ERAD deficiency disrupts mitochondrial bioenergetics by altering MAM Ca2+ in human hepatic cells

ERAD deficiency disrupts mitochondrial bioenergetics by altering MAM Ca2+ in human hepatic cells | 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 Research Article ERAD deficiency disrupts mitochondrial bioenergetics by altering MAM Ca2+ in human hepatic cells Yabing Hu, Yongfeng Bai, Beier Jiang, Mingming Wang, Feng Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4939621/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Graphical Abstract Abstract Mitochondria and the endoplasmic reticulum (ER) physically and functionally interact, regulating each other’s function, but the molecular mechanisms remain not fully understood. In this study, we revealed that ER-associated degradation (ERAD), an ER protein quality control mechanism, governs ER Ca2+ entry into mitochondria by mitochondria-associated ER membrane (MAM) in hepatic cells. Inhibition of ERAD by pharmaceuticals or genetic ablation of the key ERAD protein SEL1L resulted in altered mitochondrial morphology, reduced mitochondrial energy production, and increased Ca2+ transfer from ER to mitochondria. Additionally, SEL1L absence caused an increase in the number of MAM. In ERAD-deficient hepatic cells, a reduction in the number of MAM or knockdown of the inositol 1,4,5-trisphosphate receptor (IP3R), which is responsible for ER Ca2+ release, partially restored mitochondrial Ca2+ signaling and bioenergetics. Together, these results suggest that ERAD plays a crucial role in regulating mitochondrial bioenergetics, suggesting the potential to improve cellular and organismal mitochondrial function by increasing cellular ERAD activity. hepatic cells ERAD mitochondrial function MAM mitochondrial calcium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Interorganelle communication occurs through membrane contact sites and is crucial for the maintenance of organelle homeostasis and organismal health (Arora et al. 2022 ; Henne et al. 2021;Wu et al. 2018 ). Interactions between endoplasmic reticulum (ER) and mitochondria are excellent examples of the importance of this partnership. The close physical contact (~ 30 nm) between the ER and mitochondria occurs in a region known as the mitochondria- associated ER membrane (MAM). MAM effectively integrates signal transduction with metabolic pathways to regulate ER-mitochondrial communication. It plays important regulatory roles in several key cellular processes including Ca 2+ exchange, lipid synthesis, ER stress (ERS), mitochondrial dynamics and function, autophagy, and apoptosis (Rowland et al. 2012; Phillips et al. 2016; Jang et al. 2022 ). Evidence indicates that dysfunction in MAM contributes to conditions such as neurodegenerative diseases, fatty liver disease, and diabetes (Liu et al. 2022; Beaulant et al. 2022 ; Elwakiel et al. 2024 ). There is still considerable uncertainty regarding how interorganelle communication operates and the physiological consequences of miscommunication. Quality control systems normally maintain homeostasis and proteostasis in the ER throughout an organism's lifespan, and ER-associated degradation (ERAD) is an important branch. The primary function of ERAD is to identify, sort, and degrade misfolded proteins to prevent the harmful effects caused by their buildup. This is particularly important in secretory cells (Kang et al. 2021). The Sel1L-Hrd1 protein complex represents the most highly conserved branch of ERAD from yeast to humans. Recent studies have found that the Sel1L-Hrd1 ERAD may have a constitutive function in maintaining optimal concentrations of key proteins that control mitochondrial dynamics (Zhou et al. 2020 ). However, little is known about the pathophysiological effects of defective ER quality control systems on mitochondrial function and vice versa in secretory cells such as hepatocytes. We had previously shown that disrupting ERAD genetically and pharmacologi- cally impairs glucose-stimulated insulin secretion in β-cells in vitro and in mice (Hu et al. 2019 ). In the current study, we investigated the impact of ERAD deficiency on mitochondria both structurally and energetically using an ERAD-deficient hepatic cell line model. The consequences of ERAD deficiency were linked to aberrant MAM levels, which, in turn, caused mitochondrial Ca 2+ excess. Our results indicate that ERAD is critical for maintaining normal mitochondrial energy production and raise the potential for enhancing cellular ERAD capacity to improve mitochondrial function at the organismal and cellular levels. 2. Materials and methods 2.1. Cell culture and treatment HepG2 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% FBS(Gibco) and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. The cells were treated with ERAD inhibitor eeyarestatin I (8 µM; MedChemExpress, USA), cycloheximide (50 µg/mL; Abmole, China), 2-APB (20 µM; Sigma-Aldrich, USA), ATP (10 µM; MedChemExpress), palmitic acid (0.5 mM; Sigma-Aldrich), si-con or siIP3R (10 nM; Gemma-Pharma, China) for the corresponding times. Cells between passages five and eight were used for all experiments. 2.2. CRISPR/Cas9 mediated Sel1l gene editing in HepG2 cells Cas9-mediated editing of Sel1l was performed as previously described (Liu et al. 2020 ). Briefly, sgRNAs flanking exon 6 were designed using the Optimized CRISPR Design tool ( http://crispr.mit.edu/ ) and cloned into the expression vector pGL3- U6 -2sgRNA. The sgRNA expression vector and Cas9 expression vector pST1374-NLS-3xFlag- linker-Cas9 were both inserted into HepG2 cells using Lipofectamine 3000 (Baillat et al. 2016 ). After transfection, 5 µg/mL of puromycin was added to the growth medium to specifically choose cells that are not killed by puromycin. Approximately one week later, puromycin-resistant HepG2 colonies were selected, expanded, and tested by PCR. 2.3. Mitochondrial morphology, mitochondrial membrane potential and ATP production assay To investigate the effect of ERAD on mitochondria, we examined the mitochondrial morphology, mitochondrial membrane potential, and cellular ATP levels.Mitochondria were visualized using a MitoTracker Green fluorescent probe (Invitrogen, USA) or Tom20, followed by observation with laser confocal microscopy and analysis using ImageJ software. The mitochondrial membrane potential was determined using a JC-1 assay kit (Beyotime,China). Briefly, HepG2 cells were cultured in 6-well plates at a density of 2x10*5 cells per well and treated as required (EerI, 2APB, or siIP3R). The cells were then incubated with 2 µM JC-1 for 30 min at 37°C in the dark, washed with PBS, and the fluorescence was measured using flow cytometry (BD,USA) and/or a fluorescence microscope (Olympus, JAPAN). Cellular ATP levels were measured using a Firefly Luciferase ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, treated cells were lysed and centrifuged for 5 min at 12000 g to obtain the cell supernatant. After mixing 100 µL of cell supernatant with 100 µL of ATP assay working solution in a 96-well plate, the reaction was allowed to proceed for 5 min at room temperature. The fluorescence intensity was quantified using a microplate reader (Molecular Devices), and the ATP value was normalized to the protein concentration. 2.4. Western blot analysis As previously mentioned (Sha et al. 2009 ), cell lysates were prepared and western blotting was performed. Briefly, cells were scraped and resuspended in 10 mM Tris–HCl pH 7.5, 1 mM PMSF, and incubated at 4 ℃ for 20 min. The lysate was centrifuged at 10,000 g for 10 min at 4°C to obtain the supernatant. The protein concentration in the supernatant was determined using a BCA kit (Thermo Fisher, USA). Denaturing buffer was added to the protein supernatant, which was boiled for 5 min before separation by SDS-PAGE. The following antibodies were used in this study: GFP (1:500 rabbit) from Santa Cruz Biotechnology, β-actin (1:1,000 mouse) from Sigma-Aldrich, Sel1L (1:1,000 rabbit) and IP3R (1:500 rabbit) from Abcam. Anti-rabbit or anti-mouse secondary antibodies were incubated for 2 h at room temperature and band density was quantified using ImageJ software. 2.5. Cytosolic, ER and mitochondrial Ca 2+ measurement HepG2 cells were seeded into 6-well glass-bottomed chambers at a density of 1×10 5 cells/well and cultured in DMEM for 24 h. Ca 2+ in the cytoplasm, ER, and mitochondria was detected using Fluo4, AM, Mag-Fluo4, and Rhod2 probes. The probes were then added to Ca 2+ -free PBS and incubated for 30–60 min at room temperature. Using a fluorescence microscope, fluorescent images were collected every 50 s, background-corrected, and analyzed using the ImageJ software. 2.6. Cell immunofluorescence HepG2 cells were cultured overnight in a 6 cm dish. After fixation with 4% paraformaldehyde for 15 min, the cells were permeabilized for 5 min with 1% Triton X-100 in PBS. To prevent non-specific binding, cells were incubated in 10% donkey serum in TBST for 1 h at 37°C. Then, primary antibodies calnexin (1:200, Santa Cruz Biotechnology) and Tom20 (1:200, Absin) were introduced to the cells for 1 h at 37 ℃. The cells were then incubated with secondary antibodies in 5% donkey serum for 2 h at room temperature and washed. Stained cells were imaged using laser confocal microscopy, and the intensity and colocalization of fluorescence were analyzed using ImageJ software. 