Magnesium Accumulation Upon Cyclin M4 Silencing Activates Microsomal Triglyceride Transfer Protein Improving NASH

Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH Graphical abstract Highlights CNNM4 acts as a magnesium exporter in the liver. Its upregulation in NASH leads to elevated magnesium levels in serum. Liver-specific CNNM4 targeting alleviates steatosis, inflammation, and fibrosis in preclinical NASH models. siRNA-mediated CNNM4 downregulation promotes hepatic magnesium accumulation and reduces endoplasmic reticulum stress. Silencing CNNM4 enhances microsomal triglyceride transfer protein activity leading to VLDL assembly and secretion. Authors Jorge Simón, Naroa GoikoetxeaUsandizaga, Marina Serrano-Maciá, ., Teresa Cardoso Delgado, Luis Alfonso Martínez-Cruz, María Luz Martínez-Chantar Correspondence [email protected] (M.L. Martínez-Chantar) Lay summary Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein. https://doi.org/10.1016/j.jhep.2021.01.043 © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Hepatol. 2021, 75, 34–45 Research Article NAFLD and Alcohol-Related Liver Diseases Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH Jorge Simón 1,2 , Naroa Goikoetxea-Usandizaga 1 , Marina Serrano-Maciá 1 , David Fernández-Ramos 1,2,3 , Diego Sáenz de Urturi 4 , Jessica J. Gruskos 5 , Pablo Fernández-Tussy 1 , Sofía Lachiondo-Ortega 1 , Irene González-Recio 1 , Rubén Rodríguez-Agudo 1 , Virginia Gutiérrez-de-Juan 1 , Begoña Rodríguez-Iruretagoyena 1 , Marta Varela-Rey 1,2 , Paula Gimenez-Mascarell 1 , María Mercado-Gomez 1 , Beatriz Gómez-Santos 4 , Carmen Fernandez-Rodriguez 1 , Fernando Lopitz-Otsoa 1,3 , Maider Bizkarguenaga 1,3 , Sibylle Dames 6 , Ute Schaeper 6 , Franz Martin 7,8 , Guadalupe Sabio 9 , Paula Iruzubieta 10,11 , Javier Crespo 10,11 , Patricia Aspichueta 2,4,12 , Kevan H.-Y. Chu 5 , Daniela Buccella 5 , César Martín 13 , Teresa Cardoso Delgado 1 , Luis Alfonso Martínez-Cruz 1,† , María Luz Martínez-Chantar 1,2, * ,† 1 Liver Disease Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain; 2 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160, Bizkaia, Spain; 3 Precision Medicine and Metabolism Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain; 4 Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain; 5 Department of Chemistry, New York University, New York, NY, USA; 6 Silence Therapeutics GmbH, Berlin, Germany; 7 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad Pablo de Olavide, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain; 8 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain; 9 Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain; 10 Gastroenterology and Hepatology Department, Marqués de Valdecilla University Hospital, Santander, Spain; 11 Clinical and Translational Digestive Research Group, Instituto de Investigación Sanitaria Valdecilla (IDIVAL), Santander, Spain; 12 Biocruces Health Research Institute, Barakaldo, Bizkaia, Spain; 13 Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain Background & Aims: Perturbations of intracellular magnesium (Mg 2+ ) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg 2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). Methods: Serum Mg 2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine ® or conjugated to Nacetylgalactosamine. Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg 2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg 2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg 2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. Lay summary: Cyclin M4 (CNNM4) is overexpressed in nonalcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein. © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Dietary imbalances, such as a low intake of magnesium, are recognised as the root cause of diseases. Although daily magnesium recommended intake is 300–400 mg/day, there is a growing concern about decreased intake in last decade, attributable to changes in dietary habits and food processing. 1,2 Its ionic form, Mg 2+ , is the most abundant divalent cation in the cell and it is required as a cofactor for over 300 enzymatic reactions. 3 Keywords: Non-alcoholic steatohepatitis; NASH; Cyclin M4; CNNM4; Magnesium; Therapy; siRNA; Endoplasmic reticulum stress; Microsomal triglyceride transfer protein; MTP. Received 20 April 2020; received in revised form 18 January 2021; accepted 19 January 2021; available online 9 February 2021 *Corresponding author. Address: Liver Disease Laboratory, Building 801A, Technologic Park of Biscay, Derio (Biscay), Spain. Tel.: +34-944-061-318; Fax: +34944-061-301. E-mail address: [email protected] (M.L. Martínez-Chantar). † Senior authorship. https://doi.org/10.1016/j.jhep.2021.01.043 Journal of Hepatology 2021 vol. 75 j34–45 Research Article NAFLD and Alcohol-Related Liver Diseases An intricate network of Mg 2+ transporters participate in its uptake and excretion, allowing its flux across cellular membranes, thus impacting Mg 2+ homeostasis and distribution. 1 Mg 2+ supplementation reduces mortality from hepatic complications arising from alcohol intake or steatosis. 