Investigating the regulatory mechanism of glucose metabolism by ubiquitin-like protein MNSFβ

Investigating the regulatory mechanism of glucose metabolism by ubiquitin-like protein MNSFβ | 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 Investigating the regulatory mechanism of glucose metabolism by ubiquitin-like protein MNSFβ Megumi Kono, Kyoko Yamasaki, Morihiko Nakamura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4720952/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Background Monoclonal nonspecific suppressor factor β (MNSFβ), a ubiquitously expressed member of the ubiquitin-like protein family, is associated with diverse cell regulatory functions. It has been implicated in glycolysis regulation and cell proliferation enhancement in the macrophage-like cell line Raw264.7. This study aims to show that HIF-1α regulates MNSFβ-mediated metabolic reprogramming. Methods and results In Raw264.7 cells, MNSFβ siRNA increased the oxygen consumption rate and ROS production but decreased ATP levels. Cells with MNSFβ knockdown showed a markedly increased ATP reduction rate upon the addition of oligomycin, a mitochondrial ATP synthase inhibitor. In addition, MNSFβ siRNA decreased the expression levels of mRNA and protein of HIF-1α—a regulator of glucose metabolism. Evaluation of the effect of MNSFβ on glucose metabolism in murine peritoneal macrophages revealed no changes in lactate production, glucose consumption, or ROS production. Conclusion MNSFβ affects both glycolysis and mitochondrial metabolism, suggesting HIF-1α involvement in the MNSFβ-regulated glucose metabolism in Raw264.7 cells. Ubiquitin-like protein MNSFβ HIF-1α Metabolism Metabolic reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Monoclonal nonspecific suppressor factor β (MNSFβ), a member of the ubiquitin-like protein family, is involved in various biological functions, such as cell proliferation, immunological responses, and apoptosis. MNSFβ presents a 57% amino acid sequence homology and 36% similarity with ubiquitin [ 1 ]. The C-terminal glycine doublet of ubiquitin, which participates in the isopeptide bond formation during conjugation, is a conserved sequence even in MNSFβ [ 2 ]. MNSFβ forms covalent bonds with certain lysines of specific target proteins, including endophilin Ⅱ and Bcl-G. While MNSFβ covalently attaches to endophilin Ⅱ and downregulates phagocytosis in macrophages [ 3 , 4 ], it also covalently binds to Bcl-G and markedly promotes apoptosis induced by lipopolysaccharide (LPS)/interferon γ (IFNγ) in macrophages [ 5 ]. Unlike ubiquitin, MNSFβ may not be involved in the degradation of targeted proteins. While MNSFβ regulates glycolysis, its effects is related to GLUT1 [ 6 ]. Hypoxia-inducible factor-1α (HIF-1α) controls GLUT1 expression in multiple cell lines, but whether MNSFβ affects HIF-1α remains unclear. The heterodimeric transcription factor HIF-1 increases the expression of enzymes implicated in glucose metabolism, such as hexokinase, phosphofructokinase-1, and pyruvate kinase M2 [ 7 ]. HIF-1 comprises two subunits: an oxygen-labile HIF-1α subunit and a constitutively stable HIF-1β subunit. HIF-1α has an oxygen-dependent degradation domain (ODDD) that binds the E3 ubiquitin ligase von Hippel Lindau protein (pVHL). Prolyl hydroxylases (PHDs) hydroxylate the two proline residues (Pro 402 , Pro 564 ) within the ODDD, which recruits pVHL, and HIF-1α is ubiquitinated and degraded by the 26S proteasome. In hypoxia, PHD inhibition impedes pVHL binding. This is followed by HIF-1α stabilization, nuclear translocation, dimerization with HIF-1β, coactivator recruitment, and binding to hypoxia response elements in the promoter region of target genes [ 7 , 8 ]. The asparaginyl hydroxylase factor-inhibiting HIF-1α (FIH) suppresses HIF-1α transcriptional activity. FIH causes hydroxylation of HIF-1α at the Asn 803 in the carboxy-terminal transactivation domain (C-TAD), which prevents HIF-1α binding to transcriptional coactivators, such as CREB-binding protein (CBP)/p300 [ 9 ]. FIH overexpression suppresses HIF-1α transcriptional activity [ 10 ]. The histone acetyltransferase CBP/p300 are relevant to HIF-1α/β heterodimer formation. Acetylated CBP/p300 interacts with the C-TAD of HIF-1α to enhance gene expression [ 8 ]. Certain cancer cells showed overexpressed CBP/p300 mRNAs [ 11 ]. Hypoxia, alongside many oncogenic and inflammatory stimuli, promotes HIF-1α activation and accumulation [ 12 , 13 ]. HIF-1α overexpression has been found in various cancer types and is related to tumor aggressiveness [ 13 – 15 ]. In this study, MNSFβ alters both glycolysis and mitochondrial metabolism, markedly affecting glucose metabolism and cytokine production, and HIF-1α may be involved in this regulatory mechanism. Materials and methods Antibodies and chemicals Antibodies used included anti-HIF-1α (Novus Biologicals, Littleton, CO, USA), anti-Acetyl-CBP (Lys1535)/p300 (Lys1499) (Cell Signaling Technology, Beverly, MA, USA), anti-FIH (Cell Signaling Technology), anti-PHD2 (GeneTex, San Antonio, TX), anti-β-actin (Medical & Biological laboratories), and peroxidase-conjugated rabbit IgG antibody (Cappel, Solon, OH, USA). Sigma (St. Louis, MO, USA) supplied LPS and oligomycin A. Cell culture, siRNAs, and transfection of cells The macrophage-like cell line Raw264.7 was obtained from ATCC (Manassas, VA). Cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin/streptomycin (1:100). siRNAs were purchased from Qiagen (Chatsworth, CA, USA). The target sequence of siRNAs was as follows: MNSFβ 5′-CCACCCTGCCATGCTAATAAA-3′ and the scrambled negative control 5′-AATTCTCCGAACGTGTCACGT-3′. siRNA transfection was performed with HiPerFect Transfection Reagent (Qiagen) following the manufacturer’s instructions. cDNAs and transfection cells cDNAs were prepared as previously described [ 2 ]. cDNA encoding MNSFβ was subcloned into the vector pcDNA3.1(+) (Invitrogen). DNA transfection was performed using Lipofectamine LTX reagent with Plus reagent (Invitrogen), following the manufacturer’s instructions. Cells were seeded and cultured until 70–80% confluent. Lipofectamine LTX and Plus reagent was mixed with cDNA. The DNA mixture was incubated for 5 min at room temperature to form a DNA-lipid complex and added to cells. The final volume of cDNA was 500 ng per well. Western blotting Protein samples were subjected to SDS-PAGE and electro-transferred from gels to polyvinylidene fluoride membranes. Membranes were blocked for 1 h at room temperature using Tris-buffered saline with 0.02% Tween 20 (TBST) and 5% skim milk or 5% bovine serum albumin and probed with primary antibodies overnight at 4 ℃ in blocking buffer. The membranes were washed four times for 5 min each with TBST and incubated with horseradish peroxidase secondary antibodies for 2 h at room temperature. The secondary antibodies were used at 1:15000 dilutions in blocking buffer. Membranes were washed four times for 5 min each with TBST and detected with ECL reagents (Amersham Biosciences) and analyzed with ImageJ software. RT-PCR RT-PCR was performed with Premix Taq (Ex Taq Version 2.0) (Takara) following the manufacturer’s instructions. The PCR conditions used were as follows: initial denaturation at 95 ℃ for 1 min, 40 cycles of 98 ℃ for 10 s, 55 ℃ for 30 s, and 72 ℃ for 1 min. Primers for HIF-1α were 5′-TGATGTGGGTGCTGGTGTC-3′ (sense) and 5′-TTGTGTTGGGGCAGTACTG-3′ (anti-sense). Primers for GAPDH were 5′-CAACTACATGGTTTACATGTTC-3′ (sense) and 5′-GCCAGTGGACTCCACGAC-3′ (anti-sense). We amplified GAPDH as an internal standard. PCR products were electrophoresed on 2% agarose gels with ethidium bromide and detected using a UV transilluminator. The signals were quantified with ImageJ software. ROS detection Cells were seeded at 2.0 × 10 4 cells per well in a 96-well microplate. Intracellular ROS was evaluated with a ROS Assay Kit -Highly sensitive DCFH-DA- (Dojindo) according to the manufacturer’s instructions. Cells were washed twice using Hanks’ Balanced Salt Solution (HBSS) (Sigma) and incubated with the highly sensitive DCFH-DA dye working solution at 37 ℃ for 30 min. The cells were washed twice, and each well was refilled with HBSS. Fluorescence was read using a microplate reader (Beckman Coulter). Electrophoretic mobility shift assay (EMSA) Cells were seeded at 1.0 × 10 6 cells per well in a 6-well plate. Nuclear extracts were prepared with EzSubcell Fraction (ATTO) according to the manufacturer’s instructions. HIF-1α binds to hypoxia response element (HRE) sequences within the promoter region. HIF-1α-DNA interactions were detected using biotin end-labeled DNA probes containing HRE. The sequence of the probe was 5′-CACCCCACCCCCGTGAGGAGGAGGGTGAGGAAAC-3′. The binding reaction was performed using a Gelshift Chemiluminescent EMSA kit (Active Motif) following the manufacturer’s instructions. The reaction products