2.7. SiRNA-mediated knockdown of IP3R in HepG2 cells A scrambled negative control siRNA for hIP3R was predesigned and purchased from Gemma Pharma (China). HepG2 cells were seeded in 6-well plates. When the cells were 80% confluent, Opti-MEM was mixed with either control or IP3R siRNA together with Lipofectamine 3000 (Invitrogen) and incubated for 6 h. After transfection, the medium was replaced with DMEM containing 10% FBS and the cells were cultured for an additional 48 h. Cell lysates were collected to detect IP3R expression levels by western blotting. 2.8. Mitochondrial respiration assay Mitochondrial respiration was assessed by measuring the oxygen consumption rate (OCR) using a Seahorse XFe96 Analyzer as described previously (Mthembu et al. 2024 ). HepG2 cells were grown overnight in Seahorse XFe96 microtiter wells at a density of 3×10 4 cells/well, and treated as required. On the first day of the experiment, the culture medium was replaced with Seahorse XF Base Medium (Agilent Technologies) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. The following mitochondrial stress test compounds (1 µM Oligomycin, 0.5 µM FCCP, 0.5 µM Rotenone, and 0.5 µM antimycin A) were sequentially injected into the wells at specified time points. The OCR values were normalized to the total protein content in each well and determined using the BCA Protein Assay Kit (Beyotime). 2.9. Statistical analysis Data are shown as mean ± S.E. Differences between compared groups were evaluated using SPSS software (version 16.0) to perform Student’s t-test or analysis of variance (ANOVA). The significance level was set at P < 0.05. 3. Results 3.1. ERAD dysfunction impairs HepG2 cells mitochondrial bioenergetics Our previous study showed that disrupting ERAD decreases ATP production in the INS-1 rat pancreatic β-cell line, suggesting that a deficiency in ERAD compromises the mitochondrial respiratory function(Hu et al. 2019 ). To investigate whether this phenomenon extends to other mammalian cells, we treated human hepatic HepG2 cells with the ERAD inhibitor eeyarestatin I (EerI). EerI treatment reduced ATP levels in a time-and dose-dependent manner (Fig. 1 A, B), altered mitochondrial morphology (Fig. 1 C- 1 F, and S1 A-F), and mitochondrial membrane potential (MMP) was reduced (Fig. 1 G- 1 L). SEL1L is essential for the formation of the ERAD complex on the ER membrane in mammals. To further investigate the effect of ERAD on ATP levels, we used CRISPR /Cas9-based gene editing to generate SEL1L-deficient (Sel1L −/− ) HepG2 cells (Fig. 2 A, B). Next, we used cycloheximide (CHX)-based pulse-chase analysis for α1-antitrypsin null Hong Kong variant (NHK) in WT and Sel1L −/− HepG2 cells to determine the ERAD-degrading function. As shown in Fig. 2 C and 2 D, the folding-deficient mutant NHK displayed efficient degradation in WT HepG2 cells, but not in Sel1L −/− HepG2 cells. This result indicates that the ERAD deficiency cell model was successfully constructed using the sel1L knockout. For ATP and MMP, Sel1L −/− HepG2 cells were lower than WT HepG2 cells (Fig. 2 E, F). Overall, our findings suggest that ERAD deficiency (treatment with EerI or deletion of Sel1L) impairs mitochondrial morphology and bioenergetics in hepatic cells. 3.2. ERAD deficiency causes increased mitochondrial Ca 2+ mainly originated from ER It is well established that mitochondrial Ca 2+ concentration is a critical determinant in mediating mitochondrial respiration (Sun et al. 2023 ; Santulli et al. 2015 ). Given our findings in pancreatic β-cells that ERAD deficiency causes changes in mitochondrial Ca 2+ levels (Hu et al. 2019 ), we tested whether a similar change occurs in ERAD-defective HepG2 cells. In ERAD-defective HepG2 cells, we used the fluorescent probe Rhod2 AM to detect mitochondrial Ca 2+ . As shown in Fig. 3 A, B, and S2 A, both Sel1L deletion and EerI treatment produced a significant increase in Ca 2+ levels in the mitochondria of HepG2 cells. Since ERAD is an ER-associated degradation mechanism, and the ER is a major Ca 2+ storage reservoir in eukaryotic cells (Christianson et al. 2023 ), we investigated whether ER Ca 2+ contributes to increased mitochondrial Ca 2+ in ERAD-deficient cells. First, we measured ER Ca 2+ using the Mag-Fluo4 probe and found that ER Ca 2+ levels in Sel1L −/− cells were higher than in WT cells (Fig. 3 E, F). Next, we treated WT and ERAD-deficient cells with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor, 2APB, which blocks ER Ca 2+ release. As shown in Fig. 3 C and 3 D, ERAD-deficient cells treated with 2APB showed a marked reduction in mitochondrial Ca 2+ levels. When stimulated with ATP, which promotes the release of ER Ca 2+ , ERAD-deficient cells displayed higher mitochondrial Ca 2+ uptake than the WT cells (Fig. 3 G). Upon addition of 2APB, there was a significant and abrupt decrease in mitochondrial Ca 2+ levels in both ERAD-deficient and WT cells (Fig. 3 G). Together, these findings indicate that increased mitochondrial Ca 2+ originates mostly from the ER in ERAD-deficient hepatic cells. 3.3. ER Ca 2+ enters mitochondria via the MAM connections in ERAD-deficient cells. To evaluate whether increased mitochondrial Ca 2+ is the result of direct transport through MAM junctions or IP3R-mediated ER Ca 2+ release into the cytosol, followed by mitochondrial uptake, we analyzed cytosolic Ca 2+ dynamics in response to IP3R stimulation in WT and Sel1L −/− HepG2 cells using Fluo4-AM. The baseline cytosolic Ca 2+ concentration was significantly (P < 0.05) higher in Sel1L −/− HepG2 cells (Fig. 4 A). However, ATP stimulation caused a comparable increase in cytosolic Ca 2+ levels in WT and Sel1L −/− HepG2 cells (Fig. 4 B), despite the higher mitochondrial Ca 2+ peak in ERAD-deficient cells (Fig. 3 G, 4 C). These findings suggest that ERAD deficiency leads to increased Ca 2+ transport from the ER through MAM connections, resulting in elevated mitochondrial Ca 2+ in HepG2 cells. To investigate how MAM affects Ca 2+ fluctuations between the ER and mitochondria in ERAD-deficient cells, we labeled the ER with calnexin and the mitochondria with TOM20 and then quantified MAM changes by analyzing the degree to which calnexin and TOM20 colocalized. As shown in Fig. 4 D- 4 H, the amount of MAM in Sel1L −/− cells was significantly (p < 0.05) higher than that in WT cells. Correlative studies have demonstrated that an increased number of MAM promotes higher mitochondrial Ca 2+ levels (Arruda et al. 2014 ). As a result, ERAD deficiency may lead to an increase in mitochondrial Ca 2+ concentration by altering the number of MAM in hepatic cells. 3.4. Reduced MAM Ca 2+ partially restores mitochondrial bioenergetics in ERAD deficient cells The effect of an imbalance in mitochondrial Ca 2+ homeostasis on mitochondrial respiration is widely recognized. Given the finding that ERAD deficiency caused a substantial influx of ER Ca 2+ into mitochondria, leading to an increase in mitochondrial Ca 2+ , we tested whether altered mitochondrial Ca 2+ levels contribute to defective mitochondrial bioenergetics in ERAD-deficient liver cells. To decrease ER Ca 2+ influx into mitochondria, we introduced 2APB into HepG2 cells. 2APB partially reversed the EerI-induced reduction in ATP levels (Fig. 5 A). IP3R is an ER Ca 2+ release receptor; IP3R siRNA was transfected into WT and SEl1L −/− cells, and the knockdown effect is shown in Fig. 5 B. In Sel1L −/− cells, we found that the knockdown of IP3R expression partially restored MMP (Fig. 5 C) and ATP levels (Fig. 5 D). Given the finding that ERAD deletion may boost ER Ca 2+ influx into the mitochondria by increasing the number of MAM (Fig. 4 D- 4 H), we used an outer mitochondrial membrane (OMM) linker plasmid that decreased the number of MAM (Fig. S2B). We found that OMM linker expression reduced mitochondrial Ca 2+ levels (Fig. 5 E) and improved OCR (Fig. 5 F) in Sel1L −/− cells. Together, these findings show that ERAD deficiency compromises mitochondrial bioenergetics by increasing MAM and encouraging a substantial input of ER Ca 2+ into mitochondria. 