serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). Next, we measured gene expression patterns related to lipid homeostasis in the liver in samples extracted from each group of animals. Aging altered lipid metabolism to promote TG accumulation, and vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation led to the opposite results only in aged mice, corresponding with histological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). In particular, we observed profound alterations in the expression of the following genes: peroxisome proliferator-activated receptor (PPAR)γ and cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) involved in DNL \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Moreover, the expression of PPARα and carnitine palmitoyltransferase 1 (CPT-1), which are key regulators of β-oxidation, was reduced\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. A marked increase in the expression of CD36, very low density of lipoprotein receptor (Vldlr) and monoacylglycerol \u003cem\u003eO\u003c/em\u003e-acyltransferase 1 (Mgat-1), a diacylglycerol (DAG) and TG synthesis catalase, was observed\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAging and a decline in mitochondrial functions are closely associated\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and mitochondrial dynamics and MQC are important for the maintenance of optimal mitochondrial function\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Therefore, we measured changes in the levels of proteins related to mitochondrial dynamics and MQC. In aged mice, the expression of proteins involved in OXPHOS (Supplementary Fig.\u0026nbsp;1) and mitochondrial fusion was reduced, and this decrease was reversed by vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Previously, Ryan Z. C. \u003cem\u003eet al.\u003c/em\u003e reported similar effects of vitamin D, which increased the levels of mitochondrial fusion proteins, such as mitofusin1 (Mfn1) and Opa1, and reduced the levels of fission proteins, mitochondrial fission protein 1 (fis1) and Oma1 in skeletal muscle\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In agreement with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the marked compensatory effects of vitamin D\u003csub\u003e3\u003c/sub\u003e were observed only in the aged mice. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and lon protease 1 (LonP1) are representative MQC proteins located in the mitochondrial matrix\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We observed that only the level of LonP1 was specifically reduced in the aged mice and that the level was restored by vitamin D\u003csub\u003e3\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMICOS is a relatively newly discovered mitochondrial protein complex; therefore, its role, especially in diseases, is largely unknown. To determine whether the level of MICOS was altered in our animal model system, we performed real-time PCR and Western blot analysis. Interestingly, we found that the expression of Mic60 was specifically reduced by aging, and the level was restored by vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation only in the aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To confirm these results, we performed immunohistochemistry with an anti-Mic60 antibody and determined that the expression of Mic60 was inversely correlated with aging and vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Considering these results, we demonstrated that aging, mitochondrial dysfunction, and NAFLD are closely related and that vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation is effective only when the vitamin D\u003csub\u003e3\u003c/sub\u003e concentration in the blood is too low, as it is in aged mice. In particular, we observed a reduction in Mic60 and LonP1 levels due to aging and found that vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation restored these levels in aged mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDepletion of Mic60 specifically induced TG accumulation in the liver\u003c/h2\u003e \u003cp\u003eTo examine whether the decrease in Mic60 or Lonp1 level with aging is related to fat accumulation in the liver, we first transfected HepG2 human hepatoma cells with siRNAs against \u003cem\u003eImmt\u003c/em\u003e and \u003cem\u003elonp1\u003c/em\u003e. In all cases, transfection of both siRNAs led to specific depletion of individual genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), and the cellular ATP production level and mitochondrial membrane potential were gradually decreased (Supplementary Fig.\u0026nbsp;2). Next, we examined whether the expression pattern of MICOS subunits is altered by depletion of Mic60 or LonP1. Significant changes were observed in MICOS subunits, except Mic27, after \u003cem\u003eImmt\u003c/em\u003e silencing, but no profound reduction in MICOS subunit levels was observed after \u003cem\u003esiLonP1\u003c/em\u003e silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Interestingly, we observed opposite changes in the Sam50 and SLC25A46 levels after the depletion of Mic60 or LonP1. We therefore examined the amount of both proteins in our animal models. