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• W2018291804 abstract "•HFD feeding represses rRNA transcription in mouse livers by recruiting NML•NML-KO mice show elevated rRNA level and reduced ATP concentration•Hepatic NML deficiency results in altered hepatic lipid metabolism•Hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage. Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage. Energy homeostasis requires the coordinated regulation of energy intake, storage, and expenditure. ATP, which is produced as an energy source, is consumed by most anabolic reactions (e.g., protein synthesis and fat synthesis), active transport of molecules and ions, nerve impulses, and muscle contraction. Ribosome biosynthesis in the nucleolus among them is the most energy-consuming process within proliferating eukaryotic cells, and it adapts to changes in the intracellular energy status (Grummt and Grummt, 1976Grummt I. Grummt F. Control of nucleolar RNA synthesis by the intracellular pool sizes of ATP and GTP.Cell. 1976; 7: 447-453Abstract Full Text PDF PubMed Scopus (90) Google Scholar, Moss et al., 2007Moss T. Langlois F. Gagnon-Kugler T. Stefanovsky V. A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis.Cell. Mol. Life Sci. 2007; 64: 29-49Crossref PubMed Scopus (211) Google Scholar). Mammalian cells quickly adjust the rate of ribosome synthesis on the basis of the availability of nutrients and growth-promoting mitogens. In addition, cells that exit the division cycle into a quiescent state greatly limit the ribosome production and overall protein synthesis. Ribosome biogenesis involves rRNA transcription, rRNA processing, and the assembly of maturated rRNA and ribosomal proteins. The rate-limiting step of ribosome biosynthesis is rRNA transcription in the nucleolus. Therefore, the control of rRNA transcription in the nucleolus is thought to regulate intracellular energy consumption. rRNA genes are present in multiple copies (approximately 400 rRNA genes per mammalian cell). However, all of the rRNA genes in the human diploid genome are not transcriptionally active. rRNA synthesis is modulated by varying the transcription rate per gene or by varying the number of actively transcribed rRNA genes (Grummt, 2010Grummt I. Wisely chosen paths—regulation of rRNA synthesis: delivered on 30 June 2010 at the 35th FEBS Congress in Gothenburg, Sweden.FEBS J. 2010; 277: 4626-4639Crossref PubMed Scopus (56) Google Scholar, Grummt and Pikaard, 2003Grummt I. Pikaard C.S. Epigenetic silencing of RNA polymerase I transcription.Nat. Rev. Mol. Cell Biol. 2003; 4: 641-649Crossref PubMed Scopus (243) Google Scholar). The basal transcription factors, transcription initiation factor IA (TIF-IA), selectivity factor 1 (SL1), and upstream binding factor (UBF) are essential for transcription by RNA polymerase I (Pol I) and appear to be modulated by different signaling pathways in response to changes in environmental conditions. For example, the extracellular signal-regulated kinase, mammalian target of rapamycin, and c-Jun N-terminal kinase pathways regulate Pol I transcription via the activities of UBF, SL1, and TIF-IA (Grummt, 2010Grummt I. Wisely chosen paths—regulation of rRNA synthesis: delivered on 30 June 2010 at the 35th FEBS Congress in Gothenburg, Sweden.FEBS J. 2010; 277: 4626-4639Crossref PubMed Scopus (56) Google Scholar). In addition, recent findings point toward the existence of additional regulatory pathways such as epigenetic regulation of rRNA transcription. Santoro et al., 2002Santoro R. Li J. Grummt I. The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription.Nat. Genet. 