3.5. ERAD mediates palmitic acid-induced mitochondrial dysfunction in HepG2 cells Palmitic acid (PA) is a significant contributor to the development of nonalcoholic fatty liver disease (NAFLD) and can induce lipid accumulation in the liver through its impact on mitochondrial function (Silva Figueiredo et al. 2017 ; Marra et al. 2018). A recent proteomic analysis revealed that treatment of HepG2 cells with PA damaged the protein quality control machinery (autophagy, ERAD, and the ubiquitin-proteasomal system) (Fig. 6 A) (Saha et al. 2022 ). We tested whether ERAD mediated the impairment of mitochondrial function in hepatocytes caused by PA. We first performed cycloheximide (CHX)-based pulse-chase analysis for folding -deficient mutant NHK in HepG2 cells treated with and without PA. Folding-deficient mutant NHK showed efficient degradation in HepG2 cells without PA, but not in HepG2 cells treated with PA (Fig. 6 B, C). Next, we investigated the effects of PA on MAM and mitochondrial Ca 2+ . As shown in Fig. 6 D- 6 G, PA exposure increased MAM and mitochondrial Ca 2+ levels in HepG2 cells. According to other studies, PA lowers the OCR (Mthembu et al. 2024 ). This study confirmed that PA impairs OCR and combining it with the ERAD inhibitor EerI further decreases OCR; yet the OMM linker restored the PA-induced OCR reduction (Fig. 6 H). Together, these data suggest that PA exposure in HepG2 cells impairs ERAD function, and that ERAD is likely a mediator of PA impairment of mitochondrial function. 4. Discussion The ER and mitochondria interact both physically and functionally, allowing them to control each other's functions. A recent study revealed that ER protein quality control (ERAD) plays a crucial role in maintaining optimal levels of sigma receptor 1 (SigmaR1) to regulate mitochondrial dynamics in brown adipose cells (Zhou et al. 2020 ). In this study, we investigated whether the protein quality control system influences mitochondrial function in hepatic cells. Our research showed that ERAD facilitates Ca 2+ signaling in the mitochondria by controlling the quantity of MAM. ERAD deficiency results in altered mitochondrial structure, reduced mitochondrial energy production, and increased Ca 2+ transfer from the ER to the mitochondria by increasing the number of MAM. Knockdown of IP3R or a reduction in the number of MAM partially restores mitochondrial Ca 2+ signaling and bioenergetics in ERAD-deficient hepatic cells. Moreover, we observed that PA disrupted ERAD protein degradation, leading to impaired mitochondrial function in hepatocytes. Collectively, these results suggest that ERAD is an important regulator of mitochondrial function, at least partially, by maintaining ER-mitochondrial Ca 2+ homeostasis. The physiological significance of ERAD in mitochondrial function in hepatic cells was first illustrated through in vitro experiments using the human hepatocellular carcinoma cell line HepG2. HepG2 cells treated with the ERAD inhibitor EerI showed a significant decrease in mitochondrial energy production and membrane potential (Fig. 1 A, B and 1 G, L). A similar reduction in mitochondrial energy production was observed in HepG2 cells with targeted disruption of the key ERAD protein, SEL1L (Fig. 2 E). Overall, these data strongly suggested that ERAD plays a crucial role in facilitating mitochondrial bioenergetics in hepatic cells. To the best of our knowledge, this is the first direct experimental study linking ERAD deficiency to faulty mitochondrial bioenergetics. The central focus of the present study was to clarify how ERAD deficiency mechanistically impairs mitochondrial function in hepatic cells. To this end, we first investigated whether changes in intracellular Ca 2+ levels affect mitochondrial bioenergetics in ERAD-deficient hepatic cells. This investigation was prompted by the following: 1) Our previous study found that ERAD-defective β-cells exhibit aberrant intracellular Ca 2+ levels; and 2) Numerous studies have demonstrated that intracellular Ca 2+ is a critical signal mediating mitochondrial bioenergetics (Vecellio et al. 2024; Huo et al. 2024; Dridi et al. 2023 ). The present study found a significant increase in mitochondrial Ca 2+ levels (Fig. 3 A, B) and a decrease in ER Ca 2+ (Fig. 3 E, F) in ERAD-deficient HepG2 cells induced by SEL1L knockout. These SEL1L knockout-induced changes in ER and mitochondrial Ca 2+ levels were prevented by the ER Ca 2+ channel blocker 2-APB (Fig. 3 G). More importantly, the negative impact of ERAD deficiency on mitochondrial bioenergetics was reversed by treatment with the ER Ca 2+ channel inhibitor 2-APB and siRNA against IP3R (Fig. 5 A, D). These results suggest that a deficiency in ERAD function hinders mitochondrial bioenergetics by increasing ER Ca 2+ entry into the mitochondria. Ca 2+ fluorescent probes and immunofluorescence experiments further revealed that ERAD deficiency promotes ER Ca 2+ entry into the mitochondria via MAM in hepatic cells. Analysis using a Ca 2+ fluorescent probe demonstrated that the absence of SEL1L led to an increase in cytoplasmic Ca 2+ in the basal state (Fig. 4 A). However, the increase in cytoplasmic Ca 2+ is similar in both WT and SEL1L −/− HepG2 cells after ATP stimulation (Fig. 4 B). These findings suggest that ER Ca 2+ is transported into the mitochondria through MAM in ERAD-deficient hepatic cells. Immunofluorescence analysis showed that SEL1L deficiency resulted in increasing the number of MAM in HepG2 cells (Fig. 4 D–H). Increased amounts of MAM in hepatocytes under high-fat conditions promote the transfer of ER Ca 2+ into mitochondria (Arruda et al. 2014 ). Although the precise molecular mechanisms underlying the increase in MAM numbers remain to be elucidated, the promotion of excessive ER Ca 2+ entry into mitochondria Ca 2+ is likely through an increase in MAM number in ERAD-deficient hepatic cells. There is a link between mitochondrial dysfunction and many chronic metabolic diseases, such as type 2 diabetes, fatty liver, and cardiovascular disease, and PA plays an important role in this process (Ly et al. 2017 ; Liu et al. 2023 ; Riccardi et al. 2004 ). Recent studies have shown that PA can disrupt protein quality control machinery and mitochondrial function in liver cells. Importantly, our study demonstrated that the reduction in mitochondrial oxidative phosphorylation caused by PA was magnified in the absence of ERAD, and this effect could be blocked by a reduction in the number of MAM (Fig. 6 H). Furthermore, we found that PA disrupted the function of ERAD, increased MAM number, and increased mitochondrial Ca 2+ levels in hepatic cells (Fig. 6 B-G). These findings suggested that the absence of ERAD exacerbated the effects of PA on mitochondrial dysfunction. Although it is necessary to elucidate the precise molecular mechanisms underlying the observed synergy between PA and ERAD deficiency in mitochondrial dysfunction, ERAD is likely to play an important role in PA-induced mitochondrial dysfunction by affecting MAM Ca 2+ . In conclusion, our findings from pharmacological and genetic experiments highlight the critical role of ERAD in controlling mitochondrial function in human hepatic cells. Mechanistically, ERAD deficiency induces functional damage to mitochondria by impairing MAM Ca 2+ homeostasis. Additionally, ERAD mediates PA-induced mitochondrial damage in liver cells. This study offers novel insights into the interactions between the ER and mitochondria and has important implications for understanding the molecular mechanisms of mitochondrial dysfunction-related diseases such as NAFLD. Declarations Acknowledgments We thank Yuanyuan Gao, Qiaocheng Zhai, and Lin Zhang for technical assistance; QiWu, and Lu Guo for expression plasmids or other reagents. Dr. Feng Wang, Qiaoming Long for helpful discussions and critical comments on the manuscript. Author Contributions Y.H. generated Sel1L-deficient HepG2 cell lines, performed the vast majority of in vitro experiments, drafted the initial version. Y.B. performed Immunofluorescence experiments. J.B and M.W provided key reagents and helpful discussions. F.Z. designed the experiments and wrote the manuscript. All authors reviewed and approved the manuscript. F.Z. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Funding This work was supported by National Natural Science Foundation of China grant 82100893. Data availability The article and supplementary material contain the data that back up the findings of this study. The corresponding author will provide access to all relevant raw data upon reasonable request. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Consent for publication All authors approved this manuscript for publication. References Arora A, Taskinen JH, Olkkonen VM. Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins. Prog Lipid Res. 2022;86:101146. Arruda AP, Pers BM, Parlakgül G, Güney E, Inouye K, Hotamisligil GS. 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Endoplasmic reticulum-associated degradation regulates mitochondrial dynamics in brown adipocytes. Science. 2020;368(6486):54–60. Additional Declarations No competing interests reported. Supplementary Files floatimage8.png Fig. S1 Fluorescence Mitochondrial morphology A-F, Fluorescent images of Sel1L +/+ , Sel1L -/- , and EerI-treated Sel1L +/+ cells after stainning with mito-Tracker Green . Inset at the bottom right of each panel represents a magnified view of the dash line marked area. floatimage9.png Fig. S2 Mitochondrial Ca 2+ and MAM quantity assays A, detection of mitochondrial Ca 2+ in HepG2 cells treated with EerI. B, detection of ER-Mitochondrial co-localization after OMM linker expression in cells. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4939621","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347531396,"identity":"710b9440-70b7-4357-8eb2-8ad01ab7d7bf","order_by":0,"name":"Yabing Hu","email":"","orcid":"","institution":"Quzhou Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yabing","middleName":"","lastName":"Hu","suffix":""},{"id":347531400,"identity":"4b376395-25c1-4bf4-b507-8b4af6537b62","order_by":1,"name":"Yongfeng Bai","email":"","orcid":"","institution":"Quzhou Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongfeng","middleName":"","lastName":"Bai","suffix":""},{"id":347531402,"identity":"6402e238-13e5-43cf-b1bd-fe19bb9c8dcc","order_by":2,"name":"Beier Jiang","email":"","orcid":"","institution":"Quzhou Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Beier","middleName":"","lastName":"Jiang","suffix":""},{"id":347531403,"identity":"6aa38259-47fa-4f3b-950e-9061f07f694f","order_by":3,"name":"Mingming Wang","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingming","middleName":"","lastName":"Wang","suffix":""},{"id":347531404,"identity":"91d2eeaf-bcf4-4d22-ab87-60091e53c539","order_by":4,"name":"Feng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBAC9gYgwdgAY0HZeAHPAZgyMIs0LRIJxGph7z388ucOuzz5yDemm3kYbGQ3HGB+9gCvFp5zaRaSZ5KLDW/nmN3mYUgz3nCAzdwAnxZ7iRwzA8M25sSNs3O3AbUcTtxwgIdNAq8t8m/MDBLb6hM3zjwL0vKfCC0SPMYPDrYdTpwvwQvScoAILTw5ZoyNbccTN/Dkf7s5xyDZeOZhNjP8WtjPGH/82VadOL/9WNqNNxV2sn3Hm5/h1QIEEGcYHACTQMxMQD1IyQcQKd9AWOUoGAWjYBSMUAAAf3ZMyLv10nYAAAAASUVORK5CYII=","orcid":"","institution":"Quzhou Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-08-19 15:14:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4939621/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4939621/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65070131,"identity":"01b50098-2897-404d-8807-25f0a4b90942","added_by":"auto","created_at":"2024-09-23 09:44:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":541505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological inhibition of ERAD alters the morphology and bioenerge -tics of mitochondria in HepG2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepG2 cells were seeded in 96-well and 6-well plates at densities of 1×10\u003csup\u003e4\u003c/sup\u003e and 1×10\u003csup\u003e5\u003c/sup\u003e cells/well, respectively, and cultured for 12 h before EerI treatment. Following treatment, HepG2 cells were analyzed for A and B, the dose-and time-dependent effects of EerI on the cellular ATP concentration. C-F, Mitochondria were labeled with TOM20, and the inset represents a magnified view of the dashed line marked area. G-L, Mitochondrial membrane potential (MMP) was detected by JC-1; When MMP was high, JC-1 aggregated in the mitochondrial matrix to form polymers (J-aggregates) that emitted red fluorescence. When MMP was low, JC-1 monomers produced green fluorescence. All data are mean ± S.E. (n =3). **, p＜0.01, ***, p＜0.001 by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/44086647f35119e2f9cd5bf4.png"},{"id":65070079,"identity":"99b2c64c-e284-430c-b595-489aa56e066d","added_by":"auto","created_at":"2024-09-23 09:44:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic ablation of ERAD core protein Sel1L impairs mitochondrial bioenergetics of HepG2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Schematic illustration of the CRISPR/Cas9-mediated editing of Sel1L gene in HepG2 cells. g1,2 indicates the sgRNA-binding sites. Black bars represent the exons. B Western blot analysis of SeL1L expression in Sel1L\u003csup\u003e+/+\u003c/sup\u003e and Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells. C and D: NHK degradation in the presence or absence of SEL1L in HepG2 cells. NHK indicates α1-antitrypsin null Hong Kong variant (NHK). E, Dual-channel FACS analysis of MMP (high/low [H/L]) in Sel1L\u003csup\u003e+/+ \u003c/sup\u003eand Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells. The two channels were labeled with FITC and PE. F, ATP level in SEL1L\u003csup\u003e+/+\u003c/sup\u003e and SEL1L\u003csup\u003e-/- \u003c/sup\u003ecells. All data are mean ± S.E. (n =3). **, p＜0.01 by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/4ece2bef5d6d0bafe15da4d3.png"},{"id":65070123,"identity":"cb19e4a3-5ecf-4ea3-80c6-3d63009c8ffd","added_by":"auto","created_at":"2024-09-23 09:44:20","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":736057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERAD deficiency causes increased mitochondrial Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e levels originating from ER\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-D, FACS analysis of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in Sel1L\u003csup\u003e+/+\u003c/sup\u003e and Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells after 2APB treatment. Mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels were measured using Rhod2 (4.5 µM) probe. N and P represent negative and positive cells, respectively. E-F, Steady-state [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eER\u003c/sub\u003e in Sel1L\u003csup\u003e+/+\u003c/sup\u003e and Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells was measured using Mag-Fluo4 probe (4 µM). G. Measurement of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e level using Rhod2 in Sel1L\u003csup\u003e+/+\u003c/sup\u003e and Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells following 2APB treatment. The fluorescence was recorded every 50 s. 10 µM ATP was injected at 60 s. [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003em\u003c/sub\u003e represents mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e. All data are mean ± S.E. (n =3).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/d085bdb6c48d51b304b57368.jpeg"},{"id":65071053,"identity":"e9925842-66b1-4e2f-890f-8e5d4a13d58c","added_by":"auto","created_at":"2024-09-23 09:52:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":436251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e enters mitochondria via the MAM route in ERAD-deficient cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepG2 cells were grown overnight in 96-well plates at a density of 1×10\u003csup\u003e4 \u003c/sup\u003ecells/well. Cytosolic and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e were then detected by using Ca\u003csup\u003e2+\u003c/sup\u003e-dependent fluorescent probes Fluo4 (2 uM) and Rhod\u003csub\u003e2\u003c/sub\u003e (4.5 uM). A, Basal cytosolic Ca\u003csup\u003e2+ \u003c/sup\u003eassay in Sel1L\u003csup\u003e+/+\u003c/sup\u003e and Sel1L\u003csup\u003e-/-\u003c/sup\u003e cells; [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eC\u003c/sub\u003e represents cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e. B, ATP-stimulated cytosolic Ca2+ detection, 10 µM ATP was injected at 60 s. C, ATP-stimulated mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e detection in HepG2 cell after EerI treatment. D-G, Confocal microscopic analysis of ER and mitochondria using calnexin (red) and TOM20 (green) antibodies, respectively, in Sel1L\u003csup\u003e+/+ \u003c/sup\u003eand Sel1L\u003csup\u003e-/-\u003c/sup\u003e HepG2 cells. Colocalized portions of the ER and Mitochondria are shown in white. H, Quantification of co-localization between calnexin and TOM20. All data are mean ± S.E. (n =3). **, p＜0.01 by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/b6ba35ed100e41380edb7a05.png"},{"id":65070119,"identity":"fd06348c-2bb9-4a69-9a72-d6d24e4e4107","added_by":"auto","created_at":"2024-09-23 09:44:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced MAM Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003epartially restores mitochondrial bioenergetics in ERAD deficiency cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. ATP levels were measured in HepG2 cells after pretreatment with 2APB and co-incubation with EerI for 24 h. HepG2 cells were grown in 6-well plates. IP3R siRNA was introduced when cell density reached approximately 90%. Subsequently, HepG2 cells were analyzed for B, Western blot analysis of\u0026nbsp;IP3R expression in cells; C, mitochondrial membrane potential; D, cellular ATP levels. E and F, The OMM-linker was overexpressed in both Sel1L WT and KO cells, and mitochondrial Ca\u003csup\u003e2+ \u003c/sup\u003e(E) and oxygen consumption rate (OCR) (F) were measured. All data are mean ± S.E. (n =3). *, p＜0.05, **, p＜0.01 by Student’ s t test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/bbfaa24d4e8ca4e0d6777ade.png"},{"id":65070127,"identity":"0974cb6e-81bd-4b16-9c8c-d56f0b430836","added_by":"auto","created_at":"2024-09-23 09:44:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":552363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERAD mediates palmitic acid-induced mitochondrial dysfunction in HepG2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Major pathways significantly altered in Hepg2 cells treated with 0.5 mM palmitic acid (PXD029010). *ER quality control systems are indicated. HepG2 cells were treated with 0.5 mM palmitic