were loaded on a 6% polyacrylamide gel in 0.5% Tris borate/EDTA at 4 ℃, transferred to a nylon membrane, and fixed on the membrane by UV cross-linking. The biotin-labeled probe was detected using chemiluminescence. ATP measurement Cells were seeded at 1.0 × 10 4 cells per well in a 96-well microplate. Intracellular ATP was detected using an ATP Assay Kit-Luminescence (Dojindo) based on the manufacturer’s instructions. Cells were incubated with working solution at 25 ℃ for 10 min. Luminescence was read with a microplate reader (Promega). The ATP concentrations were calculated from a calibration curve generated from the ATP standard. OCR detection Cells were seeded at 1.0 × 10 4 cells per well in a 96-well microplate. The mitochondrial oxygen consumption rate (OCR) was evaluated using an Extracellular OCR Plate Assay Kit (Dojindo) based on the manufacturer’s instructions. Cells were incubated with working solution at 37 ℃ for 30 min. Then, one drop of mineral oil was added to each well, and the cells were incubated at 37 ℃ for 5 min. Fluorescence was read using a microplate reader at 10-min intervals for 200 min. The OCR was calculated from the intensity using the calculation sheet provided by the manufacturer. Glucose and lactate assay Cells were seeded at 2.0 × 10 5 cells per well in a 24-well plate. Glucose and lactate in the supernatant were detected using a glucose assay kit-WST (Dojindo) and glycolysis cell-based assay kit (Cayman) based on the manufacturer’s instructions, respectively. The concentrations were calculated from a calibration curve generated from the standard. The glucose assay had a detection limit of 0.02 mmol/l glucose. Proteome profiler mouse cytokine array Cells were seeded at 1.0 × 10 5 cells per well in a 24-well plate. The cytokine expressions in cell culture supernatants were detected using a Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems) following the manufacturer’s instructions. Cell culture supernatants were cleared of particulates by centrifugation for 5 min at 1,000 ×g and then used for the cytokine array. The signal was quantified using ImageJ software. Metabolome analysis The cells were washed with PBS and lysed using 70% (v/v) methanol. Metabolome analysis by gas chromatography-mass spectrometry (GC/MS) was performed at the Integrated Center for Mass Spectrometry, Graduate School of Medicine, Kobe University. Isolation of peritoneal macrophages Peritoneal macrophages were harvested from female BALB/c mice (Japan SLC, Inc.) 4 days after intraperitoneal injection of 1.5 ml of sterile 4% Brewer’s thioglycollate medium (Difco Laboratories). The cells were collected by centrifugation at 400 ×g for 5 min, washed and seeded in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The collected macrophages were used in various experiments. All animal studies were approved by the Animal Care Committee of Shimane University. Statistical analysis Statistical significance was analyzed by Student’s t-test and expressed as P values. p < 0.05 was considered statistically significant. Results MNSFβ siRNA increased oxidative phosphorylation in Raw264.7 cells We most recently reported that MNSFβ regulates the glycolytic system [ 6 ]. Mitochondrial respiration is closely involved in the mechanism of intracellular glycometabolism. For instance, glycolytic suppression enhances mitochondrial oxidative phosphorylation (OXPHOS) [ 16 , 17 ]. Thus, we examined the effect of MNSFβ on metabolism in mitochondria. First, we determined whether glycolytic suppression induced by MNSFβ siRNA affects OXPHOS in Raw264.7 cells. We next measured the OCR, an indicator of metabolic activity in mitochondria, and reactive oxygen species (ROS), a byproduct of oxidative phosphorylation. After 48 h transfection, MNSFβ siRNA significantly increased both OCR and ROS production in Raw264.7 cells (Fig. 1 a, 1 b). Conversely, 18 h of MNSFβ overexpression reduced ROS production, strongly indicating that this ubiquitin-like protein positively regulates ROS production (Fig. 1 c). In addition, we measured ATP levels in Raw264.7 cells transfected with MNSFβ siRNA and treated with the ATP synthase inhibitor oligomycin. MNSFβ siRNA transfection markedly reduced ATP levels. Upon oligomycin treatment, cells with MNSFβ knockdown exhibited a greater ATP reduction rate than the controls (Fig. 1 d), suggesting that MNSFβ siRNA treatment may alter the main ATP source from glycolysis to OXPHOS. MNSFβ affects HIF-1α expression and HIF-1α–DNA interactions in Raw264.7 cells In macrophages, HIF-1α disruption facilitates OXPHOS and decreases the glycolysis level [ 18 ]. MNSFβ siRNA can inhibit the expression of GLUT1, GLUT3, and the key molecules in glycolysis [ 6 ]. HIF-1α enhances glycolysis by inducing the expression of glycolytic enzymes and glucose transporters. To examine whether HIF-1α mediates MNSFβ-regulated glycolysis, we analyzed the expression of mRNA and protein of HIF-1α. After 48 h transfection of MNSFβ siRNA, HIF-1α mRNA and protein expressions were significantly decreased (Fig. 2 a). Evaluation of the DNA binding property of HIF-1α by EMSA revealed that MNSFβ siRNA impaired DNA–HIF-1α interaction (Fig. 2 b). These results strongly indicate that MNSFβ knockdown-triggered reduction in HIF-1α transcriptional activity may be implicated in glycolysis suppression. MNSFβ siRNA decreases acetyl-CBP/p300 expression in Raw264.7 cells The stability and transcriptional activity of HIF-1α are regulated by its post-translational modifications such as ubiquitination, hydroxylation, and acetylation [ 7 ]. Therefore, we examined whether MNSFβ affects factors involved in HIF-1α stabilization and complex formation. After 48 h transfection, MNSFβ siRNA reduced acetyl-CBP/p300 expression (Fig. 3 a) without altering that of FIH and PHD2 (Fig. 3 b, 3 c). These results indicate that MNSFβ siRNA-induced decrease in HIF-1α transcriptional activity is affected not only by HIF-1α but also by decreased expression of acetyl-CBP/p300, which suppresses glycolysis through decreased expression of HIF-1α target genes. MNSFβ siRNA changes intracellular metabolite levels in Raw264.7 cells In cancer cells, HIF-1α activation diminishes the tricarboxylic acid (TCA) cycle and OXPHOS [ 19 , 20 ]. To investigate further MNSFβ knockdown-triggered glycolytic changes, intermediate metabolites were detected by GC-MS. After 48 h transfection, MNSFβ siRNA reduced pyruvate and lactate, TCA cycle intermediates, especially citrate (Table 1 ). Among the amino acids, proline and branched chain amino acids were greatly reduced, while only serine was increased. These data suggest that MNSFβ affects overall glucose metabolism and partially alters amino acid metabolism as well. Table 1 Metabolome analysis Metabolite name MNSFβ siRNA /Control siRNA Metabolite name MNSFβ siRNA /Control siRNA Coniferyl alcohol 8.020 α-ketoglutarate 0.706 GABA 7.347 Succinate 0.682 2,3-Bisphospho-glycerate 2.348 Threonine 0.646 Sorbitol 6-phosphate 1.797 Mannitol 0.639 Serine 1.569 Cadaverine 0.600 Gluconic acid 1.567 beta-Alanine 0.593 Elaidic acid 1.494 Hypotaurine 0.586 Stearic acid 1.312 Alanine 0.586 Ethanolamine 1.283 Pyruvate 0.568 Glucosamine 6-phosphate 1.261 Terephthalic acid 0.556 Palmitoleic acid 1.139 Glycine 0.554 N-Methylethanolamine 1.063 Norleucine 0.544 Aspartate 1.062 Phenylalanine 0.537 n-Butylamine 1.057 Glycerol 0.531 Oxalic acid 0.991 trans-4-Hydroxyproline 0.528 Glutamate 0.911 Urea 0.509 Malate 0.858 Pyroglutamate 0.506 Inositol 0.837 Pyrophosphoric acid 0.494 2-Hydroxypyridine 0.836 Leucine 0.466 Oxamic acid 0.830 Valine 0.447 2-Aminoisobutyrate 0.815 Sorbose 0.428 n-Propylamine 0.800 Isoleucine 0.424 beta-Hydroxybutyrate 0.773 Citrate 0.416 1,5-Anhydro-D-glucitol 0.770 Proline 0.383 Fumarate 0.721 Lactate 0.326 Tagatose 0.711 Galactose 0.289 Raw264.7 cells were treated with siRNAs for 48 h, and metabolites were detected by GC/MS. MNSFβ changes the pattern of cytokine production in Raw264.7 cells HIF-1α can alter cytokine expression, which markedly contributes to the tumor microenvironment [ 21 , 22 ]. Metabolites can affect cytokine production [ 23 ]. Since MNSFβ knockdown altered HIF-1α expression as described above, we investigated the relationship between MNSFβ and cytokine expression. After 48 h transfection, the cells were treated with 1 µg/ml LPS for 24 h, and cytokines in the supernatants were analyzed by antibody arrays, resulting in 16 cytokines (Fig. 4 ). Among them, CCL2 and IL-10 were reported to be decreased by HIF-1α inhibition [ 24 ]. ICAM-1 is related to HIF-1α and GLUT1 expression during endotoxemia [ 25 ]. Thus, MNSFβ may modulate its effects on cytokine expression via HIF-1α. MNSFβ affects HIF-1α expression in peritoneal macrophages To further investigate the effect of MNSFβ on glucose metabolism, murine peritoneal macrophages were used. MNSFβ knockdown