Abstract
Hibernating bears survive up to 6 months without feeding while yet maintaining metabolic homeostasis. We previously reported expression changes in energy metabolism-related genes in the liver of hibernating Japanese black bears. The present study examined the role of microRNAs in the regulation of hepatic gene expression during hibernation. The quantitative analyses revealed significant increases in the expression of 4 microRNAs (miR-221-3p, miR-222-3p, miR-455-3p, and miR-195a-5p) and decreases of 2 microRNAs (miR-122-5p and miR-7a-1-5p) during hibernation. RNA sequencing and in silico target prediction regarding 3 upregulated microRNAs (miR-221-3p, miR-222-3p and miR-455-3p) found 13 target mRNAs with significantly decreased expression during hibernation. The transfection of microRNA mimics into cells showed that miR-222 and miR-455 reduced solute carrier family 16 member 4 (SLC16A4) and fatty acid synthase (FASN) mRNA expression, respectively. Our results suggest that the increased levels of hepatic miRNA during hibernation (miR-222-3p and miR-455-3p) negatively regulate the expression of targeted genes predicted to be involved in the transport of energy source and de novo fatty acid synthesis, is consistent with a regulatory role of these miRNAs in energy metabolism in hibernating black bears.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of data and material
The data will be made available upon reasonable request.
References
Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355. https://doi.org/10.1038/nature02871
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Can Res 64(15):5245–5250. https://doi.org/10.1158/0008-5472.Can-04-0496
Andrews S (2010) FastQC; a quality control tool for high throughput sequence data. https://bioinformatics.babraham.ac.uk/projects/fastqc/
Andrews MT, Russeth KP, Drewes LR, Henry PG (2009) Adaptive mechanisms regulate preferred utilization of ketones in the heart and brain of a hibernating mammal during arousal from torpor. Am J Physiol Regul Integr Comp Physiol 296(2):R383–R393. https://doi.org/10.1152/ajpregu.90795.2008
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297. https://doi.org/10.1016/S0092-8674(04)00045-5
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. https://doi.org/10.1016/j.cell.2009.01.002
Carey HV, Andrews MT, Martin SL (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83(4):1153–1181. https://doi.org/10.1152/physrev.00008.2003
Chen GA, Gharib TG, Huang CC, Taylor JMG, Misek DE, Kardia SLR, Beer DG (2002) Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteom 1(4):304–313. https://doi.org/10.1074/mcp.M200008-MCP200
Choi SA, Choi HS, Kim KJ, Lee DS, Lee JH, Park JY, Kim MK (2013) Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells. Vitro Cell Dev Biol Anim 49(1):42–51. https://doi.org/10.1007/s11626-012-9569-x
Deng HX, Guo YN, Song HJ, Xiao BX, Sun WL, Liu Z, Guo JM (2013) MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer. Gene 518(2):351–359. https://doi.org/10.1016/j.gene.2012.12.103
Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS (2004) MicroRNA targets in Drosophila. Genome Biol 5(1):R1. https://doi.org/10.1186/gb-2003-5-1-r1
Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Monia BP (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 3(2):87–98. https://doi.org/10.1016/j.cmet.2006.01.005
Fedorov VB, Goropashnaya AV, Toien O, Stewart NC, Gracey AY, Chang CL, Barnes BM (2009) Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus). Physiol Genom 37(2):108–118. https://doi.org/10.1152/physiolgenomics.90398.2008
Fedorov VB, Goropashnaya AV, Toien O, Stewart NC, Chang C, Wang HF, Barnes BM (2011) Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus). BMC Genom 12:171. https://doi.org/10.1186/1471-2164-12-171
Frank CL, Brooks SPJ, Harlow HJ, Strorey KB (1998) The influence of hibernation patterns on the critical enzymes of lipogenesis and lipolysis in prairie dogs. Exp Biol Online 3:1–8. https://doi.org/10.1007/s00898-998-0009-z
Garofalo M, Quintavalle C, Romano G, Croce CM, Condorelli G (2012) miR221/222 in cancer: their role in tumor progression and response to therapy. Curr Mol Med 12(1):27–33. https://doi.org/10.2174/156652412798376170