4 Hypomagnesaemia is present in several comorbidities of nonalcoholic fatty liver disease (NAFLD) such as insulin resistance and type 2 diabetes, 5 cardiovascular complications, 6 and obesity. 7 Moreover, deficiencies in Mg 2+ are related to inflammatory responses, mitochondrial dysfunction, and decreased activity of the antioxidant system, all features of liver diseases. 8 The term NAFLD encompasses a group of pathologies characterised by chronicity with serial progression from steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis. 9 Such progression is mediated by the increased production of reactive oxygen species (ROS), lipotoxicity, mitochondrial dysfunction, and the development of endoplasmic reticulum (ER) stress. 10 Remarkably, ER stress has been proposed to be an important mechanism because it disrupts calcium (Ca 2+ ) transport through ATPases 11 and leads to protein misfolding. 12 It has been widely characterised in both NAFLD and NASH patients, together with impaired VLDL assembly and secretion. 13,14 NAFLD is a global health problem with an estimated prevalence of around 25% among the adult population, being expected to increase. 9 Changes in lifestyle habits are the most common recommendations for the clinical management of NAFLD, but it is difficult to achieve the long-term compliance of patients, making pharmacological alternatives attractive. Among the various magnesiotropic proteins, the cyclin M (CNNM) family and their Bateman-module-mediated interactions with phosphatases of regenerating liver (PRLs) are of particular interest. 15 PRLs are implicated in tumour progression and metastasis, 16,17 including liver cancer, with Mg 2+ perturbations reported in such. 18 Although the CNNMs emerged as interacting molecular partners of PRLs, 17 a new perspective proposes targeting CNNMs to modulate Mg 2+ homeostasis more specifically. 19 The functions of hepatic CNNMs in this regard are unknown, and their significance as pharmacological targets remains underexplored. In the present study, we demonstrated the relevance of the differential expression of CNNM4 in NASH development, showing its functional role in transporting hepatic Mg 2+ .Moreover,we identified CNNM4 as a potential target for NASH treatment. Patients and methods Patients Measurements of serum Mg 2+ and hepatic CNNM1-4 mRNA, and immunohistochemical CNNM4 analysis were performed in different cohorts recruited at the Hospital Marqués de Valdecilla, Santander, Spain (Fig. S1A and B). Animal maintenance and preclinical studies Mice were maintained with ad libitum access to water and a choline-deficient diet with 0.1% methionine (0.1% MCDD) or a choline-deficient, high-fat diet (CD-HFD). A control group was maintained on a regular diet (SC diet). Treatment of primary mouse hepatocytes and THLE-2 cells Upon attachment, isolated mouse primary hepatocytes were transfected by overnight incubation with siRNA using DharmaFECT 1 or a CNNM4 expression plasmid using jetPRIME ® . Cells were maintained and incubated for an additional 24 h under different conditions. Cnnm4 silencing in vivo Mice fed a 0.1% MCDD for 2 weeks or a CD-HFD for 3 weeks were repeatedly administered an siRNA using Invivofectamine ® 3.0 Reagent through tail vein injection. Mice were sacrificed after 4 weeks on 0.1% MCDD and 6 weeks on CD-HFD, respectively. For Cnnm4 GalNAc-siRNA delivery, mice fed for 3 weeks on 0.1% MCDD were treated once, subcutaneously, and sacrificed after a total of 6 weeks on the diet. Samples of liver, white adipose tissue (WAT), and serum were collected. Statistical analysis All the experiments were performed at least in triplicate, with n = 3 (in vitro)andn=4(in vivo). The data are expressed as mean ± SEM and represent the fold change vs. control mean value when indicated. Statistical significance was determined using Prism 8 (GraphPad Software, San Diego, CA, USA). Groups were compared by 1-way analysis of variance (ANOVA) followed by post hoc Bonferroni tests (for 3 or more groups) or the Student ttest (for 2 groups). Results CNNM4 is overexpressed in clinical and preclinical NASH To investigate Mg 2+ dysregulation in NASH, we determined Mg 2+ levels in sera from a cohort of 50 ageand BMI-matched patients (Fig. S1A), observing increased levels of the cation in NASH patients (Fig. 1A). Mg 2+ levels did not correlate with other NASH biomarkers (data not shown). Considering the magnesiotropic role of the CNNM family, 19 we sought to assess whether dysregulated CNNM expression could be the cause of the elevated serum Mg 2+ . The hepatic mRNA levels of each CNNM were assessed in another cohort of 40 patients (Fig. S1B), showing a significant overexpression of CNNM1 and CNNM4 in NASH patients (Fig.1B). In addition, an increase in Cnnm4 mRNA expression was observed in primary hepatocytes treated with methionine and choline-deficient (MCD) medium (Fig. 1C), an in vitro model that displays features of NASH such as ROS overproduction and lipid accumulation. 