人体生理学 (7).pdf

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1、PHYSIOLOGYCopyright 2018The Authors,somerights reserved;exclusive licenseeAmerican Associationfor the Advancementof Science.No claim tooriginalU.S.GovernmentWorks.Distributedunder a CreativeCommons AttributionNonCommercialLicense 4.0(CC BY-NC).Acute sleep loss results in tissue-specific alterationsi

2、n genome-wide DNA methylation state andmetabolic fuel utilization in humansJonathan Cedernaes1*,Milena Schnke2,Jakub Orzechowski Westholm3,Jia Mi4,5,Alexander Chibalin2,Sarah Voisin1,Megan Osler2,Heike Vogel6,Katarina Hrnaeus4,Suzanne L.Dickson7,Sara Bergstrm Lind4,Jonas Bergquist4,8,9,Helgi B Schit

3、h1,Juleen R.Zierath2,Christian Benedict1Curtailed sleep promotes weight gain and loss of lean mass in humans,although the underlying molecularmechanisms are poorly understood.We investigated the genomic and physiological impact of acute sleep lossin peripheral tissues by obtaining adipose tissue and

4、 skeletal muscle after one night of sleep loss and after onefull night of sleep.We find that acute sleep loss alters genome-wide DNA methylation in adipose tissue,andunbiased transcriptome-,protein-,and metabolite-level analyses also reveal highly tissue-specific changes that arepartially reflected

5、by altered metabolite levels in blood.We observe transcriptomic signatures of inflammationin both tissues following acute sleep loss,but changes involving the circadian clock are evident only in skeletalmuscle,and we uncover molecular signatures suggestive of muscle breakdown that contrast with an a

6、nabolicadipose tissue signature.Our findings provide insight into how disruption of sleep and circadian rhythms maypromote weight gain and sarcopenia.INTRODUCTIONChronic sleep loss,social jet lag,and shift workwidespread in ourmodern24/7societiesareassociatedwithanincreasedriskofnumer-ous metabolic

7、pathologies,including obesity,metabolic syndrome,andtype2diabetes(14).Evenminorweeklyshiftsinsleeptiming,orasfewas five consecutive nights of short sleep,have been associated with anincreased risk of weight gain in healthy humans(4,5).Many of the adverse effects attributed to sleep loss and circadia

8、nmisalignment might arise due to tissue-specific metabolic perturba-tions in peripheral tissues such as skeletal muscle and adipose tissue(69).Recurrent sleep loss combined with moderate calorie restric-tion in humans increases the loss of fat-free body mass,whiledecreasing the proportion of weight

9、lost as fat(10),suggesting thatsleep loss can promote adverse tissue-specific catabolism and anab-olism.Human cohort studies and interventional sleep restrictionstudies in animals also suggest that sleep loss specifically promoteslossofmusclemass(1113),buttheunderlyingmolecularmechanismsremain elusi

10、ve.Notably,sleep restriction studies controlling for caloric intakeprovide evidence that sleep loss reduces the respiratory exchange ratio(8,14),indicating a shift toward non-glucose,that is,fatty acid,oxida-tion.Animal studies have elegantly shown that metabolic fuel selectionand overall anabolic v

11、ersus catabolic homeostasis are regulated bytissue-specific rhythms driven by the core circadian clock(15).Keymetabolic processes,for example,glycolysis and mitochondrial oxida-tive metabolism,exhibit 24-hour rhythms in tissues such as skeletalmuscle(1618).This is,to a significant extent,orchestrate

12、d throughcircadianregulationofkeytranscriptionfactorsandenzymessuchaspyruvate dehydrogenase kinase 4(Pdk4),Ldhb,and phosphofructo-kinase2(Pfk2),whichbelongtosomeofthemosthighlyrhythmictran-scripts in skeletal muscle across circadian data sets in mice(19).Correspondingly,ablationofthecoreclockgeneBma

13、l1altersmetabolicfuel utilization in mice(20,21),and circadian desynchrony in humansresults in decreased resting metabolic rate(22).Furthermore,even asingle night of sleep loss has been shown to induce tissue-specific tran-scriptional and DNA methylation(an epigenetic modification thatcan regulate c