acid for 24 h and then analyzed for: B and C, degradation levels of NHK-GFP; D-F, Analysis of Colocalized portion of ER and mitochondria using calnexin (red) and TOM20 (green), Colocalized portion of ER and Mitochondria is shown in white; G, mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e; H, Oxygen consumption rate assay of cells treated with PA, OMM linker, and EerI. All data are mean ± S.E. (n =3). *, p＜0.05, **, p＜0.01 by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/ca70182c68fa951bb729b7da.png"},{"id":65070129,"identity":"d3b69666-523d-4e79-9f28-0d28b3e6ce2c","added_by":"auto","created_at":"2024-09-23 09:44:20","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":31098,"visible":true,"origin":"","legend":"Mitochondria and the endoplasmic reticulum (ER) physically and functionally interact, regulating each other\u0026rsquo;s function, but the molecular mechanisms remain not fully understood. In this study, we revealed that ER-associated degradation (ERAD), an ER protein quality control mechanism, governs ER Ca entry into mitochondria by mitochondria-associated ER membrane (MAM) in hepatic cells. Inhibition of ERAD by pharmaceuticals or genetic ablation of the key ERAD protein SEL1L resulted in altered mitochondrial morphology, reduced mitochondrial energy production, and increased Ca transfer from ER to mitochondria. Additionally, SEL1L absence caused an increase in the number of MAM. In ERAD-deficient hepatic cells, a reduction in the number of MAM or knockdown of the inositol 1,4,5-trisphosphate receptor (IP3R), which is responsible for ER Ca release, partially restored mitochondrial Ca signaling and bioenergetics. Together, these results suggest that ERAD plays a crucial role in regulating mitochondrial bioenergetics, suggesting the potential to improve cellular and organismal mitochondrial function by increasing cellular ERAD activity.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/ddec929e7ed4a1573dce3ace.png"},{"id":67603415,"identity":"c2221f26-7d13-4fda-9e9d-21ff66ce0bf9","added_by":"auto","created_at":"2024-10-28 03:54:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3099742,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/a8fbe276-5f49-4cb1-8527-4766011a3680.pdf"},{"id":65070133,"identity":"c2f2772f-0284-495c-9920-2738902dba65","added_by":"auto","created_at":"2024-09-23 09:44:21","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":545718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Fluorescence Mitochondrial morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-F, Fluorescent images of Sel1L\u003csup\u003e+/+\u003c/sup\u003e, Sel1L\u003csup\u003e-/-\u003c/sup\u003e, and EerI-treated Sel1L\u003csup\u003e+/+\u003c/sup\u003e cells after stainning with mito-Tracker Green . Inset at the bottom right of each panel represents a magnified view of the dash line marked area.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/5e2b60bbd5de95bcfc98b65f.png"},{"id":65070086,"identity":"e7627f11-ff8f-4ab1-9c8a-10b89fd4be07","added_by":"auto","created_at":"2024-09-23 09:44:19","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 Mitochondrial Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and MAM quantity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA,\u003cstrong\u003e \u003c/strong\u003edetection of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in HepG2 cells treated with EerI. B, detection of ER-Mitochondrial co-localization after OMM linker expression in cells.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4939621/v1/565c07ea4aae8ef238d851f8.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"ERAD deficiency disrupts mitochondrial bioenergetics by altering MAM Ca2+ in human hepatic cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInterorganelle communication occurs through membrane contact sites and is crucial for the maintenance of organelle homeostasis and organismal health (Arora et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Henne et al. 2021;Wu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Interactions between endoplasmic reticulum (ER) and mitochondria are excellent examples of the importance of this partnership. The close physical contact (~\u0026thinsp;30 nm) between the ER and mitochondria occurs in a region known as the mitochondria- associated ER membrane (MAM). MAM effectively integrates signal transduction with metabolic pathways to regulate ER-mitochondrial communication. It plays important regulatory roles in several key cellular processes including Ca\u003csup\u003e2+\u003c/sup\u003e exchange, lipid synthesis, ER stress (ERS), mitochondrial dynamics and function, autophagy, and apoptosis (Rowland et al. 2012; Phillips et al. 2016; Jang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Evidence indicates that dysfunction in MAM contributes to conditions such as neurodegenerative diseases, fatty liver disease, and diabetes (Liu et al. 2022; Beaulant et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Elwakiel et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There is still considerable uncertainty regarding how interorganelle communication operates and the physiological consequences of miscommunication.\u003c/p\u003e \u003cp\u003eQuality control systems normally maintain homeostasis and proteostasis in the ER throughout an organism's lifespan, and ER-associated degradation (ERAD) is an important branch. The primary function of ERAD is to identify, sort, and degrade misfolded proteins to prevent the harmful effects caused by their buildup. This is particularly important in secretory cells (Kang et al. 2021). The Sel1L-Hrd1 protein complex represents the most highly conserved branch of ERAD from yeast to humans. Recent studies have found that the Sel1L-Hrd1 ERAD may have a constitutive function in maintaining optimal concentrations of key proteins that control mitochondrial dynamics (Zhou et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, little is known about the pathophysiological effects of defective ER quality control systems on mitochondrial function and vice versa in secretory cells such as hepatocytes.\u003c/p\u003e \u003cp\u003eWe had previously shown that disrupting ERAD genetically and pharmacologi- cally impairs glucose-stimulated insulin secretion in β-cells in vitro and in mice (Hu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the current study, we investigated the impact of ERAD deficiency on mitochondria both structurally and energetically using an ERAD-deficient hepatic cell line model. The consequences of ERAD deficiency were linked to aberrant MAM levels, which, in turn, caused mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e excess. Our results indicate that ERAD is critical for maintaining normal mitochondrial energy production and raise the potential for enhancing cellular ERAD capacity to improve mitochondrial function at the organismal and cellular levels.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell culture and treatment\u003c/h2\u003e \u003cp\u003eHepG2 cells (ATCC) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Gibco, USA) supplemented with 10% FBS(Gibco) and 1% penicillin/streptomycin in a 5% CO2 incubator at 37\u0026deg;C. The cells were treated with ERAD inhibitor eeyarestatin I (8 \u0026micro;M; MedChemExpress, USA), cycloheximide (50 \u0026micro;g/mL; Abmole, China), 2-APB (20 \u0026micro;M; Sigma-Aldrich, USA), ATP (10 \u0026micro;M; MedChemExpress), palmitic acid (0.5 mM; Sigma-Aldrich), si-con or siIP3R (10 nM; Gemma-Pharma, China) for the corresponding times. Cells between passages five and eight were used for all experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. CRISPR/Cas9 mediated Sel1l gene editing in HepG2 cells\u003c/h2\u003e \u003cp\u003eCas9-mediated editing of Sel1l was performed as previously described (Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, sgRNAs flanking exon 6 were designed using the Optimized CRISPR Design tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.mit.edu/\u003c/span\u003e\u003cspan address=\"http://crispr.mit.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and cloned into the expression vector pGL3- U6 -2sgRNA. The sgRNA expression vector and Cas9 expression vector pST1374-NLS-3xFlag- linker-Cas9 were both inserted into HepG2 cells using Lipofectamine 3000 (Baillat et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). After transfection, 5 \u0026micro;g/mL of puromycin was added to the growth medium to specifically choose cells that are not killed by puromycin. Approximately one week later, puromycin-resistant HepG2 colonies were selected, expanded, and tested by PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Mitochondrial morphology, mitochondrial membrane potential and ATP production assay\u003c/h2\u003e \u003cp\u003eTo investigate the effect of ERAD on mitochondria, we examined the mitochondrial morphology, mitochondrial membrane potential, and cellular ATP levels.Mitochondria were visualized using a MitoTracker Green fluorescent probe (Invitrogen, USA) or Tom20, followed by observation with laser confocal microscopy and analysis using ImageJ software.