20,21 Similarly, hepatic CNNM4 overexpression in NASH was confirmed by immunohistochemical protein determination in clinical samples (Fig. 1D) and animal models –mice fed a 0.1% MCDD (Fig. 1E) and CD-HFD (Fig. 1F). These animal models also showed elevated Cnnm4 mRNA, as did primary hepatocytes (Fig. S1C and D). By contrast, Cnnm1 overexpression was observed in neither the in vitro nor in vivo NASH models. To evaluate protein stability, human THLE-2 cells were treated with MLN4924 to inhibit NEDDylation, which prevents proteasome-mediated degradation, 22 resulting in decreased CNNM4 expression (Fig. S1E). The expression of other Mg 2+ transporters was determined in animal models (Fig. S1F) and clinical samples (Fig. S1G), showing that CNNM4 is unique among Mg 2+ transporters in its upregulation in all conditions. Targeting CNMM4 ameliorates NASH The possible role of CNNM4 in NASH development was examined by in vitro screening by specifically silencing each Cnnm in primary hepatocytes using small interfering RNA (siRNA). Targeting Cnnm4 (siCnnm4) alone effectively reduced MCD-induced lipid accumulation (Fig. 2A). The expression of each Cnnm and Prl Journal of Hepatology 2021 vol. 75 j34–45 35 F 0 1 2 3 4 5 SC diet Cnnm4 area (a.u.) 2 weeks *** 4 weeks **** CD-HFD SC diet 3 weeks 6 weeks CD-HFD CNNM4 E 0 5 10 15 Cnnm4 area (a.u.) SC diet * 2 weeks **** 4 weeks 0.1% MCDD SC diet 2 weeks 4 weeks 0.1% MCDD CNNM4 D 0 5 10 15 20 25 Cnnm4 area (a.u.) Healthy * Steatosis *** NASH Healthy NASHSteatosis CNNM4 C 0.0 0.5 1.0 1.5 2.0 Ctrl MCD * RelativemRNA expression (fold-change) Cnnm1 Cnnm2 Cnnm3 Cnnm4 B 0 1 2 3 4 5 Relative mRNA expression (fold-change) **** *** Healthy (n = 5) Steatosis (n = 20) NASH (n = 15) CNMM1 CNNM2 CNNM3 CNNM4 Healthy (n = 18) NASH (n = 18) Steatosis (n = 14) 0 1 2 3 4* Magnesiumin serum (mg/dl) A Fig. 1. CNNM4 is overexpressed in NASH patients and preclinical animal models. (A) Magnesium determination in serum and (B) hepatic levels of CNNM1-4 mRNA in different cohorts of healthy individuals, patients with steatosis and patients with NASH. (C) Levels of Cnnm1-4 mRNA in primary hepatocytes stimulated with MCD medium. (D) Liver immunohistochemical staining and quantification for CNNM4 in a cohort of healthy individuals, patients with steatosis and patients with NASH, as well as mice fed (E) a 0.1% MCDD or (F) a CD-HFD. Scale bar corresponds to 100 l m. *p<0.05, ***p<0.001, and ****p<0.0001 vs. healthy/ctrl/SC diet. CD-HFD, choline-deficient high-fat diet; MCD, methionine and choline deficient; MCDD, MCD diet; NASH, non-alcoholic steatohepatitis; SC diet, regular diet. B 0 3 6 9 12 CNNM4 area (a.u.) * #20 30 40 0.1% MCDD siCtrl SC diet 0.1% MCDD siCnnm4 siCnnm4 siCtrl SC diet 0 10 # *** Suran red area (a.u.) CNNM4SUDAN RED 0.1% MCDD siCnnm4siCtrlSC diet CNNM4 area (a.u.) 0 1 2 3 4 ** ## 5 10 15 CD-HFD siCtrl SC diet CD-HFD siCtrl SC diet Suran red area (a.u.) 0 * # CD-HFD CNNM4SUDAN RED siCnnm4siCtrlSC diet C siCnnm4 siCnnm4 A 0 1 2 3 4 5 MCD *** siCnnm1 *** siCnnm2 *** siCnnm3 ### siCnnm4 Ctrl *** siCtrl Intensity of lipid bodies/ cells (fold-change) BODIPY Ctrl MCD + siCtrl MCD + siCnnm2 MCD + siCnnm3 MCD + siCnnm1 MCD + siCnnm4 Fig. 2. Targeting CNNM4 reduces lipid accumulation in in vitro and in vivo NASH models. (A) BODIPY staining micrographs and quantification in primary hepatocytes transfected with siRNA against Cnnm1-4 (siCnnm1-4) and stimulated with MCD for 24 h. Micrographs of CNNM4 and Sudan Red staining and respective quantification in mice fed (B) a 0.1% MCD diet (0.1% MCDD) or (C) a CD-HFD, and treated with Cnnm4 siRNA (siCnnm4) or an unrelated control (siCtrl). Scale bar corresponds to 50 l m. *p<0.05, **p<0.01, and ***p<0.001 vs. Ctrl/SC diet; # p<0.05, ## p<0.01, and ### p<0.001 vs. MCD + siCtrl/0.1% MCDD + siCtrl/CDHFD + siCtrl. CD-HFD, choline-deficient high-fat diet; MCD, methionine and choline-deficient; MCDD, MCD diet; NASH, non-alcoholic steatosis; SC diet, regular diet; siRNA, small interfering RNA. 36 Journal of Hepatology 2021 vol. 75 j34–45 Research Article NAFLD and Alcohol-Related Liver Diseases mRNA was evaluated, confirming the specific and effective silencing of Cnnm (Fig. S2A) and eliminating the possibility of Prl regulation when targeting Cnnm4 (Fig. S2B). Additionally, CNNM4 silencing in THLE-2 cells also reduced MCD-induced lipid accumulation (Fig. S2C and D). Taking into consideration the CNNM4 overexpression observed in NASH, and the siCnnm4-derived reduction of lipid accumulation, the therapeutic potential of silencing Cnnm4 in vivo was explored. Mice were fed a 0.1% MCDD to induce steatosis development, increased oxidative stress, inflammation, and fibrosis in a short period of time. 23 At 2 weeks, by which steatosis 21 and CNNM4 overexpression are observable (Fig. 1E), 0.1% MCDD-fed mice were separated into 2 groups and treated repeatedly with either an siRNA against Cnnm4 (0.1% MCDD + siCnnm4) or an unrelated control (0.1% MCDD + siCtrl), using Invivofectamine 3.0 ® . The mice were sacrificed at the fourth week. Interestingly, the mice fed the 0.1% MCDD showed hepatic CNNM4 overexpression, whereas Cnnm4 silencing reduced dietinduced steatosis (Fig. 2B and Fig. S2E). Alpha-smooth muscle actin ( a SMA) staining revealed that fibrosis, another NASH hallmark, was induced by 0.1% MCDD and attenuated by siCnnm4 (Fig. S2F). Furthermore, the treatment also reduced serum alanine aminotransferase (ALT) levels, a marker of liver damage (Fig. S2G). In a different NASH murine model, mice were fed a CD-HFD, as this leads to a pattern of pathology more similar to that observed in humans. 