14、hromatin structure and gene expression)changes to corecircadianclockgenesinhumans(23),butthedownstreamtissue-specificimpactonmetabolicpathwaysremainstobedetermined.Moreover,itispresentlyunknowntowhatextentDNAmethylationmaybemodulatedthroughoutthehumangenomeinmetabolictissuesinresponsetoacutesleep lo

15、ss,and whether metabolic tissues respond in a tissue-specificmanner across multiple genomic and molecular levels.Onthebasisoftheaboveobservations,andasa modelofshiftworkthat often entails overnight wakefulness,we hypothesized that acutesleep loss(that is,overnight wakefulness)would induce tissue-spe

16、cificalterations at the genomic and physiological levels in pathways regulat-ing metabolic substrate utilization and anabolic versus catabolic state.Specifically,we expected acute sleep loss to increase non-glycolyticoxidationandproteinbreakdowninskeletalmuscle(12,13),withtheformer favoring hypergly

17、cemia.Sincerecurrentsleep losshasalsobeenlinkedtoadverseweightgain(2,10,24),wealsohypothesizedthatacutesleep loss would promote signatures of increased adipogenesis and thatsome of these tissue-specific changes would be reflected at the DNAmethylation level,indicating altered“metabolic memory.”To th

18、isend,we carried out a range of molecular analyses in subcutaneousadipose tissue and skeletal muscle samples,complemented by analysesin blood,in samples obtained from healthy young men both after anight of sleep loss and after a night of full sleep.1Department of Neuroscience,Uppsala University,Upps

19、ala,Sweden.2Departmentof Molecular Medicine and Surgery,Karolinska Institutet,Solna,Sweden.3Sciencefor Life Laboratory,Department of Biochemistry and Biophysics,Stockholm Uni-versity,Stockholm,Sweden.4Department of ChemistryBMC,Uppsala University,Uppsala,Sweden.5Medicine and Pharmarcy Research Cente

20、r,Binzhou MedicalUniversity,Yantai,China.6Department of Experimental Diabetology,German In-stitute of Human Nutrition Potsdam-Rehbruecke,Potsdam,Germany.7Depart-ment of Physiology/Endocrinology,Institute of Neuroscience and Physiology,Sahlgrenska Academy,University of Gothenburg,Gothenburg,Sweden.8D

21、epart-ment of Pathology,University of Utah,Salt Lake City,UT 84132,USA.9PrecisionMedicine,Binzhou Medical University,Yantai,China.*Corresponding author.Email:jonathan.cedernaesneuro.uu.seThese authors contributed equally to this work.SCIENCE ADVANCES|RESEARCH ARTICLECedernaes et al.,Sci.Adv.2018;4:e

22、aar859022 August 20181 of 14RESULTSAcute sleep loss results in tissue-specific DNA methylationand transcriptomic changesToexaminewhetheracutesleeplossinducesgenome-widealterationsinepigenetic modifications,we used the Infinium HumanMethylation450BeadChip(485,764probes)tointerrogatechangesinDNAmethyl

23、ationin adipose tissue and skeletal muscle samples obtained from 15 healthyparticipants in the morning fasting state,both after one night of sleeploss and after a night of normal sleep(age,22.3 0.5 years;body massindex,22.6 0.5 kg/m2;further characteristics and sleep data arepresented in table S1,an

24、d experimental design is shown in Fig.1A).Wefoundthatsleeplossresultedin148significantdifferentiallymethy-lated regions(DMRs)false discovery rate(FDR)0.05 in sub-cutaneous adipose tissue(Fig.1B and table S2,A and B),most ofwhich were hypermethylated(92 DMRs)and within 5 kilobase pairsof the transcri

25、ption start site(TSS)(129 DMRs or 87%).To investigatewhich gene pathways were associated with altered methylation statusfollowing sleep loss,we used gene ontology(GO)analyses to identifywhich biological pathways were enriched for genes close to our signif-icantDMRs(Fig.1C).WhenthedirectionalityofDNA