2002; 32: 393-396Crossref PubMed Scopus (336) Google Scholar revealed that the chromatin-remodeling complex nucleolar chromatin-remodeling complex (NoRC), which consists of the transcription-termination-factor-1-interacting protein 5 and the ATPase sucrose nonfermenting protein 2 homolog, recruits histone deacetylases, histone methyltransferases, and DNA methyltransferases to inactive rRNA gene repeats (Mayer et al., 2006Mayer C. Schmitz K.M. Li J. Grummt I. Santoro R. Intergenic transcripts regulate the epigenetic state of rRNA genes.Mol. Cell. 2006; 22: 351-361Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, Santoro et al., 2002Santoro R. Li J. Grummt I. The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription.Nat. Genet. 2002; 32: 393-396Crossref PubMed Scopus (336) Google Scholar). Furthermore, lysine-specific demethylase 2A (KDM2A), KDM2B, KDM4C, and PHF8 are members of the JmjC family of demethylases that have been epigenetically implicated in rRNA transcription. KDM2A and KDM2B repress rRNA transcription (Frescas et al., 2007Frescas D. Guardavaccaro D. Bassermann F. Koyama-Nasu R. Pagano M. JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes.Nature. 2007; 450: 309-313Crossref PubMed Scopus (205) Google Scholar, Tanaka et al., 2010Tanaka Y. Okamoto K. Teye K. Umata T. Yamagiwa N. Suto Y. Zhang Y. Tsuneoka M. JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation.EMBO J. 2010; 29: 1510-1522Crossref PubMed Scopus (79) Google Scholar). In contrast, KDM4C and PHF8 activate rRNA transcription (Feng et al., 2010Feng W. Yonezawa M. Ye J. Jenuwein T. Grummt I. PHF8 activates transcription of rRNA genes through H3K4me3 binding and H3K9me1/2 demethylation.Nat. Struct. Mol. Biol. 2010; 17: 445-450Crossref PubMed Scopus (166) Google Scholar, Liu et al., 2010Liu W. Tanasa B. Tyurina O.V. Zhou T.Y. Gassmann R. Liu W.T. Ohgi K.A. Benner C. Garcia-Bassets I. Aggarwal A.K. et al.PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression.Nature. 2010; 466: 508-512Crossref PubMed Scopus (290) Google Scholar). However, the biological role of these factors in the regulation of rRNA transcription is not completely understood. Previously, we identified the nucleolar protein nucleomethylin (NML) that binds to histone H3 dimethylated at Lys9 (H3K9me2) and recruits sirtuin1 (SIRT1) and the suppressor of variegation 3-9 homolog 1 (Suv39h1) to the rRNA gene loci, thereby establishing the structural characteristics of silent chromatin (Grummt and Ladurner, 2008Grummt I. Ladurner A.G. A metabolic throttle regulates the epigenetic state of rDNA.Cell. 2008; 133: 577-580Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Murayama et al., 2008Murayama A. Ohmori K. Fujimura A. Minami H. Yasuzawa-Tanaka K. Kuroda T. Oie S. Daitoku H. Okuwaki M. Nagata K. et al.Epigenetic control of rDNA loci in response to intracellular energy status.Cell. 2008; 133: 627-639Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). This NML/SIRT1/Suv39h1 complex, designated as energy-dependent nucleolar silencing complex (eNoSC), regulates energy balance in proliferative cells by epigenetically limiting rRNA transcription. NML binds to H3K9me2 throughout the rRNA gene transcription unit. A change in the NAD+/NADH ratio induced by a reduction in the intracellular energy status may promote the activity of SIRT1, leading to the deacetylation of histone H3 and dimethylation at Lys9 of histone H3 by Suv39h1, thus establishing silent chromatin. Thus, eNoSC links the cellular energy balance to the epigenetic silencing of rRNA genes, thereby protecting cells from energy starvation-dependent apoptosis (Grummt and Ladurner, 2008Grummt I. Ladurner A.G. A metabolic throttle regulates the epigenetic state of rDNA.Cell. 2008; 133: 577-580Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Murayama et al., 2008Murayama A. Ohmori K. Fujimura A. Minami H. Yasuzawa-Tanaka K. Kuroda T. Oie S. Daitoku H. Okuwaki M. Nagata K. et al.Epigenetic control of rDNA loci in response to intracellular energy status.Cell. 2008; 133: 627-639Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Subsequently, we reported that, in response to glucose starvation, eNoSC suppresses rRNA transcription, which results in a reduction in the nucleolar RNA content. The reduced nucleolar RNA