Geiser F (2013) Hibernation. Curr Biol 23(5):R188–R193. https://doi.org/10.1016/j.cub.2013.01.062
Geiser F, Ruf T (1995) Hibernation versus daily torpor in mammals and birds—physiological variables and classification of torpor patterns. Physiol Zool 68(6):935–966. https://doi.org/10.1086/physzool.68.6.30163788
Gill RK, Saksena S, Alrefai WA, Sarwar Z, Goldstein JL, Carroll RE, Dudeja PK (2005) Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol 289(4):C846–C852. https://doi.org/10.1152/ajpcell.00112.2005
Gingras AC, Raught B, Sonenberg N (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68:913–963. https://doi.org/10.1146/annurev.biochem.68.1.913
Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8(8):1494–1512. https://doi.org/10.1038/nprot.2013.084
Hadj-Moussa H, Moggridge JA, Luu BE, Quintero-Galvis JF, Gaitan-Espitia JD, Nespolo RF, Storey KB (2016) The hibernating South American marsupial, Dromiciops gliroides, displays torpor-sensitive microRNA expression patterns. Sci Rep 6:24627. https://doi.org/10.1038/srep24627
Halestrap AP (2012) The monocarboxylate transporter family—structure and functional characterization. IUBMB Life 64(1):1–9. https://doi.org/10.1002/iub.573
Harlow HJ, Lohuis T, Beck TDI, Iaizzo PA (2001) Muscle strength in overwintering bears—unlike humans, bears retain their muscle tone when moribund for long periods. Nature 409(6823):997–997. https://doi.org/10.1038/35059165
Harlow HJ, Lohuis T, Anderson-Sprecher RC, Beck TDI (2004) Body surface temperature of hibernating black bears may be related to periodic muscle activity. J Mammal 85(3):414–419. https://doi.org/10.1644/1545-1542(2004)085%3c0414:Bstohb%3e2.0.Co;2
Hellgren EC (1998) Physiology of hibernation in bears. Ursus 10:467–477
Hilgendorf C, Ahlin G, Seithel A, Artursson P, Ungell AL, Karlsson J (2007) Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug Metab Dispos 35(8):1333–1340. https://doi.org/10.1124/dmd.107.014902
Huang PY, He ZY, Ji SY, Sun HW, Xiang D, Liu CC, Hui LJ (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475(7356):386-U142. https://doi.org/10.1038/nature10116
Inagaki A, Hayashi M, Andharia N, Matsuda H (2019) Involvement of butyrate in electrogenic K+ secretion in rat rectal colon. Pflugers Archiv Eur J Physiol 471(2):313–327. https://doi.org/10.1007/s00424-018-2208-y
Jansen HT, Trojahn S, Saxton MW, Quackenbush CR, Evans Hutzenbiler BD, Nelson OL, Kelley JL (2019) Hibernation induces widespread transcriptional remodeling in metabolic tissues of the grizzly bear. Commun Biol 2:336. https://doi.org/10.1038/s42003-019-0574-4
Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12(9):735–739. https://doi.org/10.1016/S0960-9822(02)00809-6
Lang-Ouellette D, Morin PJ (2014) Differential expression of miRNAs with metabolic implications in hibernating thirteen-lined ground squirrels Ictidomys tridecemlineatus. Mol Cell Biochem 394(1–2):291–298. https://doi.org/10.1007/s11010-014-2105-4
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20. https://doi.org/10.1016/j.cell.2004.12.035
Lin YW, Wu J, Chen H, Mao YQ, Liu YF, Mao QQ, Xie LP (2012) Cyclin-dependent kinase 4 is a novel target in micoRNA-195-mediated cell cycle arrest in bladder cancer cells. FEBS Lett 586(4):442–447. https://doi.org/10.1016/j.febslet.2012.01.027
Liu YT, Hu WC, Wang HF, Lu MH, Shao CX, Menzel C, Yan J (2010) Genomic analysis of miRNAs in an extreme mammalian hibernator, the Arctic ground squirrel. Physiol Genom 42(1):39–51. https://doi.org/10.1152/physiolgenomics.00054.2010
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Logan SM, Storey KB (2016) Tissue-specific response of carbohydrate-responsive element binding protein (ChREBP) to mammalian hibernation in 13-lined ground squirrels. Cryobiology 73(2):103–111. https://doi.org/10.1016/j.cryobiol.2016.09.002
Lustig Y, Barhod E, Ashwal-Fluss R, Gordin R, Shomron N, Baruch-Umansky K, Kanety H (2014) RNA-binding protein PTB and microRNA-221 coregulate AdipoR1 translation and adiponectin signaling. Diabetes 63(2):433–445. https://doi.org/10.2337/db13-1032
Luu BE, Lefai E, Giroud S, Swenson JE, Chazarin B, Gauquelin-Koch G, Storey KB (2020) MicroRNAs facilitate skeletal muscle maintenance and metabolic suppression in hibernating brown bears. J Cell Physiol 235(4):3984–3993. https://doi.org/10.1002/jcp.29294