24 Although studies using this model tend to be performed for longer than 6 weeks, 24 our group previously showed that mice developed early NASH phenotypes comprising weight gain, steatosis, and inflammation as early as this point in time. 21 After 3 weeks on the diet, at which point CNNM4 was already overexpressed (Fig. 1F), the CD-HFD-fed group was divided and treated with siCnnm4 (CD-HFD + siCnnm4) or siCtrl (CD-HFD + siCtrl). They were sacrificed after 6 weeks and, similarly to in the previous results, the mice fed the CD-HFD showed hepatic CNNM4 overexpression, while the siRNA therapy reduced steatosis (Fig. 2C and Fig. S2H) and fibrosis development (Fig. S2I). Serum ALT levels remained unaltered (Fig. S2J). G Healthy liver Other Mg2+ exporters Other Mg2+ exporters Other Mg2 + exporters NASH liver Mg2+ Mg 2+ Mg2+ Mg2+ Mg2+ Mg2+ siCnnm4 siCnnm4-treated liver Cnnm4 Cnnm4 F E 2.0 Magnesium in culture medium (mg/dl) 0.0 0.5 1.0 1.5 siCnnm1 siCnnm2 siCnnm3 siCtrl 0.0 0.2 0.4 0.6 siCnnm1 siCnnm2 siCnnm3 siCtrl Non-targeted fluorescence ratio (a.u.) 0.0 0.2 0.4 0.6 siCnnm1 siCnnm2 siCnnm3 siCtrl Mitochondrial-targeted fluorescence ratio (a.u.) D 0.0 0.3 0.6 0.9 1.2 siCtrl siCnnm4 Magnesium in culture medium (mg/dl) # C Mitotracker REDMerge siCnnm4 siCtrl Non-targeted labeling Mitotracker RED Merge siCnnm4 siCtrl Mitochondrial targeted labeling 0.00 0.15 0.30 0.45 0.60 Non-targeted fluorescence ratio (a.u.) # siCtrl siCnnm4 0.00 0.15 0.30 0.45 Mitochondrial-targeted fluorescence ratio (a.u.) siCtrl siCnnm4 ## B MagS Non targeted MagS-TPP Mitochondria targeted A 0.0 0.3 0.6 0.9 1.2 0.1% MCDD siCnnm4 # siCtrl SC diet Magnesium in serum (mg/dl) 0.0 0.5 1.0 1.5 2.0 CD-HFD siCnnm4 # ** siCtrl SC diet Magnesium in serum (mg/dl) Fig. 3. Magnesium distribution after specific silencing of Cnnm4.(A) Magnesium in serum from mice fed a 0.1% MCDD or CD-HFD with specificCnnm4 silencing (siCnnm4) compared with non-treated (siCtrl) mice. (B) Biochemical structure of non-targeted MagS and MagS-TPP probes. (C) Micrographs and relative intracellular magnesium levels and (D) extracellular magnesium levels in primary hepatocytes treated with an siRNA against Cnnm4 (siCnnm4). Scale bar corresponds to 100 l m. (E) Intracellular magnesium and (F) extracellular magnesium levels in primary hepatocytes treated with siCnnm1-3. (G) Schematic representation of CNNM4-dependent magnesium fluctuations in liver and in circulation **p<0.01 vs. SC diet; # p<0.05, and ## p<0.01 vs. 0.1% MCDD + siCtrl/CD-HFD + siCtrl/siCtrl. CD-HFD, choline-deficient high-fat diet; CNNM4, cyclin M4; MagS, magnesium-specific; MagS-TPP, mitochondrion-targeted magnesium-specific; MCDD, methionine and choline deficient diet; SC diet, regular diet; siRNA, small-interfering RNA. Journal of Hepatology 2021 vol. 75 j34–45 37 CNNM4 acts in the liver as a magnesium exporter Despite the magnesiotropic role of CNNM4 in kidney epithelia, 25 its physiological function in the liver remains largely unknown. The possible relationship between increased serum Mg 2+ levels and hepatic CNNM4 overexpression (Fig. 1A and D) led us to anticipate a modulation of the cation in the studied NASH animal models. The measurement of Mg 2+ in the serum revealed an increase in CD-HFD-fed mice and a decrease under siCnnm4 treatment in both the CD-HFD and 0.1% MCDD models (Fig. 3A). To confirm the hypothesis of CNNM4 being a hepatic Mg 2+ exporter, CNNM4-related Mg 2+ flux in hepatocytes was assessed in vitro. Fluorescent staining with the ratiometric probe MagS was used to estimate the relative levels of cytosolic free Mg 2+ in live cells treated with siCnnm4. MagS is an analogue of MagFURA-2, developed by the Buccela group, 26 which displays similar metal recognition but enhanced optical properties. Furthermore, taking into consideration the key role of mitochondria in hepatocyte function, 27,28 we sought to explore the mitochondrial Mg 2+ levels in the aforementioned siCnnm4 conditions. For this purpose, we developed MagS-TPP, a targeted variant functionalised with a phosphonium group for delivery to the mitochondrial matrix (Fig. 3B). The new dye exhibits a selectivity profile similar to MagS and binds to Mg 2+ with an apparent dissociation constant suitable for the detection of typical intracellular concentrations of free Mg 2+ (Fig. S3). Mitochondrionand non-targeted fluorescent indicators were applied, revealing an increase in both cytosolic and mitochondrial free Mg 2+ levels in cells with reduced Cnnm4 expression (Fig 3C), combined with a decrease