content induces cell-cycle arrest and apoptosis due to p53 activation. These findings indicate that eNoSC may act as a sensor in the nucleolus that connects the intracellular energy status with the cell-cycle machinery (Kumazawa et al., 2011Kumazawa T. Nishimura K. Kuroda T. Ono W. Yamaguchi C. Katagiri N. Tsuchiya M. Masumoto H. Nakajima Y. Murayama A. et al.Novel nucleolar pathway connecting intracellular energy status with p53 activation.J. Biol. Chem. 2011; 286: 20861-20869Crossref PubMed Scopus (22) Google Scholar, Kuroda et al., 2011Kuroda T. Murayama A. Katagiri N. Ohta Y.M. Fujita E. Masumoto H. Ema M. Takahashi S. Kimura K. Yanagisawa J. RNA content in the nucleolus alters p53 acetylation via MYBBP1A.EMBO J. 2011; 30: 1054-1066Crossref PubMed Scopus (54) Google Scholar). Furthermore, NML regulates hepatic ATP levels during liver regeneration after partial hepatectomy (Mikogai et al., 2009Mikogai A. Yanagisawa J. Yasuzawa-Tanaka K. Murayama A. The nucleolar protein NML regulates hepatic ATP levels during liver regeneration after partial hepatectomy.Biochem. Biophys. Res. Commun. 2009; 390: 591-596Crossref PubMed Scopus (2) Google Scholar). However, whether eNoSC in the nucleolus is linked to whole-body energy metabolism remains unknown. In addition, whether rRNA transcription levels in vivo are linked to energy metabolism remains unknown. In this study, we found that high-fat-diet (HFD) feeding induced the repression of rRNA transcription in livers. To clarify the relationship between hepatic rRNA transcriptional repression and obesity, we generated NML-null (NML-KO) mice. We found that NML deficiency increased the pre-rRNA level and the AMP/ATP ratio in the liver and subsequently induced the activation of AMP-activated protein kinase (AMPK). Furthermore, NML deficiency increased fatty acid oxidation (FAO) activity and decreased lipid synthesis activity in the liver. We found that the NML-KO mice had an increased O2 consumption compared with the wild-type (WT) mice. The NML-KO mice were leaner than the WT mice. Furthermore, in the NML-KO mice after HFD feeding, hepatic pre-rRNA levels remained higher than that in the WT mice. And the NML-KO mice were resistant to HFD-induced obesity. Finally, using liver-specific NML-KO (liver-NML-KO) mice, we confirmed that hepatic NML deficiency caused the resistance to HFD-induced obesity. These results suggest that the repression of hepatic rRNA transcription induced by HFD is important for fat accumulation and energy storage. Ribosome biosynthesis is a major intracellular energy-consuming process. However, the relationship between ribosome biosynthesis and whole-body energy metabolism remains unknown. First, we evaluated hepatic pre-rRNA levels in WT mice (C57BL6) fed two different HFDs because the liver plays a major role in metabolism. Hepatic pre-rRNA levels decreased in the HFD-fed obese mice compared with the normal chow diet (NC)-fed mice (Figures 1A, 1B, and S1A). To examine the relationship between obesity and hepatic rRNA transcription, we analyzed hepatic pre-rRNA levels using obese model mice (ob/ob mice). The hepatic pre-rRNA levels in the ob/ob mice were also lower than those in the WT mice (Figures 1C and 1D). Next, to investigate the mechanism of repression of hepatic rRNA transcription in obese mice, we compared the epigenetic status of rRNA gene loci in the livers of NC- and HFD-fed mice using a chromatin immunoprecipitation (ChIP) assay. As shown in Figures 1F and 1G, HFD-induced obesity increased the dimethylation level of histone H3K9 and decreased the acetylation level of histone H3 on the rRNA gene loci in the liver. Furthermore, we examined the recruitment of NML and SIRT1 to the rRNA gene loci in the liver using the ChIP assay to investigate whether NML or SIRT1 induce repression of rRNA transcription in the livers of HFD-fed mice. The recruitment of NML and SIRT1 to the rRNA gene loci was promoted in the livers of HFD-fed mice (Figures 1H and 1I). However, NML and SIRT1 expression levels were not different between the livers of NC- and HFD-fed mice (Figures 1J, S1B, and S1C). Taken together, these results suggest that HFD feeding repressed rRNA transcription in the mouse liver by recruiting NML and SIRT1 in an epigenetic manner. Next, to clarify the relationship between repression of hepatic rRNA transcription and HFD-induced obesity, we generated NML-KO mice using standard homologous recombination techniques (Figures S2A–S2C). Some of the NML-KO mice died before birth (Figure S2D). Surviving NML-KO mice were fertile and grew normally (Figures S2E–S2G). Furthermore, we confirmed that NML deficiency did not affect liver histology or serum concentrations of ALT, AST, and albumin (Figures S2H and S2I). Therefore, the surviving NML-KO mice were subjected to further analysis. To examine the influence of hepatic NML deficiency, we evaluated the pre-rRNA expression level in the WT and NML-KO livers; pre-rRNA levels increased significantly in the NML-KO livers compared with the WT livers (Figures 2A and S2J). In contrast, the pre-rRNA level in other tissues did not change between the WT and NML-KO mice (Figure S2K). Coexpression of eNoSC (NML, SIRT1, and Suv39h1) was dominant in the liver (Figures 2B and S2L). These results indicate that eNoSC mainly functions in the liver. To confirm that NML deficiency affects chromatin modification on the rRNA gene loci, we performed the ChIP assay; the result indicated that NML deficiency reduced the amount of H3K9me2 associated with the rRNA gene loci in the liver (Figure 2C). In contrast, NML deficiency increased the amount of H3Ace associated with the rRNA gene loci in the liver (Figure 2D). Furthermore, NML deficiency reduced the recruitment of SIRT1 to the rRNA gene loci in the liver (Figure 2E). Consistent with these results, we confirmed that NML deficiency increased the recruitment of Pol I to the rRNA gene loci in the liver (Figure 2F). These results demonstrate that NML is involved in the epigenetic regulation of rRNA transcription in the liver. Because activation of rRNA transcription affects energy consumption in cultured cells, we hypothesized that NML deficiency induces acceleration of energy consumption in the liver. We measured the quantities of AMP and ATP in the liver using high-performance liquid chromatography (HPLC). We found that ATP concentration was lower in the NML-KO livers than in the WT livers, which resulted in a higher AMP/ATP ratio in the NML-KO livers (Figure 2G). In contrast, ATP and AMP concentrations in other tissues were not affected by NML deficiency (Figure S2M). An increase in the AMP/ATP ratio induces phosphorylation of and activates AMPK, which is a major cellular energy sensor and a master regulator of metabolic homeostasis (Hardie, 2011Hardie D.G. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function.Genes Dev. 2011; 25: 1895-1908Crossref PubMed Scopus (1078) Google Scholar, Viollet et al., 2009Viollet B. Athea Y. Mounier R. Guigas B. Zarrinpashneh E. Horman S. Lantier L. Hebrard S. Devin-Leclerc J. Beauloye C. et al.AMPK: lessons from transgenic and knockout animals.Front. Biosci. 2009; 14: 19-44Crossref PubMed Scopus (217) Google Scholar, Zhang et al., 2009Zhang B.B. Zhou G. Li C. AMPK: an emerging drug target for diabetes and the metabolic syndrome.Cell Metab. 2009; 9: 407-416Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). Therefore, we confirmed that the level of AMPKα phosphorylation at Thr172 (pAMPK) increased in the NML-KO livers compared with the WT livers (Figure 2H). Furthermore, the phosphorylation level of acetyl-coenzyme A (CoA) carboxylase (ACC), a direct target of AMPK activation (Carling et al., 1989Carling D. Clarke P.R. Zammit V.A. Hardie D.G. Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3- methylglutaryl-CoA reductase kinase activities.Eur. J. Biochem. 