decreased HIF-1α mRNA. Unlike Raw264 cells, in unstimulated peritoneal macrophages, the expression level of HIF-1α protein is very low [ 26 ]. However, since the expression of HIF-1α protein is increased by LPS stimulation [ 26 ], we examined the effect of MNSFβ on HIF-1α expression in LPS-stimulated murine peritoneal macrophages. MNSFβ knockdown markedly inhibited LPS-stimulated increase in HIF-1α protein expression (Fig. 5 b). Since our previous study showed glucose consumption and lactate secretion by MNSFβ knockdown in Raw264.7 cells [ 6 ], we performed the same verification in peritoneal macrophages. Unlike Raw264.7 cells, peritoneal macrophages treated with MNSFβ siRNA showed no difference in glucose and lactate levels in the culture supernatant or in ROS production (Fig. 5 c, 5 d and 5 e). Discussion In macrophages, metabolic characteristics are closely related to phenotypes and functions [ 27 ]. MNSFβ is involved in glycolytic regulation [ 6 ]. Therefore, in this study, we first focused on metabolic changes in mitochondria. MNSFβ knockdown increased OCR and ROS production in Raw264.7 cells (Fig. 1 a, 1 b), which showed a markedly decreased ATP level upon oligomycin treatment (Fig. 1 d). In addition, MNSFβ siRNA reduces lactate in the culture supernatant and glucose consumption in Raw264.7 cells [ 6 ], suggesting that MNSFβ knockdown shifts the primary ATP production pathway from glycolysis to OXPHOS. Many cancer cells rely on the glycolytic system for much of their ATP production, even though mitochondrial function is maintained under aerobic conditions [ 28 ]. This phenomenon is widely known as the Warburg effect, in which HIF-1α is closely involved [ 29 ]. HIF-1α promotes the glycolytic system and causes metabolic reprogramming by inhibiting pyruvate influx into the TCA cycle and OXPHOS [ 19 ]. Pyruvate dehydrogenase kinase 1 (PDK1) is a particularly important enzyme in the metabolic shift from mitochondria to the glycolytic system, and PDK1 is a target gene of HIF-1α. Increased PDK1 by HIF-1α activation inhibits pyruvate dehydrogenase (PDH), thereby reducing mitochondrial respiration and ROS production and preventing cell death due to excess ROS [ 20 , 30 ]. Thus, the results shown in Fig. 1 are consistent with reports that MNSFβ knockdown reduces PDK1 expression [ 6 ]. HIF-1α and autoacetylation activate CBP/p300, which is involved in various gene expression [ 31 ]. MNSFβ siRNA decreased HIF-1α expression and transcriptional activity (Fig. 2 ) and acetyl-CBP/p300 expression (Fig. 3 a). The decreased HIF-1α transcriptional activity probably resulted from decreased HIF-1α and acetyl-CBP/p300 expression. Further experiments are needed to determine how MNSFβ affects HIF-1α and acetyl-CBP/p300 expression. The TCA cycle is indirectly involved in energy production by producing NADH and FADH2 for transfer to the electron transport chain. In addition, TCA cycle metabolites become building biomolecules or participate in chromatin modification and post-translational protein modifications [ 32 , 33 ]. HIF-1α activation suppresses the influx of pyruvate into the TCA cycle, but TCA cycle intermediates in the TCA cycle are compensated for by other metabolic pathways [ 34 ]. The results of reduced lactate and pyruvate in Raw264.7 cells with MNSFβ knockdown in metabolomic analysis corroborate our previous findings [ 6 ]. In addition, TCA cycle intermediates, especially citrate, were decreased, possibly due to glycolytic inhibition in MNSFβ-knockdown cells. Metabolic reprogramming markedly contributes to the adaptive immune response by affecting cytokine secretion. LPS-stimulated macrophages enhance glycolysis, the pentose phosphate pathway, and fatty acid synthesis via the activation of transcription factors such as HIF-1α and STAT1/3 [ 23 , 35 ]. In human monocyte-derived macrophages, LPS promotes the secretion of pro-inflammatory cytokines mediated by Akt kinases. This is inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) [ 36 ]. In a mouse model of inflammatory disease induced by LPS administration, 2-DG also inhibits the secretion of cytokines such as IL-6, IL-1β, and TNFα, reducing inflammatory symptoms in mice [ 37 ]. In murine myeloid leukemia cells, nuclear factor-kappa B (NF-κB) binds to the promoter region of IL-6 and promotes IL-6 production [ 38 ]. MNSFβ knockdown markedly promotes LPS-induced degradation of IκBα, as we have reported [ 39 ]. Therefore, this suggests NF-κB involvement in the MNSFβ knockdown-triggered increase in IL-6. Zhen XX et al. reported that MNSFβ knockdown decreased TNF-α mRNA expression in the human monocyte cell line Thp1-derived macrophages stimulated with LPS for 1 h or 4 h [ 40 ]. However, in murine macrophage Raw264.7 cells stimulated with 100 ng/ml LPS for 4 h, MNSFβ knockdown increased TNFα in the culture supernatant [ 41 ]. The effect of MNSFβ on LPS-induced TNF-α production appears to vary by cell type and stimulation conditions. IL-1ra, an anti-inflammatory cytokine and IL-1 receptor antagonist, competitively inhibits IL-1α and IL-1β signaling. In Raw264.7, increased IL-1ra secretion by LPS is mediated by P2X7 receptor (P2X7R) activation [ 42 ]. P2X7R is a receptor whose ligand is extracellular ATP, which is abundantly expressed in immune cells. Extracellular ATP release is increased by stimulation of ROS, nitric oxide, TLR2, and TLR4 [ 43 ]. MNSFβ knockdown increased ROS production (Fig. 1 b), but its overexpression decreased ROS (Fig. 1 c). Although only cellular ATP was measured in this study, ROS-induced cell damage may have caused an increase in extracellular ATP, possibly affecting IL-1ra expression via P2X7R. Since the lack of P2X7R significantly reduces OXPHOS [ 44 ], the possible involvement of P2X7R in the MNSFβ knockdown-triggered metabolic changes requires investigation. Our evaluation of the effects of MNSFβ on the regulation of glucose metabolism in murine peritoneal macrophages, which are not cancer cells, revealed no changes in lactate secretion, glucose consumption, or ROS production (Fig. 5 c, 5 d and 5 e). Notably, HIF-1α expression in peritoneal macrophages remained at a low level in the unstimulated state (Fig. 5 b). These results suggest HIF-1α mediation of these MNSFβ-induced metabolic changes at least in Raw264.7 cells. The MNSFβ knockdown-triggered changes may have been difficult to observe in unstimulated peritoneal macrophages because these cells primarily depend on OXPHOS for ATP production. In macrophages, LPS promotes the glycolytic pathway. The MNSFβ knockdown-triggered reduction in LPS-stimulated HIF-1α protein expression in peritoneal macrophages suggests that MNSFβ can regulate the glycolytic pathway in LPS-stimulated peritoneal macrophages. The present study implicates MNSFβ in glucose metabolism and inflammatory responses. Overall, MNSFβ may be an important ubiquitin-like protein that regulates multiple functions of macrophages. Abbreviations CBP CREB-binding protein C-TAD carboxy-terminal transactivation domain FIH Factor-inhibiting HIF-1α HIF-1 hypoxia-inducible factor-1 IFNγ interferon γ LPS lipopolysaccharide MNSFβ monoclonal nonspecific suppressor factor β NF-κB nuclear factor-kappa B OCR oxygen consumption rate ODDD oxygen-dependent degradation domain OXPHOS oxidative phosphorylation PDK1 Pyruvate dehydrogenase kinase 1 PHD prolyl hydroxylase pVHL von Hippel Lindau protein ROS reactive oxygen species TCA tricarboxylic acid 2-DG 2-deoxy-D-glucose Declarations Funding: This study was supported by a grant-in aid for scientific research (C) to MN from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest : The authors declare no competing interests. Ethics approval: All animal experiments (IZ3-55) were approved and performed according to the guidelines of the Animal Care Committee of Shimane University. Author contributions MN and MK designed the study. 