in the cation in the extracellular medium (Fig 3D). The silencing of other Cnnm family members did not lead to significant alterations in intraor extracellular Mg 2+ (Fig. 3E and F), thus eliminating their possible contribution to Mg 2+ homeostasis in the hepatocyte. These results support the notion that the observed decrease in serum Mg 2+ of mice treated with siCnnm4 could be a consequence of its accumulation in the liver (Fig. 3G). CNNM4-mediated magnesium accumulation reduces lipid content The association between CNNM4 and magnesium efflux prompted us to characterise the contribution of Mg 2+ homeostasis to NASH development. The relative content of the cation in primary hepatocytes under MCD conditions was determined, showing an MCD-induced decrease in Mg 2+ and an increase upon silencing Cnnm4 (Fig. 4A). Taking the latter into consideration, a possible inverse relationship between hepatic Mg 2+ and lipid content was investigated. Magnesium depletion in the cell medium (0 mM Mg 2+ ) resulted in an increased hepatocyte lipid content, while silencing Cnnm4 reduced this effect (Fig. 4B). In line with the results of a clinical trial addressing the beneficial properties of magnesium, 4 Mg 2+ supplementation (5 mM Mg 2+ ) reverted MCD-induced lipid accumulation in hepatocytes without effects under normal conditions (1 mM Mg 2+ )(Fig. S4A). Neither Mg 2+ depletion nor supplementation affected Cnnm4 expression (data not shown). To eliminate the possibility of other Mg 2+ transporters contributing to siCnnm4-induced lipid reduction, their expression was characterised under various conditions. We observed an E Intensity of lipid bodies/ cell (fold-change) 0 1 2 3 4 5 Empty vector CNNM4 vector 1 mM Mg2+ 5 mM Mg2+ *** 1 mM Mg2+ *** 5 mM Mg2+ Empty vector CNNM4 vector BODIPY 1 mM Mg2+ 5 mM Mg2+ 1 mM Mg2+ 5 mM Mg2+ D 0 20 40 60 80 Empty vector CNNM4 vector *** Relative CNNM4 mRNA expression (fold-change) C 0.0 0.1 0.2 0.3 0.4 0.5 Non-targeted fluorescence ratio (a.u.) Empty vector ** CNNM4 vector 0.0 0.1 0.2 0.3 0.4 0.5 ** Mitochondrial-targeted fluorescence ratio (a.u.) Empty vector CNNM4 vector 0 1 2 3 4 5 6 7 *** ## **** B BODIPY siCnnm4siCtrlSC diet 0 mM Mg2+ media 0 mM Mg2+ siCnnm4 siCtrl Ctrl Intensity of lipid bodies/ cell (fold-change) A * * ### MCD siCtrl Ctrl 0.0 0.2 0.4 0.6 Non-targeted fluorescence ratio (a.u.) * ### * MCD siCtrl Ctrl 0.0 0.2 0.4 0.6 Mitochondrial-targeted fluorescence ratio (a.u.) siCnnm4 siCnnm4 Fig. 4. Modulation of intracellular lipid content by CNNM4. (A) Relative intracellular magnesium determination by MagS and MagS-TPP staining in primary hepatocytes under MCD stimulation and treated with a Cnnm4 siRNA (siCnnm4). (B) BODIPY staining micrographs and quantification in murine hepatocytes maintained for 24 h under magnesium depletion (0 mM Mg 2+ ) and transfected with siCnnm4. (C) Relative intracellular magnesium determination and (D) Cnnm4 mRNA levels in primary hepatocytes transfected for 6 h with an empty or CNNM4 vector. (E) BODIPY staining micrographs and respective quantification in isolated mouse hepatocytes transfected for 6 h with an empty/CNNM4 expression vector and stimulated for 24 h with magnesium (5 mM Mg 2+ )vs. a control group (1 mM Mg 2+ ). Scale bar corresponds to 100 l m. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. Ctrl/Empty vector; ## p<0.01 and ### p<0.001 vs. MCD + siCtrl/0 mM Mg 2+ + siCtrl. CD-HFD, choline-deficient high-fat diet; CNNM4, cyclin M4; MagS, magnesium-specific; MagS-TPP, mitochondrion-targeted magnesium-specific; MCD, methionine and choline deficient; siRNA, small interfering RNA. 38 Journal of Hepatology 2021 vol. 75 j34–45 Research Article NAFLD and Alcohol-Related Liver Diseases increase in the expression of Transient Receptor Protein Melastatin 6(Trpm6)(Fig. S4B), suggesting that the lipid reduction may also be the result of increased Mg 2+ entry. As there is no commercial inhibitor for TRPM6, its isoform 7 was inhibited with 2aminoethyl diphenyl borinate (2-APB) 29 to further investigate the contribution of Mg 2+ entry. We observed that the lipid accumulation induced by inhibiting Mg 2+ import was normalised by siCnnm4 (Fig. S4C). Finally, the functional relationship between cellular Mg 2+ levels and steatosis was investigated by overexpressing CNNM4 via transient transfection, which reduced Mg 2+ levels (Fig. 4C and D) and raised lipid contents in hepatocytes (Fig. 4E). Remarkably, Mg 2+ supplementation did not attenuate CNNM4-induced lipid accumulation (Fig. 4D and E). Endoplasmic reticulum and oxidative stress are reduced by silencing cyclin M4 Considering that ROS production and inflammation are major drivers of NASH progression, 10,28 mitochondrial ROS were assessed in primary hepatocytes, showing a reduction in MCDinduced ROS production upon silencing Cnnm4 (Fig. 5A). The development of ER stress is also considered a ‘second hit’, linked to oxidative stress 30 and with an existing Mg 2+ flux between the ER and mitochondria. 