1989; 186: 129-136Crossref PubMed Scopus (330) Google Scholar), increased in the NML-KO livers compared with the WT livers (Figure S2N). These results suggest that NML deficiency promotes ATP consumption in the liver. Next, to examine whether energy starvation caused by the increase in rRNA transcription affects liver metabolism, we compared the gene-expression profiles of the WT and NML-KO livers using Affymetrix microarray technology. Ingenuity pathway analysis (IPA) identified lipid metabolism as one of the main pathways affected by NML deficiency (Figure 3A). Subsequently, we examined the expression of lipid-metabolism-related genes using quantitative RT-PCR (qRT-PCR). As shown in Figure 3B, the expression of FAO- and mitochondrial-activity-related genes (PGC1α, UCP2, CPT1, and PPARα) was significantly higher in the NML-KO livers than in the WT livers. In contrast, the expression of genes involved in de novo lipid synthesis of fatty acids (SCD1, FAS, and SREBP1c) was lower in the NML-KO livers than in the WT livers (Figure 3C). The expression of genes involved in glycolysis and gluconeogenesis did not significantly differ between the WT and NML-KO livers (Figure 3D). These results suggest that NML deficiency affected hepatic lipid metabolism. To assess whether NML directly regulates the expression of these genes, we examined the recruitment of NML to the promoter of these genes using the ChIP assay. NML was not recruited to the promoters of these genes (Figure S3A). NML deficiency did not affect the status of H3K9me2 in these genes (Figure S3B). In contrast, the promoters of SCD1, FAS, and SREBP1c genes decreased H3Ace levels (Figure S3C), whereas the promoters of PGC1α, UCP2, CPT1, and PPARα genes increased the levels (Figure S3C). Furthermore, to address the relationship between NML deficiency and lipid metabolism, we performed whole-genome chromatin immunoprecipitation sequencing (ChIP-seq) to determine genome-wide NML-binding targets. We identified 1,185 binding sites (p < 10−5), the majority of which were present in intergenic regions or introns of gene loci (Figure S3D). IPA indicated that the genes overrepresented as NML-binding targets were not related to lipid metabolism (Figure S3E). Because candidate peaks mapped to repetitive regions containing the rRNA gene loci were removed in ChIP-seq experiments (Rozowsky et al., 2009Rozowsky J. Euskirchen G. Auerbach R.K. Zhang Z.D. Gibson T. Bjornson R. Carriero N. Snyder M. Gerstein M.B. PeakSeq enables systematic scoring of ChIP-seq experiments relative to controls.Nat. Biotechnol. 2009; 27: 66-75Crossref PubMed Scopus (424) Google Scholar), we compared NML binding to the rRNA gene loci with that to some NML-binding targets identified in ChIP-seq using the ChIP assay. The recruitment of NML to the rRNA gene loci was higher than that to the NML-bound regions (Figure S3F). Collectively, these findings demonstrate that NML deficiency indirectly resulted in increased FAO and decreased de novo lipid synthesis of fatty acids in the liver. Because AMPK activation in the liver induces the FAO and reduces de novo lipid synthesis of fatty acids (Hardie et al., 2012Hardie D.G. Ross F.A. Hawley S.A. AMPK: a nutrient and energy sensor that maintains energy homeostasis.Nat. Rev. Mol. Cell Biol. 2012; 13: 251-262Crossref PubMed Scopus (2570) Google Scholar), lipid metabolism observed during NML deficiency may be due to AMPK activation. Next, to further investigate the differences in lipid metabolism in detail, we isolated primary hepatocytes from WT and NML-KO mice. The levels of pre-rRNA and protein synthesis increased significantly in the NML-KO hepatocytes (Figures 4A, 4B, and S4A). Furthermore, the protein degradation level increased in the NML-KO hepatocytes (Figure S4B). These processes involving rRNA synthesis, protein synthesis, and protein degradation consume intracellular ATP (Peth et al., 2013Peth A. Nathan J.A. Goldberg A.L. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.J. Biol. Chem. 2013; 288: 29215-29222Crossref PubMed Scopus (79) Google Scholar). In addition, a SAMS peptide assay revealed that AMPK activity was significantly higher in the NML-KO hepatocytes than in the WT hepatocytes (Figure 4C). This result suggests that NML deficiency activated these processes, which resulted in a decrease in intracellular ATP concentration. Moreover, these results show that the energy status of the NML-KO hepatocytes is similar to that of the NML-KO livers. To further determine whether hepatic NML deficiency affects lipid metabolic activity, we measured hepatic FAO in primary hepatocytes from the WT and NML-KO mice. We found that the [14C]oleate oxidation ratio was higher in the NML-KO hepatocytes than in the WT hepatocytes (Figure 4D). Measuring the oxygen consumption rate (OCR) before and after the addition of palmitate resulted in an increase in the NML-KO hepatocytes (Figure S4C). These results suggest that hepatic FAO is significantly elevated in the NML-KO hepatocytes. Consistent with these data, the expression of FAO- and mitochondrial-activity-related genes was significantly higher in the NML-KO hepatocytes than in the WT hepatocytes (Figure 4E). In contrast, [14C]acetate-incorporation into total lipid fraction was significantly lower in the NML-KO hepatocytes than in the WT hepatocytes (Figure 4F). This result indicates that de novo lipid synthesis is lower in the NML-KO hepatocytes than in the WT hepatocytes. The expression of genes involved in de novo lipid synthesis of fatty acids was consistently lower in the NML-KO hepatocytes than in the WT hepatocytes (Figure 4G). Next, to evaluate the metabolic profile in hepatocytes during basal respiration, we examined the extracellular acidification rate (ECAR) and OCR in the WT and NML-KO hepatocytes. No difference was observed in the ECAR data between the WT and NML-KO hepatocytes (Figure S4D). In contrast, OCR during basal respiration was higher in the NML-KO hepatocytes than in the WT hepatocytes (Figure S4E). To clarify the cause of the elevated OCR, we examined mitochondrial respiration. As shown in Figure 4H, the maximal respiratory capacity of the mitochondria was higher in the NML-KO hepatocytes than in the WT hepatocytes. However, the mitochondrial DNA copy number did not differ between the WT and NML-KO hepatocytes (Figure 4I). These results indicate that NML deficiency results in elevated mitochondrial respiration. Furthermore, the elevated OCR after the treatment of rotenone demonstrated the elevated nonmitochondrial respiration in the NML-KO hepatocytes (Figure 4H). These results suggest that NML deficiency increased total cellular respiration without the elevation of glycolysis. To examine whether NML affects repression of hepatic rRNA transcription induced by HFD, we examined pre-rRNA levels in HFD-fed WT and NML-KO livers. Hepatic pre-rRNA levels remained higher in the NML-KO mice after HFD feeding than in the WT mice (Figures 5A and S5A). This result indicates that NML regulates HFD-dependent repression of rRNA synthesis. Next, to evaluate the effect of NML deficiency on HFD-induced fat accumulation, we examined hepatic morphology in these mice. The livers of the HFD-fed WT mice were significantly larger and seemed to exhibit a fatty liver phenotype. In contrast, the livers of the HFD-fed NML-KO mice were not enlarged and remained red in color (Figure 5B). Oil red O staining clearly revealed that lipid accumulation was lower in the livers of HFD-fed NML-KO mice than in those of the HFD-fed WT mice (Figure 5C). In addition, hepatic triglyceride (TG), total cholesterol, and total free fatty acid (TFFA) contents were significantly lower in the livers of the HFD-fed NML-KO mice than in those of the HFD-fed WT mice (Figures 5D–5F). Furthermore, we confirmed that the expression of FAO-related genes in the livers was elevated in the HFD-fed NML-KO mice compared with the HFD-fed WT mice (Figure 5G). In contrast, expression of de novo