Mandal S, Guptan P, Owusu-Ansah E, Banerjee U (2005) Mitochondrial regulation of cell cycle progression during development as by the tenured mutation revealed in Drosophila. Dev Cell 9(6):843–854. https://doi.org/10.1016/j.devcel.2005.11.006
Martin EM, Messenger KM, Sheats MK, Jones SL (2017) Misoprostol inhibits lipopolysaccharide-induced pro-inflammatory cytokine production by equine leukocytes. Front Vet Sci 4:160. https://doi.org/10.3389/fvets.2017.00160
Ma L, Tsatsos NG, Towle HC (2005) Direct role of ChREBP center dot Mlx in regulating hepatic glucose-responsive genes. J Biol Chem 280(12):12019–12027. https://doi.org/10.1074/jbc.M413063200
McCain S, Ramsay E, Kirk C (2013) The effects of hibernation and captivity on glucose metabolism and thyroid hormones in American black bear (Ursus americanus). J Zoo Wildl Med 44(2):324–332. https://doi.org/10.1638/2012-0146r1.1
McGee-Lawrence ME, Wojda SJ, Barlow LN, Drummer TD, Castillo AB, Kennedy O, Donahue SW (2009) Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation). Bone 45(6):1186–1191. https://doi.org/10.1016/j.bone.2009.08.011
Mimura S, Iwama H, Kato K, Nomura K, Kobayashi M, Yoneyama H, Masaki T (2014) Profile of microRNAs associated with aging in rat liver. Int J Mol Med 34(4):1065–1072. https://doi.org/10.3892/ijmm.2014.1892
Miyazaki M, Shimozuru M, Tsubota T (2019) Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation. PLoS ONE 14(4):e0215489. https://doi.org/10.1371/journal.pone.0215489
Morris ME, Felmlee MA (2008) Overview of the proton-coupled MCT (SLC16A) family of transporters: characterization, function and role in the transport of the drug of abuse gamma-hydroxybutyric acid. Aaps J 10(2):311–321. https://doi.org/10.1208/s12248-008-9035-6
Mostafa N, Everett DC, Chou SC, Kong PA, Florant GL, Coleman RA (1993) Seasonal-changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris). J Comp Physiol B Biochem Syst Environ Physiol 163(6):463–469
Naderi M, Pazouki A, Arefian E, Hashemi SM, Jamshidi-Adegani F, Gholamalamdari O, Soleimani M (2017) Two triacylglycerol pathway genes, CTDNEPI and LPIN1 are down-regulated by hsa-miR-122-5p in hepatocytes. Arch Iran Med 20(3):165–171
Nelson RA, Wahner HW, Jones JD, Ellefson RD, Zollman PE (1973) Metabolism of bears before, during, and after winter sleep. Am J Physiol 224(2):491–496
Nelson RA, Folk GEJ, Pfeiffer EW, Craighead JJ, Jonkel CJ, Steiger DL (1983) Behavior, biochemistry, and hibernation in black, grizzly, and polar bears. Ursus 5:284–290
Nitta S, Kusakari Y, Yamada Y, Kubo T, Neo S, Igarashi H, Hisasue M (2020) Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a. Regener Ther 14:165–176. https://doi.org/10.1016/j.reth.2020.01.003
Okamatsu-Ogura Y, Nio-Kobayashi J, Nagaya K, Tsubota A, Kimura K (2018) Brown adipocytes postnatally arise through both differentiation from progenitors and conversion from white adipocytes in Syrian hamster. J Appl Physiol 124(1):99–108. https://doi.org/10.1152/japplphysiol.00622.2017
Paulauskis JD, Sul HS (1989) Hormonal-regulation of mouse fatty-acid synthase gene-transcription in liver. J Biol Chem 264(1):574–577
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515. https://doi.org/10.1023/B:Bile.0000019559.84305.47
Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8(1):23–36. https://doi.org/10.1038/nrm2085
Rigano KS, Gehring JL, Hutzenbiler BDE, Chen AV, Nelson OL, Vella CA, Jansen HT (2017) Life in the fat lane: seasonal regulation of insulin sensitivity, food intake, and adipose biology in brown bears. J Comp Physiol B Biochem Syst Environ Physiol 187(4):649–676. https://doi.org/10.1007/s00360-016-1050-9
Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11(3):R25. https://doi.org/10.1186/gb-2010-11-3-r25
Sancho-Bru P, Roelandt P, Narain N, Pauwelyn K, Notelaers T, Shimizu T, Verfaillie C (2011) Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells. J Hepatol 54(1):98–107. https://doi.org/10.1016/j.jhep.2010.06.014
Shimozuru M, Kamine A, Tsubota T (2012) Changes in expression of hepatic genes involved in energy metabolism during hibernation in captive, adult, female Japanese black bears (Ursus thibetanus japonicus). Comp Biochem Physiol B Biochem Mol Biol 163(2):254–261. https://doi.org/10.1016/j.cbpb.2012.06.007