31 Thus, we hypothesised that ER integrity might be affected by CNNM4-induced Mg 2+ fluctuations in NASH. Therefore, cytosolic calcium (Ca 2+ ), an ER-stress indicator, was quantified in primary hepatocytes. The increased [Ca 2+ ] cytosol observed under MCD stimulation and siRNA-derived attenuation (Fig. 5B), together with the partial co-localisation of Mg 2+ and ER (Fig. S5A), suggested a protective effect of silencing Cnnm4, not only for mitochondria, but also for the ER. This was further addressed by characterising the release of Ca 2+ from the ER by stimulating primary hepatocytes with ATP, reported to promote the P2Y receptor-mediated release of Ca 2+ into the cytosol. 32 Interestingly, an MCD-induced decreased capacity and then normalisation upon silencing Cnnm4 were observed (Fig. 5C), while ER labelling was decreased under the MCD and recovered by Cnnm4 siRNA (Fig. 5D). The relationship between ER stress and hepatic lipid content was elucidated by treating primary G 0 2 4 5 10 15 0 1 2 3 4 Relative protein expression normalized (fold-change) p-eIF2 α/ elF2α ** * GRP78/ GAPDH * # *** ### **** XBP1s/ GAPDH ** ### **** * p-AMPK/ AMPK p-ERK 1/2/ ERK 1/2 p-S6/ S6 p-eIF2 α/ elF2α GRP78/ GAPDH XBP1s/ GAPDH p-AMPK/ AMPK p-ERK 1/2/ ERK 1/2 p-S6/ S6 * # ## CD-HFD + siCtrl SC diet CD-HFD + si Cnnm4 0.1%MCDD + siCtrl SC diet 0.1%MCDD + si Cnnm4 F 0 20 40 60 DHE staining (a.u.) ## 0.1%MCDD 0.1%MCDD DHE siCnnm4siCtrl siCnnm4 siCtrl E 0 2 4 6 8 Vehicle Tunicamycin 0.5 μg/ml Tunicamycin 1 μg/ml ** ## #* # MCD siCnnm4 siCtrl Ctrl Intensity of lipid bodies/ cells (fold-change) ΠΠ Π D 0.0 0.5 1.0 1.5 2.0 * # MCD siCnnm4 siCtrl Ctrl Relative ER staining (fold-change) ER-tracker Ctrl MCD + siCnnm4MCD + siCtrl A C 0.0 0.5 1.0 1.5 2.0 2.5 ## MCD siCnnm4 siCtrl Ctrl ER Ca2+ release capacity (fold-change) B 0 50 100 150 200 250 *** ### MCD siCnnm4 siCtrl Ctrl Cytosolic Ca2+ (nM) 0.0 0.5 1.0 1.5 2.0 *** ### MCD siCnnm4 siCtrl Ctrl Relative mitochondrial ROS (fold-change) Fig. 5. SpecificCnnm4 silencing reduces oxidative and ER stress in in vitro and in vivo NASH models. (A) Relative mitochondrial ROS levels, (B) cytosolic Ca 2+ levels, (C) Ca 2+ release capacity of ER over time and (D) micrographs of ER-tracker red staining and quantification in primary hepatocytes cultured for 24 h in a MCD medium and transfected with a Cnnm4 (siCnnm4) or control (siCtrl) siRNA. Scale bar corresponds to 100 l m. (E) BODIPY determination in primary hepatocytes treated for 24 h with tunicamycin, MCD, or Ctrl medium and treated with siCnnm4 or siCtrl. (F) Representative micrographs (scale bar corresponds to 50 l m) and DHE staining for liver from mice fed a 0.1% MCDD and treated with siCnnm4 or siCtrl. (G) Western blot analysis of binding immunoglobulin protein (BIP/ GRP78), x-box binding protein 1 isoform s (XBP1s), phospho Ser51 -eukaryotic initiation factor 2 a (eIF2 a ), phospho Thr172 -AMP dependent protein kinase (AMPK), phospho Thr202/Thyr204 -ERK1/2, and phospho Ser235/236 -S6 ribosomal protein (S6). Mouse livers from different groups were compared: healthy (SC diet), 0.1% MCDD, or CD-HFD treated with siCnnm4 or siCtrl. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. Ctrl/Ctrl + vehicle/SC diet; # p<0.05, ## p<0.01, and ### p<0.001 vs. MCD + siCtrl/0.1% MCDD + siCtrl/MCD + vehicle + siCtrl/CDHFD + siCtrl; p p<0.05 and pp p<0.01 for vehicle vs. tunicamycin. CD-HFD, choline-deficient high-fat diet; CNNM4, cyclin M4; DHE, dihydroxyethyl; ER, endoplasmic reticulum; MCD, methionine and choline deficient; MCDD, MCD diet; NASH, non-alcoholic steatohepatitis; ROS, reactive oxygen species; SC, regular diet; siRNA, small interfering RNA. Journal of Hepatology 2021 vol. 75 j34–45 39 hepatocytes with tunicamycin, an ER-stress inducer. 33 A dosedependent lipid accumulation was observed after 24 h of treatment, while siCnnm4 led to a decrease in lipid content (Fig. 5E and Fig. S5B). When characterising ROS production in vivo,siCnnm4-treated mice showed reduced dihydroxiethidium (DHE) staining (Fig. 5F), probably as a result of the oxidative activity measured by various pathways (Fig. S5C–F). Although oxidative stress was not determined in the study with CD-HFD mice, the observed reduction in fatty acid oxidation (Fig. S5G) is consistent with a ROS reduction. The regulation of the oxidative stress response prompted us to evaluate ER stress in the in vivo models of NASH using different markers, 34 demonstrating upregulation in both NASH models and reduction under siCnnm4, suggesting that the latter diminished the ER-stress response (Fig. 5G and Fig. S5G). ER-stress associated signalling pathways were also evaluated, showing a decreased activation of S6 ribosomal protein (S6) by phosphorylation. 35 CNNM4 inhibition increases MTP activity, promoting VLDL secretion The formation of pre-VLDL, the first step of VLDL assembly, takes place in the ER. 36 During this step, a small number of triglycerides (TGs) are associated with an apolipoprotein B (APOB) molecule and embedded in a phospholipid monolayer by the microsomal triglyceride transfer protein (MTP). 