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the protein expression of Sam50 and SLC25A46 was reduced in aged mice and restored by vitamin D\u003csub\u003e3\u003c/sub\u003e, similar to the protein expression of Mic60. Both Sam50 and SLC25A46 interact with Mic60\u003csup\u003e28,31\u003c/sup\u003e but not with LonP1. Therefore, the reduction in the Sam50 and SLC25A46 levels in aged mice seemed to be related to the reduction in the Mic60 level in aged mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we observed the influences of depleting either Mic60 or Lonp1 on mitochondrial dynamics. We found that silencing Mic60 led to upregulated Fis1, Oma1 and dynamin-related protein 1 (Drp1) expression and downregulated Mfn1 and Mfn2 expression; moreover, it led to decreased expression of Opa1, which caused changes in the directions towards to the mitochondrial fragmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, depletion of LonP1 did not lead to any significant alteration in the levels of proteins involved in mitochondrial dynamics.\u003c/p\u003e \u003cp\u003eTo determine whether these alterations in mitochondria caused by silencing of specific RNAs are related to fat accumulation in the liver, we measured the amount of TG after silencing \u003cem\u003eImmt\u003c/em\u003e or \u003cem\u003elonp1\u003c/em\u003e. Interestingly, we found that only the loss of Mic60 specifically increased TG accumulation in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). In addition, we determined that the DNL and lipid uptake rates were greatly increased by the loss of Mic60, similar to the observations in our animal model. These outcomes were obvious only in the absence of Mic60 but not in the absence of Lonp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). From these results, we suggest that there is a specific relationship between the loss of Mic60 and TG accumulation in liver cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVitamin D restores mitochondrial function and prevents TG accumulation after the loss of Mic60\u003c/h2\u003e \u003cp\u003eIn aged mice, vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation effectively prevented the reduction in Mic60 level. To test whether vitamin D\u003csub\u003e3\u003c/sub\u003e affects Mic60 expression in cell models, we treated cells with 1,25VitD\u003csub\u003e3\u003c/sub\u003e after silencing \u003cem\u003eImmt\u003c/em\u003e. Even though \u003cem\u003esiImmt\u003c/em\u003e transfection completely depleted Mic60 in cells, the 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment restored some level of the Mic60 protein and other MICOS components (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, left panel). Notably, mitochondrial fusion proteins, membrane potential and cellular ATP levels were also restored by vitamin D (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, right panel and 4b). We also observed that 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment effectively prevented TG accumulation that had been caused by a decrease in the Mic60 level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). To verify the fat accumulation specifically induced by the loss of Mic60 and the prevention of this outcome after 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment, we performed an oil red O staining assay after \u003cem\u003esiImmt\u003c/em\u003e transfection in the presence and absence of 1,25VitD\u003csub\u003e3\u003c/sub\u003e. We observed significant fat accumulation after \u003cem\u003esiImmt\u003c/em\u003e transfection and a reduction in the number of lipid droplets after 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Recently, Stephan T. \u003cem\u003eet al.\u003c/em\u003e showed that after depletion of \u003cem\u003eImmt\u003c/em\u003e in HeLa cells, normal cristae formation was largely disrupted, and most cristae formed scattered small ladder-like shapes\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We next examined whether vitamin D can restore mitochondrial morphology that had been disrupted by the depletion of Mic60. We observed mitochondrial shapes through an electron microscope 24 hr after \u003cem\u003esiImmt\u003c/em\u003e treatment in the presence or absence of 50 nM 1,25VitD\u003csub\u003e3\u003c/sub\u003e. In addition to a decrease in the Mic60 level, we found that the number of normal mitochondria was reduced, and 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment significantly restored the overall number and normal morphology of these mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that aging and a reduction in Mic60 level were related to each other, we established a cell model similar to that of the aging animal model by treating HepG2 cells with doxorubicin (Dox) as indicated\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. By increasing the concentration of Dox, the levels of representative senescence marker proteins, such as p21 and p53, were greatly increased. In contrast, the level of Mic60 was gradually decreased, as observed in our animal models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We next examined the effects of vitamin D after the induction of cellular senescence. The levels of a senescence marker protein and Mic60 were inversely correlated, and vitamin D specifically upregulated the expression of Mic60, consistent with observations in the animal model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To verify the recovery of Mic60 induced by vitamin D treatment, we measured \u003cem\u003eImmt\u003c/em\u003e expression after Dox treatment in the absence or presence of 1,25VitD\u003csub\u003e3\u003c/sub\u003e and confirmed that vitamin D specifically upregulated \u003cem\u003eImmt\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). We also measured the alteration of proteins involved in mitochondrial dynamics after Dox treatment and observed changes similar to those observed after the depletion of Mic60: A decrease in mitochondrial fusion and an increase in fission was observed, and these effects were reversed by 1,25VitD\u003csub\u003e3\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Next, we examined whether silencing \u003cem\u003eImmt\u003c/em\u003e triggered a counteraction to cellular senescence. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, the loss of \u003cem\u003eImmt\u003c/em\u003e significantly induced several senescence markers, including p53, p21 and interleukin 1A (IL-1A). From these experiments, we confirmed that the Mic60 level was dramatically decreased with aging and cellular senescence and that vitamin D specifically restored the senescence-dependent reduction in Mic60 level. In contrast, an absence or reduction in Mic60 level may trigger cellular senescence and aging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVitamin D regulates\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eImmt\u003c/span\u003e \u003cb\u003eexpression by the direct binding of VDR-RXR to\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eImmt\u003c/span\u003e\u003c/p\u003e \u003cp\u003eVitamin D is an important regulator of Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis\u003csup\u003e10\u003c/sup\u003e, multiple immune responses and antioxidative responses \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Binding of vitamin D to VDR, together with its heterodimeric receptor retinoid X receptor (RXR), usually triggers conformational changes, which enables the recognition of vitamin D-responsive elements (VDREs) on vitamin D target genes. VDRE usually does not comprise rather than a conserved linear sequences, it is more three-dimensional and variant VDRE sequences may influence unique complexes carrying VDR-RXR\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To test whether elevation of Mic60 level by vitamin D exerts indirect effects, such as alterations in Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis or oxidative stress, or whether vitamin D directly regulates Mic60 level by VDR binding, we performed chromatin immunoprecipitation (ChIP) using anti-VDR and anti-RXR antibodies. First, we searched for possible retinoid X receptor responsive elements (RXREs) or VDREs throughout the whole \u003cem\u003eImmt\u003c/em\u003e gene, from the 5\u0026acute; intron to 3\u0026acute; tail sequence, using NCBI blast suite SRA (SRX100497; HepG2_IP RXR) and found five candidate regions (Supplementary Table\u0026nbsp;1). We assessed the recruitment of VDR and RXRα to all these candidate RXRE or VDRE sites in the regulatory region upstream in the \u003cem\u003eImmt\u003c/em\u003e promoter and found one specific high-affinity VDR-RXR binding site located in the region spanning positions \u0026minus;\u0026thinsp;3157 ~ -2323 from the transcription start site (TSS). The binding of both VDR and RXRα to this VDRE was dramatically reduced by Dox treatment but was very strong in the presence of the VDR-RXR ligand 1,25VitD\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results reveal, for the first time that VDR-RXR directly binds \u003cem\u003eto Immt\u003c/em\u003e in a region ranging from position \u0026minus;\u0026thinsp;3157 to -2323, thereby specifically upregulating \u003cem\u003eImmt\u003c/em\u003e expression mediated by vitamin D.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aging population is rapidly increasing worldwide; therefore, interest in research on aging-associated diseases, such as Parkinson\u0026rsquo;s disease and metabolic syndrome, has been increasing. The prevalence of NAFLD is also closely related to aging. In this study, we showed, for the first time, that depletion of Mic60 was directly associated with age-induced NAFLD development. In this work, we report the following interesting facts: (ⅰ) Aging triggered a specific reduction in Mic60 level. (ⅱ) Depletion of Mic60 disrupted lipid homeostasis in liver cells, which directly led to TG accumulation. (ⅲ) Vitamin D\u003csub\u003e3\u003c/sub\u003e prevented lipid accumulation in aged mice and in senescence-induced HepG2 cells, which coincided with the recovery of the Mic60 level. (ⅳ) The loss of Mic60 itself disrupted mitochondrial dynamics and triggered cellular senescence. (ⅴ) Vitamin D\u003csub\u003e3\u003c/sub\u003e upregulated Mic60 expression via the recruitment of VDR-RXRα to the promoter of the \u003cem\u003eImmt\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eAlthough mitochondrial dysfunction and concomitant elevation of oxidative stress are associated with numerous diseases, including chronic diseases and even cancer\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Even though MICOS is important for cristae formation and related mitochondrial functions, most studies on MICOS have thus far focused on MICOS structural effects, and studies on the relationship of MICOS and human diseases have recently been reported.\u003c/p\u003e \u003cp\u003eTo date, representative studies on the association between MICOS and chronic diseases include the following reports: Baseler W.A. \u003cem\u003eet al.\u003c/em\u003e reported a reduction in Mic60 level in type 1 diabetic heart\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and Thapa D. \u003cem\u003eet al.