Shimozuru M, Iibuchi R, Yoshimoto T, Nagashima A, Tanaka J, Tsubota T (2013) Pregnancy during hibernation in Japanese black bears: effects on body temperature and blood biochemical profiles. J Mammal 94(3):618–627. https://doi.org/10.1644/12-Mamm-a-246.1
Shimozuru M, Nagashima A, Tanaka J, Tsubota T (2016) Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears. Comp Biochem Physiol B Biochem Mol Biol 196:38–47. https://doi.org/10.1016/j.cbpb.2016.02.001
Si-Tayeb K, Noto FK, Nagaoka M, Li JX, Battle MA, Duncan SA (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51(3):1094–1094. https://doi.org/10.1002/hep.23526
Smolarek TA, Higgins CV, Amacher DE (1990) Metabolism and cytotoxicity of acetaminophen in hepatocyte cultures from rat, rabbit, dog, and monkey. Drug Metab Dispos 18(5):659–663
Song JL, Ouyang YM, Che JY, Li XM, Zhao Y, Yang KJ, Yuan WE (2017) Potential value of miR-221/222 as diagnostic, prognostic, and therapeutic biomarkers for diseases. Front Immunol 8:56. https://doi.org/10.3389/fimmu.2017.00056
Srere HK, Wang LCH, Martin SL (1992) Central role for differential gene-expression in mammalian hibernation. Proc Natl Acad Sci USA 89(15):7119–7123. https://doi.org/10.1073/pnas.89.15.7119
Tavoni SK, Champagne CD, Houser DS, Crocker DE (2013) Lactate flux and gluconeogenesis in fasting, weaned northern elephant seals (Mirounga angustirostris). J Comp Physiol B Biochem Syst Environ Physiol 183(4):537–546. https://doi.org/10.1007/s00360-012-0720-5
Tøien Ø, Blake J, Edgar DM, Grahn DA, Heller HC, Barnes BM (2011) Hibernation in black Bears: independence of metabolic suppression from body temperature. Science 331(6019):906–909. https://doi.org/10.1126/science.1199435
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511-U174. https://doi.org/10.1038/nbt.1621
Wong RHF, Sul HS (2010) Insulin signaling in fatty acid and fat synthesis: a transcriptional perspective. Curr Opin Pharmacol 10(6):684–691. https://doi.org/10.1016/j.coph.2010.08.004
Wu CW, Storey KB (2012) Pattern of cellular quiescence over the hibernation cycle in liver of thirteen-lined ground squirrels. Cell Cycle 11(9):1714–1726. https://doi.org/10.4161/cc.19799
Wu CW, Biggar KK, Luu BE, Szereszewski KE, Storey KB (2016) Analysis of microRNA expression during the torpor-arousal cycle of a mammalian hibernator, the 13-lined ground squirrel. Physiol Genom 48(6):388–396. https://doi.org/10.1152/physiolgenomics.00005.2016
Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, Kadowaki T (2007) Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat Med 13(3):332–339. https://doi.org/10.1038/nm1557
Zhao YX, Yan MY, Yun Y, Zhang JG, Zhang RM, Li Y, Jiang HS (2017) MicroRNA-455-3p functions as a tumor suppressor by targeting eIF4E in prostate cancer. Oncol Rep 37(4):2449–2458. https://doi.org/10.3892/or.2017.5502
Acknowledgements
We wish to thank Dr. Takeshi Komatsu and all the staff at the Ani Mataginosato Bear Park for their generous support. This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (16K08067).
Funding
This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (16K08067).
Author information
Authors and Affiliations
Contributions
Design of the study: MS. Sample collection: KN, MS, TS and MM molecular biological analysis: KN, MS, YO, TS and MM. Interpretation of the data: KN, MS, MS and TT. Drafting of manuscript: KN and MS. All authors have read and accepted the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Ethics approval
All procedures and animals care were conducted in accordance with the Guidelines for Animal Care and Use of Hokkaido University and all procedures were approved by the Animal Care and Use Committee of the Graduate School of Veterinary Medicine, Hokkaido University (permit nos. 17006 and 18-0179).
Consent to participate
Not applicable.
Consent to publication
Not applicable.
Coda availability
Not applicable.
Additional information
Communicated by H.V. Carey.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Nishida, K., Shimozuru, M., Okamatsu-Ogura, Y. et al. Changes in liver microRNA expression and their possible regulatory role in energy metabolism-related genes in hibernating black bears. J Comp Physiol B 191, 397–409 (2021). https://doi.org/10.1007/s00360-020-01337-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00360-020-01337-7