36 The alterations of ER integrity previously observed under MCD and the reversion of such by siCnnm4 suggested a possible modulation of MTP activity. Remarkably, the protein was increased upon silencing Cnnm4 in both the primary hepatocytes (Fig. 6A) and in vivo rodent NASH models (Fig. 6B). The serum TGs of mice fed a 0.1% MCDD were found to decrease, owing to intrahepatic lipid accumulation, and were partially restored by Cnnm4 siRNA (Fig. S6A), suggesting an improved VLDL secretion. Therefore, we determined the relative APOB100 concentrations in the serum, an indicator of circulating lipoprotein particles, observing an increase under siCnnm4 F BODIPY siCtrlsiMtp MCD siCnnm4Ctrl siCnnm4Ctrl 0 1 2 3 4 * * **** * ## **** MCD + siCnnm4 Ctrl siCnnm4 MCD siCtrl siMtp Intensity of lipid bodies/ cells (fold-change) ΠΠΠΠ ΠΠΠΠ Π E BODIPY VehicleLomitapide MCD siCnnm4siCtrlCtrl 0 2 4 6 8 **** **** **** **** ### MCD + siCnnm4 MCD + siCtrl Ctrl Intensity of lipid bodies/ cells (fold-change) Vehicle Lomitapide ΠΠΠΠ 0 5 10 15 p = 0.1 D 0.1%MCDD + siCnnm4 0.1%MCDD + siCtrl SC diet 0-4 h 0-2 h # Liver TG secretion rate (mM/kg/h) * # C SC diet CD-HFD + siCnnm4 CD-HFD + siCtrl 0.0 0.5 1.0 1.5 2.0 * # 0.1%MCDD + siCnnm4 0.1%MCDD + siCtrl SC diet 0.0 0.5 1.0 1.5 2.0 Relative ApoB100 in serum (fold-change) B 0.0 0.5 1.0 1.5 2.0 * # SC diet CD-HFD + siCnnm4 CD-HFD + siCtrl 0.0 0.5 1.0 1.5 2.0 * ## 0.1%MCDD + siCnnm4 0.1%MCDD + siCtrl SC diet Relative MTP activity (fold-change) A 0.0 0.5 1.0 1.5 2.0 2.5 **** #### MCD + siCnnm4 MCD + siCtrl Ctrl Relative MTP activity (fold-change) Fig. 6. Inhibition of CNNM4 expression promotes lipid export by activating MTP activity. (A) Relative MTP activity in primary hepatocytes cultured for 24 h in a MCD medium and transfected with a Cnnm4 siRNA (siCnnm4) or unrelated control (siCtrl). (B) Relative MTP activity in livers from mice fed a 0.1% MCDD or a CDHFD and treated with siCnnm4 or siCtrl. (C) Relative apolipoprotein B100 (APOB100) levels in sera from mice fed a 0.1% MCDD or CD-HFD and treated with siCnnm4 or siCtrl. (D) Liver TG secretion rate in mice fed a 0.1% MCDD, treated with siCnnm4 or siCtrl and after poloxamer P407 administration. BODIPY staining micrographs and quantification of primary hepatocytes cultured under MCD and treated with siCtrl or siCnnm4 and (E) a vehicle (DMSO) or 600 nM lomitapide for 24 h or (F) an siRNA against Mtp (siMtp). Scale bar corresponds to 100 l m. *p<0.05 and ****p<0.0001 vs. SC diet/Ctrl + vehicle/Ctrl + siCtrl; # p<0.05, ## p<0.01, ### p<0.001, and #### p<0.0001 vs. 0.1% MCDD + siCtrl/CD-HFD + siCtrl/MCD + vehicle + siCtrl/MCD + siCtrl; p p<0.05 and pppp p<0.0001 for vehicle/siCtrl vs. lomitapide/siMtp. CD-HFD, choline-deficient high-fat diet; MCD, methionine and choline deficient; MCDD, MCD diet; MTP, microsomal triglyceride transfer protein; SC, regular diet; siRNA, small interfering RNA; TG, triacylglyceride. 40 Journal of Hepatology 2021 vol. 75 j34–45 Research Article NAFLD and Alcohol-Related Liver Diseases conditions (Fig. 6C and Fig. S6B). The contribution of increased lipid secretion mediated by VLDL was further confirmed by measuring APOB100 in serum extracted directly from the cava vein, with VLDLs as the main component, showing an increased APOB100 content (Fig. S6C). The hepatic TG secretion rate, as well as the VLDL content, were determined after administering poloxamer P407 to inhibit lipoprotein lipase. 37 Remarkably, both the secretion rate and the VLDL lipid content tended to increase upon silencing Cnnm4 (Fig. 6D and Fig. S6D). MTP was then inhibited in primary hepatocytes by 24-h lomitapide incubation, a selective inhibitor, 38 or with a specific siRNA (siMtp). As expected, the lipid reduction observed upon CNNM4 knockdown was absent under lomitapide stimulation (Fig. 6E) or Mtp silencing conditions (Fig. 6F and Fig S6E). The possible atherogenic secondary effects are a concern when it comes to promoting VLDL secretion. Furthermore, when determining the Mg 2+ content in isolated VLDL, the observed restoration in the group treated with the Cnnm4 siRNA (Fig. S7A) suggested a possible impact on the WAT. The measurement of the relative fatty acid oxidation (FAO) capacity of WAT revealed a tendency to increase with the siRNA treatment (Fig. S7B), together with an increased expression of genes involved in lipid oxidation, mitochondrial biogenesis, and thermogenesis (Fig. S7C). This result was corroborated in primary WAT adipocytes, where Mg 2+ supplementation increased glycerol production, an indicator of lipolysis without subsequent non-esterified fatty acid (NEFA) production (Fig. S7D). The same result was observed with conditioned medium obtained from primary hepatocytes cultured under MCD and siCnnm4 conditions (Fig. S7E). The siRNA conjugation with GalNAc offers a potential therapy Considerable progress has recently been made in the development of oligonucleotide-based therapeutics, whereby the silencing of target genes is being explored in clinical studies. 