\u003c/em\u003e showed that transgenic cardiac-specific overexpression of Mic60 ameliorated diabetic cardiomyopathy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Additionally, Guarani V. \u003cem\u003eet al\u003c/em\u003e. demonstrated that the Mic13-null mutant was critical to early onset fetal mitochondrial encephalopathy with liver diseases\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and that Mic26 was overexpressed in the human diabetic heart\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur finding clearly showed, for the first time, that age-dependent reduction in the Mic60 level or the depletion of Mic60 specifically induces TG accumulation, a key characteristic of NAFLD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We observed age-dependent reduction in Mfn1, Opa1 and LonP1 levels, consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Since many studies have been performed on mitochondrial dynamics, we selectively depleted Mic60 and LonP1, which are less known among the proteins reduced in our aged mice. Although deletion of both Mic60 and LonP1 led to a reduction in the cellular ATP level and mitochondrial membrane potential (Supplementary Fig.\u0026nbsp;2), only depletion of Mic60 specifically led to TG accumulation in HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings imply the possibility that a mechanism in addition to the reduction in the overall mitochondrial function is involved with TG accumulation. We observed a specific reduction not only in the level of Mic19, one of the MIC60-Mic19-Mic25 subunits, but also in the levels of sam50, SLC25A46, Opa1 and Mfn1 and an increase in the levels of Oma1 and Fis1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, d). These outcomes were observed only in the absence of Mic60, not in the absence of LonP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Recently, Tang J. \u003cem\u003eet al\u003c/em\u003e. reported that the Sam50-Mic19-Mic60 interaction is important to the connection between the OM and the IM and is involved in normal cristae shape formation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In addition, they found that Oma1 disrupted this membranous connection by cleaving Mic19. Recently, using liver-specific Sam50-knockout mice, Chen L. \u003cem\u003eet al\u003c/em\u003e. reported that depletion of Sam50 induced liver inflammation and liver injury\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In addition, the \u003cem\u003eSAMM50\u003c/em\u003e polymorphism has been reported to be associated with NAFLD\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Considering these studies, we hypothesize that a reduction in Sam50 level induced by the depletion of Mic60 might be a specific reason for the TG accumulation induced by the loss of Mic60. We also suggest that Mic60 may play a central role not only in mitochondrial cristae formation and the maintenance of mitochondrial dynamics but also in the connection between mitochondrial function and the environment outside organelles via its interactions with multiple proteins; thus, Mic60 may influence the development of NAFLD. Although Lonp1 is important to maintain overall mitochondrial function, we found that loss of Lonp1 did not induce TG accumulation. We suggest that LonP1 did not induce fat accumulation because it did not induce a change in the mitochondrial structure and thus exerted no overall influence on the connection between the OM and IM, even though Lonp1 plays a central role in MQC and is important to maintain mitochondrial function. However, many studies remain to be performed to determine the precise molecular mechanisms through which depletion of Mic60 may promote NAFLD.\u003c/p\u003e \u003cp\u003eMany reports, including those of Roth C. L. \u003cem\u003eet al.\u003c/em\u003e, suggested therapeutic effects of vitamin D on NAFLD\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In this study, we observed that vitamin D effectively prevented TG accumulation only in aged mice. Similarly, we observed the preventive effect of vitamin D\u003csub\u003e3\u003c/sub\u003e on pancreatic dysfunction and hyperinsulinemia defects only in aged mice\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Pines A. \u003cem\u003eet al.\u003c/em\u003e showed that the beneficial effects of vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation were greater on aged men than on younger men\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The optimal vitamin D\u003csub\u003e3\u003c/sub\u003e concentration varies considerably, and the normal concentration of vitamin D\u003csub\u003e3\u003c/sub\u003e in human serum is typically considered to be 25\u0026thinsp;~\u0026thinsp;80 ng/mL\u003csup\u003e13\u003c/sup\u003e. In our mouse model, the serum vitamin D\u003csub\u003e3\u003c/sub\u003e level was insufficient only in the aged mice fed a normal chow diet, and it was restored in the aged mice fed a diet supplemented with vitamin D\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Healthy human hepatocytes contain relatively low levels of VDR, but nonparenchymal cells produce a large amount of VDR\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. As Keane J. T. \u003cem\u003eet al\u003c/em\u003e. clearly described in their review, the amount of VDR in hepatocytes is increased in the disease state\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that vitamin D\u003csub\u003e3\u003c/sub\u003e restored the expression of Mic60 only in aged mice fed a diet supplemented with vitamin D\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d) and in senescence-induced HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In addition, we showed that VDR-RXR bound directly to \u003cem\u003eImmt\u003c/em\u003e to regulate its expression; that is, the effect of vitamin D on Mic60 was not indirectly mediated by alterations in the cellular environment. Considering these observations, we suggest that it is important to maintain a sufficient serum vitamin D and VDR level in the hepatocytes in aged mice to preserve a sufficient Mic60 level in the liver and prevent mitochondrial dysfunction and subsequent age-related NAFLD development.