\u003c/p\u003e \u003cp\u003eThe mitochondrial membrane potential was determined using a JC-1 assay kit (Beyotime,China). Briefly, HepG2 cells were cultured in 6-well plates at a density of 2x10*5 cells per well and treated as required (EerI, 2APB, or siIP3R). The cells were then incubated with 2 \u0026micro;M JC-1 for 30 min at 37\u0026deg;C in the dark, washed with PBS, and the fluorescence was measured using flow cytometry (BD,USA) and/or a fluorescence microscope (Olympus, JAPAN).\u003c/p\u003e \u003cp\u003eCellular ATP levels were measured using a Firefly Luciferase ATP Assay Kit (Beyotime) according to the manufacturer\u0026rsquo;s instructions. Briefly, treated cells were lysed and centrifuged for 5 min at 12000 g to obtain the cell supernatant. After mixing 100 \u0026micro;L of cell supernatant with 100 \u0026micro;L of ATP assay working solution in a 96-well plate, the reaction was allowed to proceed for 5 min at room temperature. The fluorescence intensity was quantified using a microplate reader (Molecular Devices), and the ATP value was normalized to the protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Western blot analysis\u003c/h2\u003e \u003cp\u003eAs previously mentioned (Sha et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), cell lysates were prepared and western blotting was performed. Briefly, cells were scraped and resuspended in 10 mM Tris\u0026ndash;HCl pH 7.5, 1 mM PMSF, and incubated at 4 ℃ for 20 min. The lysate was centrifuged at 10,000 g for 10 min at 4\u0026deg;C to obtain the supernatant. The protein concentration in the supernatant was determined using a BCA kit (Thermo Fisher, USA). Denaturing buffer was added to the protein supernatant, which was boiled for 5 min before separation by SDS-PAGE. The following antibodies were used in this study: GFP (1:500 rabbit) from Santa Cruz Biotechnology, β-actin (1:1,000 mouse) from Sigma-Aldrich, Sel1L (1:1,000 rabbit) and IP3R (1:500 rabbit) from Abcam. Anti-rabbit or anti-mouse secondary antibodies were incubated for 2 h at room temperature and band density was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cytosolic, ER and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e measurement\u003c/h2\u003e \u003cp\u003eHepG2 cells were seeded into 6-well glass-bottomed chambers at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured in DMEM for 24 h. Ca\u003csup\u003e2+\u003c/sup\u003e in the cytoplasm, ER, and mitochondria was detected using Fluo4, AM, Mag-Fluo4, and Rhod2 probes. The probes were then added to Ca\u003csup\u003e2+\u003c/sup\u003e-free PBS and incubated for 30\u0026ndash;60 min at room temperature. Using a fluorescence microscope, fluorescent images were collected every 50 s, background-corrected, and analyzed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell immunofluorescence\u003c/h2\u003e \u003cp\u003eHepG2 cells were cultured overnight in a 6 cm dish. After fixation with 4% paraformaldehyde for 15 min, the cells were permeabilized for 5 min with 1% Triton X-100 in PBS. To prevent non-specific binding, cells were incubated in 10% donkey serum in TBST for 1 h at 37\u0026deg;C. Then, primary antibodies calnexin (1:200, Santa Cruz Biotechnology) and Tom20 (1:200, Absin) were introduced to the cells for 1 h at 37 ℃. The cells were then incubated with secondary antibodies in 5% donkey serum for 2 h at room temperature and washed. Stained cells were imaged using laser confocal microscopy, and the intensity and colocalization of fluorescence were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. SiRNA-mediated knockdown of IP3R in HepG2 cells\u003c/h2\u003e \u003cp\u003eA scrambled negative control siRNA for hIP3R was predesigned and purchased from Gemma Pharma (China). HepG2 cells were seeded in 6-well plates. When the cells were 80% confluent, Opti-MEM was mixed with either control or IP3R siRNA together with Lipofectamine 3000 (Invitrogen) and incubated for 6 h. After transfection, the medium was replaced with DMEM containing 10% FBS and the cells were cultured for an additional 48 h. Cell lysates were collected to detect IP3R expression levels by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Mitochondrial respiration assay\u003c/h2\u003e \u003cp\u003eMitochondrial respiration was assessed by measuring the oxygen consumption rate (OCR) using a Seahorse XFe96 Analyzer as described previously (Mthembu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). HepG2 cells were grown overnight in Seahorse XFe96 microtiter wells at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, and treated as required. On the first day of the experiment, the culture medium was replaced with Seahorse XF Base Medium (Agilent Technologies) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. The following mitochondrial stress test compounds (1 \u0026micro;M Oligomycin, 0.5 \u0026micro;M FCCP, 0.5 \u0026micro;M Rotenone, and 0.5 \u0026micro;M antimycin A) were sequentially injected into the wells at specified time points. The OCR values were normalized to the total protein content in each well and determined using the BCA Protein Assay Kit (Beyotime).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E. Differences between compared groups were evaluated using SPSS software (version 16.0) to perform Student\u0026rsquo;s t-test or analysis of variance (ANOVA). The significance level was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. ERAD dysfunction impairs HepG2 cells mitochondrial bioenergetics\u003c/h2\u003e \u003cp\u003eOur previous study showed that disrupting ERAD decreases ATP production in the INS-1 rat pancreatic β-cell line, suggesting that a deficiency in ERAD compromises the mitochondrial respiratory function(Hu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To investigate whether this phenomenon extends to other mammalian cells, we treated human hepatic HepG2 cells with the ERAD inhibitor eeyarestatin I (EerI). EerI treatment reduced ATP levels in a time-and dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B), altered mitochondrial morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-F), and mitochondrial membrane potential (MMP) was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eSEL1L is essential for the formation of the ERAD complex on the ER membrane in mammals. To further investigate the effect of ERAD on ATP levels, we used CRISPR /Cas9-based gene editing to generate SEL1L-deficient (Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Next, we used cycloheximide (CHX)-based pulse-chase analysis for α1-antitrypsin null Hong Kong variant (NHK) in WT and Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells to determine the ERAD-degrading function. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, the folding-deficient mutant NHK displayed efficient degradation in WT HepG2 cells, but not in Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells. This result indicates that the ERAD deficiency cell model was successfully constructed using the sel1L knockout. For ATP and MMP, Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells were lower than WT HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Overall, our findings suggest that ERAD deficiency (treatment with EerI or deletion of Sel1L) impairs mitochondrial morphology and bioenergetics in hepatic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. ERAD deficiency causes increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e mainly originated from ER\u003c/h2\u003e \u003cp\u003eIt is well established that mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration is a critical determinant in mediating mitochondrial respiration (Sun et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Santulli et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Given our findings in pancreatic β-cells that ERAD deficiency causes changes in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels (Hu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), we tested whether a similar change occurs in ERAD-defective HepG2 cells. In ERAD-defective HepG2 cells, we used the fluorescent probe Rhod2 AM to detect mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, both Sel1L deletion and EerI treatment produced a significant increase in Ca\u003csup\u003e2+\u003c/sup\u003e levels in the mitochondria of HepG2 cells.\u003c/p\u003e \u003cp\u003eSince ERAD is an ER-associated degradation mechanism, and the ER is a major Ca\u003csup\u003e2+\u003c/sup\u003e storage reservoir in eukaryotic cells (Christianson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we investigated whether ER Ca\u003csup\u003e2+\u003c/sup\u003e contributes to increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in ERAD-deficient cells. First, we measured ER Ca\u003csup\u003e2+\u003c/sup\u003e using the Mag-Fluo4 probe and found that ER Ca\u003csup\u003e2+\u003c/sup\u003e levels in Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells were higher than in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Next, we treated WT and ERAD-deficient cells with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor, 2APB, which blocks ER Ca\u003csup\u003e2+\u003c/sup\u003e release. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, ERAD-deficient cells treated with 2APB showed a marked reduction in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels. When stimulated with ATP, which promotes the release of ER Ca\u003csup\u003e2+\u003c/sup\u003e, ERAD-deficient cells displayed higher mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake than the WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Upon addition of 2APB, there was a significant and abrupt decrease in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels in both ERAD-deficient and WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Together, these findings indicate that increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e originates mostly from the ER in ERAD-deficient hepatic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. ER Ca\u003csup\u003e2+\u003c/sup\u003e enters mitochondria via the MAM connections in ERAD-deficient cells.\u003c/h2\u003e \u003cp\u003eTo evaluate whether increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e is the result of direct transport through MAM junctions or IP3R-mediated ER Ca\u003csup\u003e2+\u003c/sup\u003e release into the cytosol, followed by mitochondrial uptake, we analyzed cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e dynamics in response to IP3R stimulation in WT and Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells using Fluo4-AM. The baseline cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, ATP stimulation caused a comparable increase in cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels in WT and Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), despite the higher mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e peak in ERAD-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that ERAD deficiency leads to increased Ca\u003csup\u003e2+\u003c/sup\u003e transport from the ER through MAM connections, resulting in elevated mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e in HepG2 cells.\u003c/p\u003e \u003cp\u003eTo investigate how MAM affects Ca\u003csup\u003e2+\u003c/sup\u003e fluctuations between the ER and mitochondria in ERAD-deficient cells, we labeled the ER with calnexin and the mitochondria with TOM20 and then quantified MAM changes by analyzing the degree to which calnexin and TOM20 colocalized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, the amount of MAM in Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher than that in WT cells. Correlative studies have demonstrated that an increased number of MAM promotes higher mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels (Arruda et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As a result, ERAD deficiency may lead to an increase in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration by altering the number of MAM in hepatic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Reduced MAM Ca\u003csup\u003e2+\u003c/sup\u003e partially restores mitochondrial bioenergetics in ERAD deficient cells\u003c/h2\u003e \u003cp\u003eThe effect of an imbalance in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis on mitochondrial respiration is widely recognized. Given the finding that ERAD deficiency caused a substantial influx of ER Ca\u003csup\u003e2+\u003c/sup\u003e into mitochondria, leading to an increase in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e, we tested whether altered mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels contribute to defective mitochondrial bioenergetics in ERAD-deficient liver cells. To decrease ER Ca\u003csup\u003e2+\u003c/sup\u003e influx into mitochondria, we introduced 2APB into HepG2 cells. 2APB partially reversed the EerI-induced reduction in ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). IP3R is an ER Ca\u003csup\u003e2+\u003c/sup\u003e release receptor; IP3R siRNA was transfected into WT and SEl1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells, and the knockdown effect is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. In Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells, we found that the knockdown of IP3R expression partially restored MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eGiven the finding that ERAD deletion may boost ER Ca\u003csup\u003e2+\u003c/sup\u003e influx into the mitochondria by increasing the number of MAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), we used an outer mitochondrial membrane (OMM) linker plasmid that decreased the number of MAM (Fig. S2B). We found that OMM linker expression reduced mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and improved OCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) in Sel1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells. Together, these findings show that ERAD deficiency compromises mitochondrial bioenergetics by increasing MAM and encouraging a substantial input of ER Ca\u003csup\u003e2+\u003c/sup\u003e into mitochondria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. ERAD mediates palmitic acid-induced mitochondrial dysfunction in HepG2 cells\u003c/h2\u003e \u003cp\u003ePalmitic acid (PA) is a significant contributor to the development of nonalcoholic fatty liver disease (NAFLD) and can induce lipid accumulation in the liver through its impact on mitochondrial function (Silva Figueiredo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Marra et al. 2018). A recent proteomic analysis revealed that treatment of HepG2 cells with PA damaged the protein quality control machinery (autophagy, ERAD, and the ubiquitin-proteasomal system) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) (Saha et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We tested whether ERAD mediated the impairment of mitochondrial function in hepatocytes caused by PA. We first performed cycloheximide (CHX)-based pulse-chase analysis for folding -deficient mutant NHK in HepG2 cells treated with and without PA. Folding-deficient mutant NHK showed efficient degradation in HepG2 cells without PA, but not in HepG2 cells treated with PA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Next, we investigated the effects of PA on MAM and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, PA exposure increased MAM and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels in HepG2 cells. According to other studies, PA lowers the OCR (Mthembu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This study confirmed that PA impairs OCR and combining it with the ERAD inhibitor EerI further decreases OCR; yet the OMM linker restored the PA-induced OCR reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Together, these data suggest that PA exposure in HepG2 cells impairs ERAD function, and that ERAD is likely a mediator of PA impairment of mitochondrial function.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe ER and mitochondria interact both physically and functionally, allowing them to control each other's functions. A recent study revealed that ER protein quality control (ERAD) plays a crucial role in maintaining optimal levels of sigma receptor 1 (SigmaR1) to regulate mitochondrial dynamics in brown adipose cells (Zhou et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, we investigated whether the protein quality control system influences mitochondrial function in hepatic cells. Our research showed that ERAD facilitates Ca\u003csup\u003e2+\u003c/sup\u003e signaling in the mitochondria by controlling the quantity of MAM. ERAD deficiency results in altered mitochondrial structure, reduced mitochondrial energy production, and increased Ca\u003csup\u003e2+\u003c/sup\u003e transfer from the ER to the mitochondria by increasing the number of MAM. Knockdown of IP3R or a reduction in the number of MAM partially restores mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e signaling and bioenergetics in ERAD-deficient hepatic cells. Moreover, we observed that PA disrupted ERAD protein degradation, leading to impaired mitochondrial function in hepatocytes. Collectively, these results suggest that ERAD is an important regulator of mitochondrial function, at least partially, by maintaining ER-mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ehomeostasis.\u003c/p\u003e \u003cp\u003eThe physiological significance of ERAD in mitochondrial function in hepatic cells was first illustrated through in vitro experiments using the human hepatocellular carcinoma cell line HepG2. HepG2 cells treated with the ERAD inhibitor EerI showed a significant decrease in mitochondrial energy production and membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, L). A similar reduction in mitochondrial energy production was observed in HepG2 cells with targeted disruption of the key ERAD protein, SEL1L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Overall, these data strongly suggested that ERAD plays a crucial role in facilitating mitochondrial bioenergetics in hepatic cells. To the best of our knowledge, this is the first direct experimental study linking ERAD deficiency to faulty mitochondrial bioenergetics.