26、methylationwasnot considered,we found that pathways associated with,for example,lipid metabolism,cell differentiation,and DNA damage response werealtered(table S2C).Next,to gain a better understanding of whetherthese pathways were driven by increased or decreased DNA methyl-ation for specific genes

27、in adipose tissue in response to sleep loss,weseparately investigated pathways associated with the identified hyper-methylated versus hypomethylated DMRs.Notably,hypermethylatedgenes were found to enrich for biological pathways such as lipid re-sponse and cell differentiation,whereas hypomethylated

28、genes wererelatedtopathwayssuchasDNAdamageresponseregulationandlipidmetabolism(table S2,D and E).We found that sleep loss resulted inhypermethylation for DMRs near the TSS of genes that have beenobservedtobeinahypermethylatedstatebeforegastricbypasssurgery,such as TNXB,TRIM2,and FOXP2(Fig.1D and tab

29、le S2A)(25).Wealso observed altered methylation near the TSS of genes involved inadipogenesis:CD36,AKR1CL1(an aldose reductase),and HOXA2,with the latter being hypermethylated.Specifically,we found thatHOXA2,a homeobox transcription factor,was hypermethylatednearitsTSS,which,throughalteredDNAmethyla

30、tionandgeneexpres-sion level,has been found to distinguish adipogenesis in subcutaneousBaseline period(26 hours with 8.5 hours sleep opportunity)Sleep intervention(22300700 h)Morning intervention(07001200 h)3 daily isocaloric meals8.5 hours sleep opportunityAcute sleep deprivationStandardized physic

31、al activity patternDNA methylationTranscriptomicsProteomicsMetabolomicsExperimental overviewOmics analysis pipelineSAT biopsyVLM biopsyOGTTABDifferentially methylated regions(DMRs)in adipose tissueOOHNH2NHGTAGTCACTTATG.ACACCCCTTTCAAACTGTAACAGTGAAAGATGACACCTACTCAGGCTTGTTCTGTGTATCGGGAGAATCAGTCTATCTTGA

32、TCACOCTACTCGTCTATCTTGDSpecific DMRs in adipose tissueSleepWakeBeta levelTSPAN32TNXBGNASGFI1TRIM2FOXP20.50.40.30.80.70.60.50.40.70.60.50.60.50.40.30.60.50.40.30.60.50.40.30.700.660.620.840.820.800.780.90.80.70.50.40.30.2INSHOXA2CD36AKR1CL1Subject1234567891011121314153210123SubjectCPathways enriched f

33、or hyper-and hypomethylatedDMRs in adipose tissue0.020.040.06Response to external stimulusResponse to lipidCell morphogenesis involved in differentiationNegative regulation of STAT cascadePlasma lipoprotein particle clearanceCellular response to lipidFatty acid transport1 1032 1033 103DE-to-Nq value

34、Positive regulation of DNA damage responsePositive regulation of signal transductionby p53 class mediatorRegulation of DNA damage response,signal transduction by p53 class mediatorRegulation of lipid metabolic processRegulation of fatty acid metabolic processApoptotic signaling pathwayPositive regul

35、ation of lipid metabolic process0.050.100.150.206 1044 1042 1048 104DE-to-Nq valuePositive regulation of response toDNA damage stimulusNegative regulation of JAK-STAT cascadeSleepWakeFig.1.Acute sleep loss induces changes in DNA methylation in adipose tissue in healthy humans.(A)Participants were in

36、vestigated both after a night of sleeploss(that is,overnight wakefulness)and after a night of normal sleep,in each condition after an in-lab baseline day and night(26 hours in total,with an 8.5-hourbaseline sleep opportunity)with standardized physical activity levels and isocaloric meals.Biopsies fr

37、om the vastus lateralis muscle(VLM)and subcutaneous adiposetissue(SAT),as well as fasting blood sampling,preceded an oral glucose tolerance test(OGTT)and subsequent blood sampling.This was followed by a pipeline of omicanalyses across tissues.(B)Differentially methylated regions(DMRs;FDR 0.05)in adi

38、pose tissue showing DNA methylation(beta levels)after sleep and sleep loss(wake)across the 15 participants,with hierarchical clustering of DMR beta levels(z scores).(C)Significant gene ontology(GO)annotations based on hypermethylated(top)and hypomethylated DMRs(bottom)in adipose tissue in response t