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J Biol Chem 295:3099-114. https://doi.org/10.1074/jbc.RA119.010589 Uehara I, Kajita M, Tanimura A, Hida S, Onda M, Naito Z, Taki S, Tanaka N (2022) 2-deoxy-d-glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation. Pharmacol Res Perspect 10:e00940. https://doi.org/10.1002/prp2.940 Kawashima T, Murata K, Akira S, Tonozuka Y, Minoshima Y, Feng S, Kumagai H, Tsuruga H, Ikeda Y, Asano S, Nosaka T, Kitamura T (2001) STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65. J Immunol 167:3652-60. https://doi.org/10.4049/jimmunol.167.7.3652 Nakamura M, Notsu K, Nakagawa M (2019) Heat shock protein 60 negatively regulates the biological functions of ubiquitin-like protein MNSFβ in macrophages. Mol Cell Biochem 456:29-39. https://doi.org/10.1007/s11010-018-3487-5 Zhen X-X, Yang L, Gu Y, Yang Q, Gu W-W, He Y-P, Wang Y-L, Wang J (2021) MNSFβ regulates TNFα production by interacting with RC3H1 in human macrophages, and dysfunction of MNSFβ in decidual macrophages is associated with recurrent pregnancy loss. Front Immunol 12:691908. https://doi.org/10.3389/fimmu.2021.691908 Nakamura M, Omura S (2008) Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages. Biosci Biotechnol Biochem 72:1915-20. https://doi.org/10.1271/bbb.80167 Wilson HL, Francis SE, Dower SK, Crossman DC (2004) Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation. J Immunol 173:1202-8. https://doi.org/10.4049/jimmunol.173.2.1202 Vultaggio-Poma V, Sarti AC, Di Virgilio F (2020) Extracellular ATP: A feasible target for cancer therapy. Cells 9:2496. https://doi.org/10.3390/cells9112496 Sarti AC, Vultaggio-Poma V, Falzoni S, Missiroli S, Giuliani AL, Boldrini P, Bonora M, Faita F, Di Lascio N, Kusmic C, Solini A, Novello S, Morari M, Rossato M, Wieckowski MR, Giorgi C, Pinton P, Di Virgilio F (2021) Mitochondrial P2X7 receptor localization modulates energy metabolism enhancing physical performance. Function (Oxf) 2:zqab005. https://doi.org/10.1093/function/zqab005 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 12 Sep, 2024 Reviews received at journal 11 Sep, 2024 Reviews received at journal 05 Sep, 2024 Reviews received at journal 28 Aug, 2024 Reviewers agreed at journal 12 Aug, 2024 Reviewers agreed at journal 12 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers invited by journal 11 Jul, 2024 Editor assigned by journal 11 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 10 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4720952","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328049867,"identity":"cf4a28eb-b8b9-4c50-b8ee-d2d6423cfc54","order_by":0,"name":"Megumi Kono","email":"","orcid":"","institution":"Shimane University","correspondingAuthor":false,"prefix":"","firstName":"Megumi","middleName":"","lastName":"Kono","suffix":""},{"id":328049869,"identity":"e3ef6569-907f-4b46-a6bd-862030b6986a","order_by":1,"name":"Kyoko Yamasaki","email":"","orcid":"","institution":"Shimane University","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Yamasaki","suffix":""},{"id":328049871,"identity":"14745aef-6fea-444d-a013-3eea0d0d2082","order_by":2,"name":"Morihiko Nakamura","email":"data:image/png;base64,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","orcid":"","institution":"Shimane University","correspondingAuthor":true,"prefix":"","firstName":"Morihiko","middleName":"","lastName":"Nakamura","suffix":""}],"badges":[],"createdAt":"2024-07-11 00:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4720952/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4720952/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-024-10009-6","type":"published","date":"2024-10-15T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61757893,"identity":"dd34a899-d68b-45c9-84c7-7a92aa246788","added_by":"auto","created_at":"2024-08-05 08:53:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMNSFβ shifts ATP production from glycolysis to oxidative phosphorylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Raw264.7 cells were transfected with siRNAs for 48 h, and OCR was measured. ROS production of Raw264.7 cells transfected with siRNAs for 48 h (\u003cstrong\u003eb\u003c/strong\u003e) and cDNAs for 18 h (\u003cstrong\u003ec\u003c/strong\u003e). (\u003cstrong\u003ed\u003c/strong\u003e) After 48 h transfection, cells were stimulated with 1 µM oligomycin for 1 h. ATP was detected as described in the Materials and Methods section. The data are presented as the mean ± SD from three independent experiments. **\u003cem\u003eP\u003c/em\u003e＜0.01, *\u003cem\u003eP\u003c/em\u003e＜0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/526a3af32863195bd8f99070.png"},{"id":61757031,"identity":"32dc5c15-730a-4f5c-b0db-4b72b150abb4","added_by":"auto","created_at":"2024-08-05 08:45:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":411381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMNSFβ regulates HIF-1α mRNA and protein expression in Raw264.7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw264.7 cells were transfected with siRNA for 48 h, and HIF-1α mRNA (\u003cstrong\u003ea\u003c/strong\u003e) and protein (\u003cstrong\u003eb\u003c/strong\u003e) expression were analyzed by RT-PCR and western blotting, respectively. (\u003cstrong\u003ec\u003c/strong\u003e) The DNA binding activity of HIF-1 was evaluated by EMSA, as described in the Materials and Methods section. The cells were stimulated with 1 µg/ml LPS for 24 h after 48 h transfection, and nuclear extracts were prepared. The data are presented as the mean ± SD from three independent experiments. **\u003cem\u003eP\u003c/em\u003e＜0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/dda1675bed5083aa4ab566b1.png"},{"id":61757033,"identity":"b99556fd-f985-4dd3-934e-ec97477d2f71","added_by":"auto","created_at":"2024-08-05 08:45:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":394657,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMNSFβ siRNA decreases acetyl-CBP/p300 expression in Raw264.7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 48 h of transfection, acetyl-p300/CBP (\u003cstrong\u003ea\u003c/strong\u003e), FIH (\u003cstrong\u003eb\u003c/strong\u003e), and PHD2 (\u003cstrong\u003ec\u003c/strong\u003e) expression were analyzed by western blotting. The data are presented as the mean ± SD from three independent experiments. **\u003cem\u003eP\u003c/em\u003e＜0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/87fac46dfd67b370732daf6a.png"},{"id":61757881,"identity":"58e3e64a-2c69-4f29-90a1-81b3638a260b","added_by":"auto","created_at":"2024-08-05 08:53:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":600165,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMNSFβ changes cytokine production patterns.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw264.7 cells were treated with siRNA for 48 h and either untreated (\u003cstrong\u003ea\u003c/strong\u003e) or treated with 1 µg/ml LPS for 24 h (\u003cstrong\u003eb\u003c/strong\u003e). Cytokines in the culture supernatants were detected. 1, G-CSF; 2, GM-CSF; 3, sICAN-1; 4, IL-1ra; 5, IL-6; 6, IL-10; 7, IL-27; 8, IP-10 (CXCL10); 9, JE (CCL2); 10, MCP-5 (CCL12); 11, MIP-1α (CCL3); 12, MIP-1β(CCL4); 13, MIP-2 (CXCL2);14, RANTES (CCL5); 15, TIMP-1; 16, TNFα.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/83eaf45e2dc27eb49bb33d22.png"},{"id":61757882,"identity":"00895e74-b9ca-46af-8125-d1e16ab339cc","added_by":"auto","created_at":"2024-08-05 08:53:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":535850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMNSFβ regulates HIF-1α expression in murine peritoneal macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeritoneal macrophages were transfected with siRNAs for 48 h and stimulated with LPS (1 µg/ml) for 24 h. MNSFβ mRNA (\u003cstrong\u003ea\u003c/strong\u003e) and protein (\u003cstrong\u003eb\u003c/strong\u003e) expression were analyzed by RT-PCR and western blotting, respectively. After 48 h transfection with siRNAs, glucose (\u003cstrong\u003ec\u003c/strong\u003e) and lactate (\u003cstrong\u003ed\u003c/strong\u003e) in the culture supernatant and ROS production (\u003cstrong\u003ee\u003c/strong\u003e) were detected. Data are presented as the mean ± SD from three independent experiments. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/b749205fbe689def56b08cdd.png"},{"id":67149601,"identity":"e0431071-db09-4ff7-9262-2fc3490df108","added_by":"auto","created_at":"2024-10-21 16:13:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2611884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4720952/v1/3ba2c43e-46b6-4e12-a3a8-328e1f164186.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the regulatory mechanism of glucose metabolism by ubiquitin-like protein MNSFβ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMonoclonal nonspecific suppressor factor β (MNSFβ), a member of the ubiquitin-like protein family, is involved in various biological functions, such as cell proliferation, immunological responses, and apoptosis. MNSFβ presents a 57% amino acid sequence homology and 36% similarity with ubiquitin [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The C-terminal glycine doublet of ubiquitin, which participates in the isopeptide bond formation during conjugation, is a conserved sequence even in MNSFβ [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MNSFβ forms covalent bonds with certain lysines of specific target proteins, including endophilin Ⅱ and Bcl-G. While MNSFβ covalently attaches to endophilin Ⅱ and downregulates phagocytosis in macrophages [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], it also covalently binds to Bcl-G and markedly promotes apoptosis induced by lipopolysaccharide (LPS)/interferon γ (IFNγ) in macrophages [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Unlike ubiquitin, MNSFβ may not be involved in the degradation of targeted proteins. While MNSFβ regulates glycolysis, its effects is related to GLUT1 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hypoxia-inducible factor-1α (HIF-1α) controls GLUT1 expression in multiple cell lines, but whether MNSFβ affects HIF-1α remains unclear.