\u003c/p\u003e \u003cp\u003eThe central focus of the present study was to clarify how ERAD deficiency mechanistically impairs mitochondrial function in hepatic cells. To this end, we first investigated whether changes in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels affect mitochondrial bioenergetics in ERAD-deficient hepatic cells. This investigation was prompted by the following: 1) Our previous study found that ERAD-defective β-cells exhibit aberrant intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels; and 2) Numerous studies have demonstrated that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e is a critical signal mediating mitochondrial bioenergetics (Vecellio et al. 2024; Huo et al. 2024; Dridi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The present study found a significant increase in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) and a decrease in ER Ca\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F) in ERAD-deficient HepG2 cells induced by SEL1L knockout. These SEL1L knockout-induced changes in ER and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels were prevented by the ER Ca\u003csup\u003e2+\u003c/sup\u003e channel blocker 2-APB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). More importantly, the negative impact of ERAD deficiency on mitochondrial bioenergetics was reversed by treatment with the ER Ca\u003csup\u003e2+\u003c/sup\u003e channel inhibitor 2-APB and siRNA against IP3R (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, D). These results suggest that a deficiency in ERAD function hinders mitochondrial bioenergetics by increasing ER Ca\u003csup\u003e2+\u003c/sup\u003e entry into the mitochondria.\u003c/p\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e fluorescent probes and immunofluorescence experiments further revealed that ERAD deficiency promotes ER Ca\u003csup\u003e2+\u003c/sup\u003e entry into the mitochondria via MAM in hepatic cells. Analysis using a Ca\u003csup\u003e2+\u003c/sup\u003e fluorescent probe demonstrated that the absence of SEL1L led to an increase in cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e in the basal state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, the increase in cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e is similar in both WT and SEL1L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e HepG2 cells after ATP stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings suggest that ER Ca\u003csup\u003e2+\u003c/sup\u003e is transported into the mitochondria through MAM in ERAD-deficient hepatic cells. Immunofluorescence analysis showed that SEL1L deficiency resulted in increasing the number of MAM in HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;H). Increased amounts of MAM in hepatocytes under high-fat conditions promote the transfer of ER Ca\u003csup\u003e2+\u003c/sup\u003e into mitochondria (Arruda et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although the precise molecular mechanisms underlying the increase in MAM numbers remain to be elucidated, the promotion of excessive ER Ca\u003csup\u003e2+\u003c/sup\u003e entry into mitochondria Ca\u003csup\u003e2+\u003c/sup\u003e is likely through an increase in MAM number in ERAD-deficient hepatic cells.\u003c/p\u003e \u003cp\u003eThere is a link between mitochondrial dysfunction and many chronic metabolic diseases, such as type 2 diabetes, fatty liver, and cardiovascular disease, and PA plays an important role in this process (Ly et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Riccardi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Recent studies have shown that PA can disrupt protein quality control machinery and mitochondrial function in liver cells. Importantly, our study demonstrated that the reduction in mitochondrial oxidative phosphorylation caused by PA was magnified in the absence of ERAD, and this effect could be blocked by a reduction in the number of MAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Furthermore, we found that PA disrupted the function of ERAD, increased MAM number, and increased mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels in hepatic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-G). These findings suggested that the absence of ERAD exacerbated the effects of PA on mitochondrial dysfunction. Although it is necessary to elucidate the precise molecular mechanisms underlying the observed synergy between PA and ERAD deficiency in mitochondrial dysfunction, ERAD is likely to play an important role in PA-induced mitochondrial dysfunction by affecting MAM Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings from pharmacological and genetic experiments highlight the critical role of ERAD in controlling mitochondrial function in human hepatic cells. Mechanistically, ERAD deficiency induces functional damage to mitochondria by impairing MAM Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis. Additionally, ERAD mediates PA-induced mitochondrial damage in liver cells. This study offers novel insights into the interactions between the ER and mitochondria and has important implications for understanding the molecular mechanisms of mitochondrial dysfunction-related diseases such as NAFLD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yuanyuan Gao, Qiaocheng Zhai, and Lin Zhang for technical assistance; QiWu, and Lu Guo for expression plasmids or other reagents. Dr. Feng Wang, Qiaoming Long for helpful discussions and critical comments on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eY.H. generated Sel1L-deficient HepG2 cell lines, performed the vast majority of in vitro experiments, drafted the initial version.\u0026nbsp;Y.B. performed Immunofluorescence experiments. J.B and M.W provided key reagents and helpful discussions. F.Z. designed the experiments and wrote the manuscript. All authors reviewed and approved the manuscript. F.Z.\u0026nbsp;is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China grant 82100893.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe article and supplementary material contain the data that back up the findings of this study. The corresponding author will provide access to all relevant raw data upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors approved this manuscript for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArora A, Taskinen JH, Olkkonen VM. Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins. Prog Lipid Res. 2022;86:101146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArruda AP, Pers BM, Parlakg\u0026uuml;l G, G\u0026uuml;ney E, Inouye K, Hotamisligil GS. 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Science. 2020;368(6486):54\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hepatic cells, ERAD, mitochondrial function, MAM, mitochondrial calcium","lastPublishedDoi":"10.21203/rs.3.rs-4939621/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4939621/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mitochondria and the endoplasmic reticulum (ER) physically and functionally interact, regulating each other’s function, but the molecular mechanisms remain not fully understood. In this study, we revealed that ER-associated degradation (ERAD), an ER protein quality control mechanism, governs ER Ca2+ entry into mitochondria by mitochondria-associated ER membrane (MAM) in hepatic cells. Inhibition of ERAD by pharmaceuticals or genetic ablation of the key ERAD protein SEL1L resulted in altered mitochondrial morphology, reduced mitochondrial energy production, and increased Ca2+ transfer from ER to mitochondria. Additionally, SEL1L absence caused an increase in the number of MAM. In ERAD-deficient hepatic cells, a reduction in the number of MAM or knockdown of the inositol 1,4,5-trisphosphate receptor (IP3R), which is responsible for ER Ca2+ release, partially restored mitochondrial Ca2+ signaling and bioenergetics. Together, these results suggest that ERAD plays a crucial role in regulating mitochondrial bioenergetics, suggesting the potential to improve cellular and organismal mitochondrial function by increasing cellular ERAD activity.","manuscriptTitle":"ERAD deficiency disrupts mitochondrial bioenergetics by altering MAM Ca2+ in human hepatic cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 09:44:12","doi":"10.21203/rs.3.rs-4939621/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5205c98-dbb8-4b54-b244-8070dba1c625","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T03:53:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-23 09:44:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4939621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4939621","identity":"rs-4939621","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}