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study, we first demonstrated that depletion of Mic60 is related to the development of age-induced NAFLD and that vitamin D can prevent NAFLD by upregulating Mic60 expression in a VDR-RXR-dependent manner. Further studies are needed to determine whether the loss of Mic60 is related to the development of age-dependent NAFLD in humans\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by an intramural research grant from the Korea National Institute of Health (2020-NG-014-02). We are appreciated to Jee Woong Kim for the technical assistance of electron microscope measurement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.H.L designed this study and wrote the manuscript. G.H.K. mainly performed experiments and analyzed data. H.J. wrote some part of this manuscript and performed experiments. Y.J.L performed animal study. S.K.K. commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information accompanies the manuscript on the Experimental \u0026amp; Molecular Medicine\u0026rsquo;s website (http://www.nature.com/emm/\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaurice, J. \u0026amp; Manousou, P. Non-alcoholic fatty liver disease. 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Hepatology \u003cb\u003e37\u003c/b\u003e, 1034\u0026ndash;1042, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1053/jhep.2003.50176\u003c/span\u003e\u003cspan address=\"10.1053/jhep.2003.50176\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2437531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2437531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNonalcoholic fatty liver disease (NAFLD) is the most common liver disease. Despite intensive research, considerable NAFLD development remains to be revealed. In this study, we examined the effects of vitamin D on age-induced NAFLD, especially in connection with mitochondrial abnormalities.\u003c/p\u003e\n\u003cp\u003eWe observed effective prevention of liver steatosis in 22-month-old C57BL/6 mice fed a vitamin D\u003csub\u003e3\u003c/sub\u003e-supplemented (20,000 IU/kg) diet, in contrast to mice fed a control (1,000 IU/kg) diet. We evaluated whether vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation enhanced mitochondrial functions and found that the mitochondrial contact site and cristae organizing system (MICOS) 60 (Mic60) level was reduced and was specifically restored by vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation in the aged mice. In addition, depletion of \u003cem\u003eImmt\u003c/em\u003e, the human gene encoding the Mic60 protein, induced changes in gene expression that led to fat accumulation in HepG2 human hepatocellular carcinoma cells, which is effectively prevented by vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation. In contrast, silencing of \u003cem\u003eLonp1\u003c/em\u003e, the main matrix protease involved in mitochondrial quality control system and also expressed at a reduced level in aged mice, did not induce triglyceride (TG) accumulation in HepG2 cells. Moreover, VDR-RXR increased Mic60 levels by directly binding to the \u003cem\u003eImmt\u003c/em\u003e 5' promoter region spanning from position − 3157 to -2323. Our study demonstrates, for the first time, that a reduction in Mic60 level due to aging may be one of the underlying mechanisms to development aging-associated NAFLD. In addition, vitamin D\u003csub\u003e3\u003c/sub\u003e could positively regulate Mic60 expression, which may be one of the important molecular mechanisms that vitamin D could ameliorate age induced NAFLD.\u003c/p\u003e","manuscriptTitle":"Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-30 18:27:30","doi":"10.21203/rs.3.rs-2437531/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-05-24T08:35:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-05-23T05:41:12+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-05-01T05:05:55+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-10T10:19:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-01-27T00:13:42+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-01-26T05:40:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-04T02:13:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2023-01-03T05:32:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-03T05:32:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8512ad7a-135a-46db-8869-960b533b2169","owner":[],"postedDate":"January 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":18711149,"name":"Health sciences/Diseases/Metabolic disorders"},{"id":18711150,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2024-01-04T08:05:51+00:00","versionOfRecord":{"articleIdentity":"rs-2437531","link":"https://doi.org/10.1038/s12276-023-01125-7","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2024-01-04 05:00:00","publishedOnDateReadable":"January 4th, 2024"},"versionCreatedAt":"2023-01-30 18:27:30","video":"","vorDoi":"10.1038/s12276-023-01125-7","vorDoiUrl":"https://doi.org/10.1038/s12276-023-01125-7","workflowStages":[]},"version":"v1","identity":"rs-2437531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2437531","identity":"rs-2437531","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}