39、o sleep loss,showing the ratio of differentially expressed gene-associated DMRs(DE)to thetotal number(N)of genes in a given pathway(“DE-to-N”)and adjusted P values(q values,FDR 0.05).(D)Beta levels across some of the most significant DMRs inadipose tissue,in proximity to the specified genes,followin

40、g sleep and sleep loss.SCIENCE ADVANCES|RESEARCH ARTICLECedernaes et al.,Sci.Adv.2018;4:eaar859022 August 20182 of 14whiteadiposetissuefromthatin,forexample,brownorvisceraladiposetissue(2628).Notably,several of the 56 DMRs that were hypomethy-latedinresponsetosleeplosswereinchromosomalregionsornear

41、theTSS of genes known to be genomically imprinted(such as TSPAN32,GNAS,INS,andGFI1;Fig.1DandtableS2B)and,throughthisorothermechanisms,have been associated with obesity(29,30).Hypomethy-lated DMRs were also found at the TSS of genes implicated in insulinresponse or type 2 diabetes(for example,INS,CPT

42、1A)as well as lipol-ysis or beiging(ADORA2A)(3133).Whereas the average fold changeinmethylation forthe significantDMRs did notexceed 3%inresponseto sleep loss(average,+2.8 0.0%and 2.4 0.0%for hyper-andhypomethylatedDMRs,respectively),themosthighlyhypermethylatedDMR(onaverage+6.9%)wasfoundforCD36(Fig

43、.1DandtableS2A),whichisinvolvedinfattyacidimportandwhoseexpressionisdysregu-lated in obese and type 2 diabetic patients(34).In contrast to adipose tissue,no significant DMRs were observed inskeletal muscle following sleep loss compared with sleep(table S2F).This finding could indicate that other epi

44、genetic modificationsthatmay also respond to environmental changes(for example,at the chro-matinlevel)regulatethetranscriptionalresponsetosleeplossinskeletalmuscle or,alternatively,that DNA methylation changes occur at,for ex-ample,earlier or later time points in our intervention.To assess genome-wi

45、de gene expression changes following acutesleep loss in humans in the morning hours,we next performed tran-scriptomicRNAsequencing(RNA-seq)analysesoftotalRNAisolatedfromthecorrespondingskeletalmuscleandadiposetissuesamples.Wefound that acute sleep loss altered expression of117(19 up-regulated,98 dow

46、n-regulated)mRNA transcripts in skeletal muscle,whereas96 transcripts(59 up-regulated,37 down-regulated)were significantlyalteredinsubcutaneousadiposetissue(tableS3,AtoD).Acomparisonof transcripts that were significantly altered either in skeletal muscle orinsubcutaneousadiposetissuerevealedthatmany

47、transcriptsexhibitedtissue-specific directionalities(that is,with regard to their fold change)in response to sleep loss compared with sleep(Fig.2,A and B).Inaddition,almost no overlap was found between the two tissues formRNA transcripts that were differentially expressed(Fig.2C),furtherhighlighting

48、thetissuespecificityoftheresponsetoacutesleeplossinhuman metabolic tissues.An untargeted analysis of all DNA methyla-tion values versus all corresponding mRNA transcript levels confirmedthat,overall,the degree of methylation was negatively correlated with thelevelofgeneexpression,aphenomenonobserved

49、inbothadiposetissue(Spearman rs=0.39)and skeletal muscle(Spearman rs=0.41;fig.S1,A and B).Notably,however,when we next compared the changes inDNA methylation and mRNA transcript levels that were observedfollowingacutesleep loss,neithertissueexhibitedanysignificantcorre-lation between DNA methylation

50、 and transcript expression levels(ad-ipose tissue,Spearman rs=0.01;skeletal muscle,Spearman rs=0.00;fig.S1,C and D).Similarly,no overlap was found when comparingCBADSATsleepSAT wakeVLM wakeVLM sleepSATsleepSAT wakeVLM wakeVLM sleepFig.2.Tissue-specific transcriptomic alterations in response to acute