lipid synthesis of fatty acids-related genes in the livers was decreased in the HFD-fed NML-KO mice compared with the HFD-fed WT mice (Figure 5H). These results indicate that NML deficiency induces resistance to HFD-induced hepatic steatosis via an increase in FAO and a reduction in lipid synthesis. Furthermore, our findings suggest that elevation of rRNA synthesis due to NML deficiency induced resistance to HFD-induced hepatic steatosis. Next, we investigated whether the change in lipid metabolism caused by NML deficiency influences whole-body energy metabolism. First, we found that the body weight of the NC- or HFD-fed NML-KO mice was lower than that of the NC- or HFD-fed WT mice (Figure 6A). Computed tomography (CT) analysis revealed that the visceral and subcutaneous fat masses decreased significantly in the HFD-fed NML-KO mice compared with the HFD-fed WT mice (Figure 6B). Furthermore, the ratio of white adipose tissue (WAT) weight to body weight was significantly lower in the HFD-fed NML-KO mice than in the HFD-fed WT mice (Figure 6C). The ratio of other tissue weights to body weight was not different between the WT and NML-KO mice (Figure S5B). In addition, the serum contents of TG, total cholesterol, and TFFA tended to be lower in the HFD-fed NML-KO mice (Figures 6D–6F). Serum glucose content was similar between the WT and NML-KO mice fed NC or HFD (Figure 6G). Food intake on NC feeding was higher in the NML-KO mice than in the WT mice (Figure 6H). However, food intake on HFD feeding did not change between the WT and NML-KO mice (Figure 6H). These results show that the NML-KO mice were leaner than the WT mice and had resistance to HFD-induced obesity. Energy expenditure in these mice was measured after feeding NC or HFD. As shown in Figure 6I, on normalizing O2 consumption to body weight, we found that O2 consumption of both the NC- and HFD-fed NML-KO mice increased significantly compared with that of both the NC- and HFD-fed WT mice. Furthermore, analysis of covariance (ANCOVA) (Tschöp et al., 2012Tschöp M.H. Speakman J.R. Arch J.R. Auwerx J. Brüning J.C. Chan L. Eckel R.H. Farese Jr., R.V. Galgani J.E. Hambly C. et al.A guide to analysis of mouse energy metabolism.Nat. Methods. 2012; 9: 57-63Crossref Scopus (488) Google Scholar) using genotype identified no differences in O2 consumption between the NC-fed NML-KO and WT mice (p = 0.296; Figure S5C). In contrast, the result of the HFD-fed mice by ANCOVA was significantly different (p = 0.049; Figure S5D). These results suggest that a highly significant increase in energy expenditure after HFD feeding was observed in the NML-KO mice compared with the WT mice. These differences in O2 consumption were not attributable to physical activity (Figure 6J) or body temperature (Figure 6K). Taken together, these findings indicate that NML deficiency may regulate whole-body energy expenditure after HFD feeding. Finally, to examine whether the increase in whole-body energy expenditure observed in the NML-KO mice was specifically caused by hepatic NML deficiency, we generated liver-specific NML-null mice (liver-NML-KO mice; Figures S6A and S6B). NML protein expression in liver-NML-KO mice decreased only in liver tissues but was detectable (Figure 7A). Because the liver-NML-KO mice are hepatocyte-specific, NML-deficient mice, their liver tissues are involved with other cells that express the NML protein. Therefore, we confirmed that primary hepatocytes purified from the liver-NML-KO mice did not express the NML protein (Figure S6C). The liver-NML-KO mice were not dead before birth, were fertile, and grew normally (Figures S6D–S6F). Hepatic NML deficiency did not affect liver histology or serum concentrations of ALT, AST, and albumin (Figures S6" @default.
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• W2018291804 title "Hepatic rRNA Transcription Regulates High-Fat-Diet-Induced Obesity" @default.
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