\u003c/p\u003e \u003cp\u003eThe heterodimeric transcription factor HIF-1 increases the expression of enzymes implicated in glucose metabolism, such as hexokinase, phosphofructokinase-1, and pyruvate kinase M2 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. HIF-1 comprises two subunits: an oxygen-labile HIF-1α subunit and a constitutively stable HIF-1β subunit. HIF-1α has an oxygen-dependent degradation domain (ODDD) that binds the E3 ubiquitin ligase von Hippel Lindau protein (pVHL). Prolyl hydroxylases (PHDs) hydroxylate the two proline residues (Pro\u003csup\u003e402\u003c/sup\u003e, Pro\u003csup\u003e564\u003c/sup\u003e) within the ODDD, which recruits pVHL, and HIF-1α is ubiquitinated and degraded by the 26S proteasome. In hypoxia, PHD inhibition impedes pVHL binding. This is followed by HIF-1α stabilization, nuclear translocation, dimerization with HIF-1β, coactivator recruitment, and binding to hypoxia response elements in the promoter region of target genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The asparaginyl hydroxylase factor-inhibiting HIF-1α (FIH) suppresses HIF-1α transcriptional activity. FIH causes hydroxylation of HIF-1α at the Asn\u003csup\u003e803\u003c/sup\u003e in the carboxy-terminal transactivation domain (C-TAD), which prevents HIF-1α binding to transcriptional coactivators, such as CREB-binding protein (CBP)/p300 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. FIH overexpression suppresses HIF-1α transcriptional activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The histone acetyltransferase CBP/p300 are relevant to HIF-1α/β heterodimer formation. Acetylated CBP/p300 interacts with the C-TAD of HIF-1α to enhance gene expression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Certain cancer cells showed overexpressed CBP/p300 mRNAs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hypoxia, alongside many oncogenic and inflammatory stimuli, promotes HIF-1α activation and accumulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HIF-1α overexpression has been found in various cancer types and is related to tumor aggressiveness [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, MNSFβ alters both glycolysis and mitochondrial metabolism, markedly affecting glucose metabolism and cytokine production, and HIF-1α may be involved in this regulatory mechanism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and chemicals\u003c/h2\u003e \u003cp\u003eAntibodies used included anti-HIF-1α (Novus Biologicals, Littleton, CO, USA), anti-Acetyl-CBP (Lys1535)/p300 (Lys1499) (Cell Signaling Technology, Beverly, MA, USA), anti-FIH (Cell Signaling Technology), anti-PHD2 (GeneTex, San Antonio, TX), anti-β-actin (Medical \u0026amp; Biological laboratories), and peroxidase-conjugated rabbit IgG antibody (Cappel, Solon, OH, USA). Sigma (St. Louis, MO, USA) supplied LPS and oligomycin A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture, siRNAs, and transfection of cells\u003c/h2\u003e \u003cp\u003eThe macrophage-like cell line Raw264.7 was obtained from ATCC (Manassas, VA). Cells were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium containing 10% fetal bovine serum and penicillin/streptomycin (1:100). siRNAs were purchased from Qiagen (Chatsworth, CA, USA). The target sequence of siRNAs was as follows: MNSFβ 5\u0026prime;-CCACCCTGCCATGCTAATAAA-3\u0026prime; and the scrambled negative control 5\u0026prime;-AATTCTCCGAACGTGTCACGT-3\u0026prime;. siRNA transfection was performed with HiPerFect Transfection Reagent (Qiagen) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ecDNAs and transfection cells\u003c/h2\u003e \u003cp\u003ecDNAs were prepared as previously described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. cDNA encoding MNSFβ was subcloned into the vector pcDNA3.1(+) (Invitrogen). DNA transfection was performed using Lipofectamine LTX reagent with Plus reagent (Invitrogen), following the manufacturer\u0026rsquo;s instructions. Cells were seeded and cultured until 70\u0026ndash;80% confluent. Lipofectamine LTX and Plus reagent was mixed with cDNA. The DNA mixture was incubated for 5 min at room temperature to form a DNA-lipid complex and added to cells. The final volume of cDNA was 500 ng per well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eProtein samples were subjected to SDS-PAGE and electro-transferred from gels to polyvinylidene fluoride membranes. Membranes were blocked for 1 h at room temperature using Tris-buffered saline with 0.02% Tween 20 (TBST) and 5% skim milk or 5% bovine serum albumin and probed with primary antibodies overnight at 4 ℃ in blocking buffer. The membranes were washed four times for 5 min each with TBST and incubated with horseradish peroxidase secondary antibodies for 2 h at room temperature. The secondary antibodies were used at 1:15000 dilutions in blocking buffer. Membranes were washed four times for 5 min each with TBST and detected with ECL reagents (Amersham Biosciences) and analyzed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRT-PCR\u003c/h2\u003e \u003cp\u003eRT-PCR was performed with Premix Taq (Ex Taq Version 2.0) (Takara) following the manufacturer\u0026rsquo;s instructions. The PCR conditions used were as follows: initial denaturation at 95 ℃ for 1 min, 40 cycles of 98 ℃ for 10 s, 55 ℃ for 30 s, and 72 ℃ for 1 min. Primers for HIF-1α were 5\u0026prime;-TGATGTGGGTGCTGGTGTC-3\u0026prime; (sense) and 5\u0026prime;-TTGTGTTGGGGCAGTACTG-3\u0026prime; (anti-sense). Primers for GAPDH were 5\u0026prime;-CAACTACATGGTTTACATGTTC-3\u0026prime; (sense) and 5\u0026prime;-GCCAGTGGACTCCACGAC-3\u0026prime; (anti-sense). We amplified GAPDH as an internal standard. PCR products were electrophoresed on 2% agarose gels with ethidium bromide and detected using a UV transilluminator. The signals were quantified with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eROS detection\u003c/h2\u003e \u003cp\u003eCells were seeded at 2.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 96-well microplate. Intracellular ROS was evaluated with a ROS Assay Kit -Highly sensitive DCFH-DA- (Dojindo) according to the manufacturer\u0026rsquo;s instructions. Cells were washed twice using Hanks\u0026rsquo; Balanced Salt Solution (HBSS) (Sigma) and incubated with the highly sensitive DCFH-DA dye working solution at 37 ℃ for 30 min. The cells were washed twice, and each well was refilled with HBSS. Fluorescence was read using a microplate reader (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eCells were seeded at 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well in a 6-well plate. Nuclear extracts were prepared with EzSubcell Fraction (ATTO) according to the manufacturer\u0026rsquo;s instructions. HIF-1α binds to hypoxia response element (HRE) sequences within the promoter region. HIF-1α-DNA interactions were detected using biotin end-labeled DNA probes containing HRE. The sequence of the probe was 5\u0026prime;-CACCCCACCCCCGTGAGGAGGAGGGTGAGGAAAC-3\u0026prime;. The binding reaction was performed using a Gelshift Chemiluminescent EMSA kit (Active Motif) following the manufacturer\u0026rsquo;s instructions. The reaction products were loaded on a 6% polyacrylamide gel in 0.5% Tris borate/EDTA at 4 ℃, transferred to a nylon membrane, and fixed on the membrane by UV cross-linking. The biotin-labeled probe was detected using chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eATP measurement\u003c/h2\u003e \u003cp\u003eCells were seeded at 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 96-well microplate. Intracellular ATP was detected using an ATP Assay Kit-Luminescence (Dojindo) based on the manufacturer\u0026rsquo;s instructions. Cells were incubated with working solution at 25 ℃ for 10 min. Luminescence was read with a microplate reader (Promega). The ATP concentrations were calculated from a calibration curve generated from the ATP standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOCR detection\u003c/h2\u003e \u003cp\u003eCells were seeded at 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 96-well microplate. The mitochondrial oxygen consumption rate (OCR) was evaluated using an Extracellular OCR Plate Assay Kit (Dojindo) based on the manufacturer\u0026rsquo;s instructions. Cells were incubated with working solution at 37 ℃ for 30 min. Then, one drop of mineral oil was added to each well, and the cells were incubated at 37 ℃ for 5 min. Fluorescence was read using a microplate reader at 10-min intervals for 200 min. The OCR was calculated from the intensity using the calculation sheet provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGlucose and lactate assay\u003c/h2\u003e \u003cp\u003eCells were seeded at 2.