39 To transfer the presented discoveries into a therapeutic approach suitable for clinical development, a newly identified Cnnm4 siRNA candidate was conjugated to an N-acetylgalactosamine (GalNAc) cluster that binds to the asialo-glycoprotein receptors predominantly expressed in hepatocytes. This technology offers a potentially safe, specific, and efficient mode of delivery for targeting therapeutic molecules to hepatocytes. 40 The specific inhibition of Cnnm4 was achieved in primary hepatocytes through receptor-mediated uptake by adding different doses of a GalNAc-conjugated siRNA against Cnnm4 (GalNAc siRNA) directly to the culture medium (Fig. 7A). The lipid accumulation induced by MCD medium was reduced by the GalNAc siRNA treatment (Fig. 7B), and Mg 2+ accumulation in hepatocytes was observed indirectly through its decrease in the extracellular medium (Fig. 7C). ROS overproduction upon MCD stimulation was also reduced by treating the hepatocytes with the conjugate (Fig. 7D). Finally, the effectiveness of Cnnm4 GalNAc siRNA was evaluated in vivo in mice fed a 0.1% MCDD for 6 weeks, leading to a more severe NASH phenotype. 21 The treatment was initiated at the 3-week point with a single injection of different doses (1 or 5 mg/kg) of Cnnm4 GalNAc siRNA (0.1% MCDD + GalNAc siRNA) or a control siRNA conjugate (0.1% MCDD + siCtrl). A specific and significant inhibition of Cnnm4 mRNA expression was detected in mice administered either 1 or 5 mg/kg (Fig. 7E), while the Mg 2+ levels in serum were reduced in line with a presumed hepatic Mg 2+ accumulation (Fig. 7F). Importantly, CNNM4 inhibition by GalNAc siRNA significantly reduced hepatic lipid accumulation and alleviated the inflammatory response and fibrosis development (Fig. 7G). Discussion The studies described herein pinpoint CNNM4 as a key regulator of Mg 2+ homeostasis in hepatocytes and a potential therapeutic target for NASH. To date, research performed on this cation in liver pathologies has shown a protective effect of Mg 2+ supplementation 4 and revealed Mg 2+ deficiencies in patients with cirrhosis or liver cancer. 18 Indeed, hypomagnesaemia is frequently observed in NASH comorbidities. 6,7 However, there is still a dearth of knowledge on the implications of Mg 2+ perturbations for the development of NASH, and nothing has been reported about magnesiotropic proteins and their modulation in the liver. Previous studies have mainly focused on PRL, the interacting partner of CNNM, associating its expression with poor prognosis in multiple cancer types including liver, 16,17 whereas studies of CNNMs have been limited to their role in transporting Mg 2+ across epithelia. 25 Herein, we demonstrate that CNNM4 is a contributor to NASH, as it is overexpressed in preclinical models and clinical samples of the pathology. The reduction in CNNM4 expression in THLE-2 cells upon inhibiting NEDDylation suggests the involvement of post-translational mechanisms in the modulation of the stability of CNNM4 during NASH. This is further confirmed by the reduction of MCD-induced lipid accumulation by the specific silencing of Cnnm4, which does not happen upon silencing other Cnnms. Regarding the effect of targeting Cnnm4 on NASH development, preclinical studies in rodent models showed promising effects of an siRNA-based therapy in mice fed either a 0.1% MCDD or CD-HFD. Significantly, both diets increase lipid accumulation and fibrosis development, but the siRNA therapy ameliorates both hallmarks of the disease. The role that CNNM4 may play in other cell populations such as hepatic stellate cells requires more research, as do the possible posttranscriptional and post-translational mechanisms that may contribute to CNNM4 overexpression. The role of the hepatocyte in NASH and its subsequent progression has been widely characterised. 27,28 The silencing of Cnnm4 may reduce oxidative activity, leading to reduced oxidative stress, which could contribute to a reduction in fibrosis development. Although the development of oxidative stress was not characterised in the CD-HFD in vivo study, the observed reduction in FAO and lipid contents prompts to expect a similar reduction. The relevance of the hepatocyte in NASH progression and the implications of modulating CNNM4 were further characterised in mice fed a 0.1% MCDD for 6 weeks. Herein, we also demonstrated that targeting CNNM4 ameliorated NASH, even that with a more severe phenotype. 21 To develop a possible therapeutic approach, an siRNA was conjugated with GalNAc allowing stable and liver-specific delivery, 40 with results similar to those observed when targeting Cnnm4 with the liposomal siRNA formulation after 4 weeks on the 0.1% MCDD. Although CNNM4 has been characterised as a Mg 2+ transporter in kidney epithelia, 41 the CNNM4-mediated flux of the cation in the liver remains unknown. Our research shows CNNM4 to be a Mg 2+ exporter in the hepatocyte, as its specific silencing increases intracellular Mg 2+ . Hepatic siCnnm4-induced Mg 2+ accumulation was characterised in all the preclinical studies through the quantification of the cation in serum, Journal of Hepatology 2021 vol. 75 j34–45 41