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 24-well plate. Glucose and lactate in the supernatant were detected using a glucose assay kit-WST (Dojindo) and glycolysis cell-based assay kit (Cayman) based on the manufacturer\u0026rsquo;s instructions, respectively. The concentrations were calculated from a calibration curve generated from the standard. The glucose assay had a detection limit of 0.02 mmol/l glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProteome profiler mouse cytokine array\u003c/h2\u003e \u003cp\u003eCells were seeded at 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 24-well plate. The cytokine expressions in cell culture supernatants were detected using a Proteome Profiler Mouse Cytokine Array Kit, Panel A (R\u0026amp;D Systems) following the manufacturer\u0026rsquo;s instructions. Cell culture supernatants were cleared of particulates by centrifugation for 5 min at 1,000 \u0026times;g and then used for the cytokine array. The signal was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMetabolome analysis\u003c/h2\u003e \u003cp\u003eThe cells were washed with PBS and lysed using 70% (v/v) methanol. Metabolome analysis by gas chromatography-mass spectrometry (GC/MS) was performed at the Integrated Center for Mass Spectrometry, Graduate School of Medicine, Kobe University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of peritoneal macrophages\u003c/h2\u003e \u003cp\u003ePeritoneal macrophages were harvested from female BALB/c mice (Japan SLC, Inc.) 4 days after intraperitoneal injection of 1.5 ml of sterile 4% Brewer\u0026rsquo;s thioglycollate medium (Difco Laboratories). The cells were collected by centrifugation at 400 \u0026times;g for 5 min, washed and seeded in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 10% fetal bovine serum. The collected macrophages were used in various experiments. All animal studies were approved by the Animal Care Committee of Shimane University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was analyzed by Student\u0026rsquo;s t-test and expressed as \u003cem\u003eP\u003c/em\u003e values. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMNSFβ siRNA increased oxidative phosphorylation in Raw264.7 cells\u003c/h2\u003e \u003cp\u003eWe most recently reported that MNSFβ regulates the glycolytic system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mitochondrial respiration is closely involved in the mechanism of intracellular glycometabolism. For instance, glycolytic suppression enhances mitochondrial oxidative phosphorylation (OXPHOS) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, we examined the effect of MNSFβ on metabolism in mitochondria. First, we determined whether glycolytic suppression induced by MNSFβ siRNA affects OXPHOS in Raw264.7 cells. We next measured the OCR, an indicator of metabolic activity in mitochondria, and reactive oxygen species (ROS), a byproduct of oxidative phosphorylation. After 48 h transfection, MNSFβ siRNA significantly increased both OCR and ROS production in Raw264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Conversely, 18 h of MNSFβ overexpression reduced ROS production, strongly indicating that this ubiquitin-like protein positively regulates ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, we measured ATP levels in Raw264.7 cells transfected with MNSFβ siRNA and treated with the ATP synthase inhibitor oligomycin. MNSFβ siRNA transfection markedly reduced ATP levels. Upon oligomycin treatment, cells with MNSFβ knockdown exhibited a greater ATP reduction rate than the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), suggesting that MNSFβ siRNA treatment may alter the main ATP source from glycolysis to OXPHOS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMNSFβ affects HIF-1α expression and HIF-1α\u0026ndash;DNA interactions in Raw264.7 cells\u003c/h2\u003e \u003cp\u003eIn macrophages, HIF-1α disruption facilitates OXPHOS and decreases the glycolysis level [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MNSFβ siRNA can inhibit the expression of GLUT1, GLUT3, and the key molecules in glycolysis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. HIF-1α enhances glycolysis by inducing the expression of glycolytic enzymes and glucose transporters. To examine whether HIF-1α mediates MNSFβ-regulated glycolysis, we analyzed the expression of mRNA and protein of HIF-1α. After 48 h transfection of MNSFβ siRNA, HIF-1α mRNA and protein expressions were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Evaluation of the DNA binding property of HIF-1α by EMSA revealed that MNSFβ siRNA impaired DNA\u0026ndash;HIF-1α interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These results strongly indicate that MNSFβ knockdown-triggered reduction in HIF-1α transcriptional activity may be implicated in glycolysis suppression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMNSFβ siRNA decreases acetyl-CBP/p300 expression in Raw264.7 cells\u003c/h2\u003e \u003cp\u003eThe stability and transcriptional activity of HIF-1α are regulated by its post-translational modifications such as ubiquitination, hydroxylation, and acetylation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, we examined whether MNSFβ affects factors involved in HIF-1α stabilization and complex formation. After 48 h transfection, MNSFβ siRNA reduced acetyl-CBP/p300 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) without altering that of FIH and PHD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These results indicate that MNSFβ siRNA-induced decrease in HIF-1α transcriptional activity is affected not only by HIF-1α but also by decreased expression of acetyl-CBP/p300, which suppresses glycolysis through decreased expression of HIF-1α target genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMNSFβ siRNA changes intracellular metabolite levels in Raw264.7 cells\u003c/h2\u003e \u003cp\u003eIn cancer cells, HIF-1α activation diminishes the tricarboxylic acid (TCA) cycle and OXPHOS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To investigate further MNSFβ knockdown-triggered glycolytic changes, intermediate metabolites were detected by GC-MS. After 48 h transfection, MNSFβ siRNA reduced pyruvate and lactate, TCA cycle intermediates, especially citrate (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the amino acids, proline and branched chain amino acids were greatly reduced, while only serine was increased. These data suggest that MNSFβ affects overall glucose metabolism and partially alters amino acid metabolism as well.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMetabolome analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolite name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMNSFβ siRNA\u003c/p\u003e \u003cp\u003e /Control siRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"26\" rowspan=\"27\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMetabolite name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMNSFβ siRNA\u003c/p\u003e \u003cp\u003e /Control siRNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConiferyl alcohol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eα-ketoglutarate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.706\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGABA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSuccinate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2,3-Bisphospho-glycerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.646\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSorbitol 6-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.639\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCadaverine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.600\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGluconic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ebeta-Alanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.593\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElaidic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypotaurine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStearic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanolamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePyruvate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.568\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucosamine 6-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTerephthalic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitoleic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.554\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-Methylethanolamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNorleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.544\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.537\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en-Butylamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.531\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxalic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etrans-4-Hydroxyproline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.528\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePyroglutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInositol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePyrophosphoric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.494\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Hydroxypyridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.466\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.447\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Aminoisobutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSorbose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.428\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en-Propylamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.424\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebeta-Hydroxybutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1,5-Anhydro-D-glucitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.383\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFumarate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLactate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.326\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTagatose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.289\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eRaw264.7 cells were treated with siRNAs for 48 h, and metabolites were detected by GC/MS.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMNSFβ changes the pattern of cytokine production in Raw264.7 cells\u003c/h2\u003e \u003cp\u003eHIF-1α can alter cytokine expression, which markedly contributes to the tumor microenvironment [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Metabolites can affect cytokine production [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Since MNSFβ knockdown altered HIF-1α expression as described above, we investigated the relationship between MNSFβ and cytokine expression. After 48 h transfection, the cells were treated with 1 \u0026micro;g/ml LPS for 24 h, and cytokines in the supernatants were analyzed by antibody arrays, resulting in 16 cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among them, CCL2 and IL-10 were reported to be decreased by HIF-1α inhibition [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ICAM-1 is related to HIF-1α and GLUT1 expression during endotoxemia [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, MNSFβ may modulate its effects on cytokine expression via HIF-1α.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMNSFβ affects HIF-1α expression in peritoneal macrophages\u003c/h2\u003e \u003cp\u003eTo further investigate the effect of MNSFβ on glucose metabolism, murine peritoneal macrophages were used. MNSFβ knockdown decreased HIF-1α mRNA. Unlike Raw264 cells, in unstimulated peritoneal macrophages, the expression level of HIF-1α protein is very low [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, since the expression of HIF-1α protein is increased by LPS stimulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we examined the effect of MNSFβ on HIF-1α expression in LPS-stimulated murine peritoneal macrophages. MNSFβ knockdown markedly inhibited LPS-stimulated increase in HIF-1α protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Since our previous study showed glucose consumption and lactate secretion by MNSFβ knockdown in Raw264.7 cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], we performed the same verification in peritoneal macrophages. Unlike Raw264.7 cells, peritoneal macrophages treated with MNSFβ siRNA showed no difference in glucose and lactate levels in the culture supernatant or in ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn macrophages, metabolic characteristics are closely related to phenotypes and functions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. MNSFβ is involved in glycolytic regulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, in this study, we first focused on metabolic changes in mitochondria. MNSFβ knockdown increased OCR and ROS production in Raw264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which showed a markedly decreased ATP level upon oligomycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In addition, MNSFβ siRNA reduces lactate in the culture supernatant and glucose consumption in Raw264.7 cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], suggesting that MNSFβ knockdown shifts the primary ATP production pathway from glycolysis to OXPHOS.\u003c/p\u003e \u003cp\u003eMany cancer cells rely on the glycolytic system for much of their ATP production, even though mitochondrial function is maintained under aerobic conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This phenomenon is widely known as the Warburg effect, in which HIF-1α is closely involved [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. HIF-1α promotes the glycolytic system and causes metabolic reprogramming by inhibiting pyruvate influx into the TCA cycle and OXPHOS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Pyruvate dehydrogenase kinase 1 (PDK1) is a particularly important enzyme in the metabolic shift from mitochondria to the glycolytic system, and \u003cem\u003ePDK1\u003c/em\u003e is a target gene of HIF-1α. Increased PDK1 by HIF-1α activation inhibits pyruvate dehydrogenase (PDH), thereby reducing mitochondrial respiration and ROS production and preventing cell death due to excess ROS [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e are consistent with reports that MNSFβ knockdown reduces PDK1 expression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHIF-1α and autoacetylation activate CBP/p300, which is involved in various gene expression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. MNSFβ siRNA decreased HIF-1α expression and transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and acetyl-CBP/p300 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The decreased HIF-1α transcriptional activity probably resulted from decreased HIF-1α and acetyl-CBP/p300 expression. Further experiments are needed to determine how MNSFβ affects HIF-1α and acetyl-CBP/p300 expression.\u003c/p\u003e \u003cp\u003eThe TCA cycle is indirectly involved in energy production by producing NADH and FADH2 for transfer to the electron transport chain. In addition, TCA cycle metabolites become building biomolecules or participate in chromatin modification and post-translational protein modifications [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. HIF-1α activation suppresses the influx of pyruvate into the TCA cycle, but TCA cycle intermediates in the TCA cycle are compensated for by other metabolic pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The results of reduced lactate and pyruvate in Raw264.7 cells with MNSFβ knockdown in metabolomic analysis corroborate our previous findings [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, TCA cycle intermediates, especially citrate, were decreased, possibly due to glycolytic inhibition in MNSFβ-knockdown cells.\u003c/p\u003e \u003cp\u003eMetabolic reprogramming markedly contributes to the adaptive immune response by affecting cytokine secretion. LPS-stimulated macrophages enhance glycolysis, the pentose phosphate pathway, and fatty acid synthesis via the activation of transcription factors such as HIF-1α and STAT1/3 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In human monocyte-derived macrophages, LPS promotes the secretion of pro-inflammatory cytokines mediated by Akt kinases. This is inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In a mouse model of inflammatory disease induced by LPS administration, 2-DG also inhibits the secretion of cytokines such as IL-6, IL-1β, and TNFα, reducing inflammatory symptoms in mice [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In murine myeloid leukemia cells, nuclear factor-kappa B (NF-κB) binds to the promoter region of IL-6 and promotes IL-6 production [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. MNSFβ knockdown markedly promotes LPS-induced degradation of IκBα, as we have reported [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, this suggests NF-κB involvement in the MNSFβ knockdown-triggered increase in IL-6.\u003c/p\u003e \u003cp\u003eZhen XX \u003cem\u003eet al.\u003c/em\u003e reported that MNSFβ knockdown decreased TNF-α mRNA expression in the human monocyte cell line Thp1-derived macrophages stimulated with LPS for 1 h or 4 h [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, in murine macrophage Raw264.7 cells stimulated with 100 ng/ml LPS for 4 h, MNSFβ knockdown increased TNFα in the culture supernatant [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The effect of MNSFβ on LPS-induced TNF-α production appears to vary by cell type and stimulation conditions.\u003c/p\u003e \u003cp\u003eIL-1ra, an anti-inflammatory cytokine and IL-1 receptor antagonist, competitively inhibits IL-1α and IL-1β signaling. In Raw264.7, increased IL-1ra secretion by LPS is mediated by P2X7 receptor (P2X7R) activation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. P2X7R is a receptor whose ligand is extracellular ATP, which is abundantly expressed in immune cells. Extracellular ATP release is increased by stimulation of ROS, nitric oxide, TLR2, and TLR4 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. MNSFβ knockdown increased ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), but its overexpression decreased ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Although only cellular ATP was measured in this study, ROS-induced cell damage may have caused an increase in extracellular ATP, possibly affecting IL-1ra expression via P2X7R. Since the lack of P2X7R significantly reduces OXPHOS [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], the possible involvement of P2X7R in the MNSFβ knockdown-triggered metabolic changes requires investigation.\u003c/p\u003e \u003cp\u003eOur evaluation of the effects of MNSFβ on the regulation of glucose metabolism in murine peritoneal macrophages, which are not cancer cells, revealed no changes in lactate secretion, glucose consumption, or ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Notably, HIF-1α expression in peritoneal macrophages remained at a low level in the unstimulated state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results suggest HIF-1α mediation of these MNSFβ-induced metabolic changes at least in Raw264.7 cells. The MNSFβ knockdown-triggered changes may have been difficult to observe in unstimulated peritoneal macrophages because these cells primarily depend on OXPHOS for ATP production. In macrophages, LPS promotes the glycolytic pathway. The MNSFβ knockdown-triggered reduction in LPS-stimulated HIF-1α protein expression in peritoneal macrophages suggests that MNSFβ can regulate the glycolytic pathway in LPS-stimulated peritoneal macrophages. The present study implicates MNSFβ in glucose metabolism and inflammatory responses. Overall, MNSFβ may be an important ubiquitin-like protein that regulates multiple functions of macrophages.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCBP \u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;CREB-binding protein\u003c/p\u003e\n\u003cp\u003eC-TAD \u0026nbsp; \u0026nbsp;carboxy-terminal transactivation domain\u003c/p\u003e\n\u003cp\u003eFIH \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Factor-inhibiting HIF-1\u0026alpha;\u003c/p\u003e\n\u003cp\u003eHIF-1 \u0026nbsp; \u0026nbsp; hypoxia-inducible factor-1\u003c/p\u003e\n\u003cp\u003eIFN\u0026gamma; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; interferon \u0026gamma;\u003c/p\u003e\n\u003cp\u003eLPS \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eMNSF\u0026beta; \u0026nbsp; \u0026nbsp;monoclonal nonspecific suppressor factor \u0026beta;\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B\u0026nbsp; \u0026nbsp;\u0026nbsp; nuclear factor-kappa B\u003c/p\u003e\n\u003cp\u003eOCR \u0026nbsp; \u0026nbsp; \u0026nbsp;oxygen consumption rate\u003c/p\u003e\n\u003cp\u003eODDD\u0026nbsp;\u0026nbsp;\u0026nbsp; oxygen-dependent degradation domain\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOXPHOS \u0026nbsp;oxidative phosphorylation\u003c/p\u003e\n\u003cp\u003ePDK1 \u0026nbsp; \u0026nbsp; Pyruvate dehydrogenase kinase 1\u003c/p\u003e\n\u003cp\u003ePHD \u0026nbsp; \u0026nbsp; \u0026nbsp;prolyl hydroxylase\u003c/p\u003e\n\u003cp\u003epVHL \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;von Hippel Lindau protein\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp;reactive oxygen species\u003c/p\u003e\n\u003cp\u003eTCA \u0026nbsp; \u0026nbsp; \u0026nbsp;tricarboxylic acid\u003c/p\u003e\n\u003cp\u003e2-DG \u0026nbsp; \u0026nbsp; 2-deoxy-D-glucose\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by a grant-in aid for scientific research (C) to MN from the Ministry of Education, Culture, Sports, Science and Technology of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics\u003c/strong\u003e \u003cstrong\u003eapproval:\u003c/strong\u003e All animal experiments (IZ3-55) were approved and performed according to the guidelines of the Animal Care Committee of Shimane University.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN and MK designed the study. MK and KY conducted the experiments and MK performed the statistical analysis. MN and MK wrote the manuscript. All authors have read and approved the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Integrated Center for Mass Spectrometry, Graduate School of Medicine, Kobe University for metabolome analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNakamura M, Xavier RM, Tanigawa Y (1996) Ubiquitin-like moiety of the monoclonal nonspecific suppressor factor beta is responsible for its activity. J Immunol 156:532-8\u003c/li\u003e\n \u003cli\u003eNakamura M, Xavier RM, Tsunematsu T, Tanigawa Y (1995) Molecular cloning and characterization of a cDNA encoding monoclonal nonspecific suppressor factor. 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Function (Oxf) 2:zqab005. https://doi.org/10.1093/function/zqab005\u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ubiquitin-like protein MNSFβ, HIF-1α, Metabolism, Metabolic reprogramming","lastPublishedDoi":"10.21203/rs.3.rs-4720952/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4720952/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMonoclonal nonspecific suppressor factor β (MNSFβ), a ubiquitously expressed member of the ubiquitin-like protein family, is associated with diverse cell regulatory functions. It has been implicated in glycolysis regulation and cell proliferation enhancement in the macrophage-like cell line Raw264.7. This study aims to show that HIF-1α regulates MNSFβ-mediated metabolic reprogramming.\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eIn Raw264.7 cells, MNSFβ siRNA increased the oxygen consumption rate and ROS production but decreased ATP levels. Cells with MNSFβ knockdown showed a markedly increased ATP reduction rate upon the addition of oligomycin, a mitochondrial ATP synthase inhibitor. In addition, MNSFβ siRNA decreased the expression levels of mRNA and protein of HIF-1α\u0026mdash;a regulator of glucose metabolism. Evaluation of the effect of MNSFβ on glucose metabolism in murine peritoneal macrophages revealed no changes in lactate production, glucose consumption, or ROS production.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMNSFβ affects both glycolysis and mitochondrial metabolism, suggesting HIF-1α involvement in the MNSFβ-regulated glucose metabolism in Raw264.7 cells.\u003c/p\u003e","manuscriptTitle":"Investigating the regulatory mechanism of glucose metabolism by ubiquitin-like protein MNSFβ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 08:45:29","doi":"10.21203/rs.3.rs-4720952/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-12T07:55:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-11T16:12:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-05T15:49:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-28T14:17:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119590105921973701390753409429371348222","date":"2024-08-12T13:57:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314096691954574961930689920537880773617","date":"2024-08-12T09:02:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39502022935476161057055413325631506035","date":"2024-08-11T13:52:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-11T11:19:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T11:00:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T11:00:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-07-11T00:27:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ee512325-837e-4825-a337-c10189b9e239","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:08:58+00:00","versionOfRecord":{"articleIdentity":"rs-4720952","link":"https://doi.org/10.1007/s11033-024-10009-6","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-10-15 15:58:00","publishedOnDateReadable":"October 15th, 2024"},"versionCreatedAt":"2024-08-05 08:45:29","video":"","vorDoi":"10.1007/s11033-024-10009-6","vorDoiUrl":"https://doi.org/10.1007/s11033-024-10009-6","workflowStages":[]},"version":"v1","identity":"rs-4720952","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4720952","identity":"rs-4720952","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}