influences of breakfast on clock - diabetes care · breakfast consumption acutely affects clock and...

Influences of Breakfast on ClockGene Expression and PostprandialGlycemia in Healthy Individualsand Individuals With DiabetesA Randomized Clinical TrialDiabetes Care 2017401573ndash1579 | httpsdoiorg102337dc16-2753

OBJECTIVE

The circadian clock regulates glucose metabolism by mediating the activity of met-abolic enzymes hormones and transport systems Breakfast skipping and nighteating have been associated with high HbA1c and postprandial hyperglycemia afterlunch and dinner Our aim was to explore the acute effect of breakfast consumptionor omission on glucose homeostasis and clock gene expression in healthy individualsand individuals with type 2 diabetes

RESEARCH DESIGN AND METHODS

In a crossover design 18 healthy volunteers and 18 volunteers with 1456 15 yearsdiabetes BMI 3076 11 kgm2 and HbA1c 766 01 (5966 08 mmolmol) wererandomly assigned to a test day with breakfast and lunch (YesB) and a test day withonly lunch (NoB) Postprandial clock and clock-controlled gene expression plasmaglucose insulin intact glucagon-like peptide 1 (iGLP-1) and dipeptidyl peptidase IV(DPP-IV) plasma activity were assessed after breakfast and lunch

RESULTS

In healthy individuals the expression level of Per1 Cry1 Rora and Sirt1 was lower(P lt 005) but Clock was higher (P lt 005) after breakfast In contrast in individualswith type 2 diabetes Per1 Per2 and Sirt1 only slightly but significantly decreasedand Rora increased (P lt 005) after breakfast In healthy individuals the expressionlevel ofBmal1Rora and Sirt1was higher (Plt 005) after lunch on YesB daywhereasthe other clock genes remained unchanged In individuals with type 2 diabetesBmal1 Per1 Per2 Rev-erba and Ampk increased (P lt 005) after lunch on the YesBday Omission of breakfast altered clock and metabolic gene expression in bothhealthy and individuals with type 2 diabetes

CONCLUSIONS

Breakfast consumption acutely affects clock and clock-controlled gene expressionleading to normal oscillation Breakfast skipping adversely affects clock and clock-controlled gene expression and is correlated with increased postprandial glycemicresponse in both healthy individuals and individuals with diabetes

1Diabetes Unit Wolfson Medical Center SacklerFaculty of Medicine Tel Aviv University HolonIsrael2Diabetes Unit Hadassah University HospitalEin Kerem Hospital Hadassah Medical SchoolThe Hebrew University of Jerusalem JerusalemIsrael3Department of Clinical Sciences Faculty ofMedicine Lund University Lund Sweden4Institute of Biochemistry Food Science and Nu-trition The Robert H Smith Faculty of AgricultureFood and Environment The Hebrew University ofJerusalem Rehovot Israel5Department of Molecular Genetics Faculty ofBiochemistry Weizmann Institute of ScienceRehovot Israel

Corresponding authors Oren Froy orenfroymailhujiacil and Daniela Jakubowicz danielajakgmailcom

Received 25 December 2016 and accepted 30July 2017

Clinical trial reg no NCT01939782 clinicaltrialsgov

This article contains Supplementary Data onlineat httpcarediabetesjournalsorglookupsuppldoi102337dc16-2753-DC1

This article is featured in a podcast available athttpwwwdiabetesjournalsorgcontentdiabetes-core-update-podcasts

DJ and JW contributed equally to this work

copy 2017 by the American Diabetes AssociationReaders may use this article as long as the workis properly cited the use is educational and notfor profit and the work is not altered More infor-mation is available at httpwwwdiabetesjournalsorgcontentlicense

Daniela Jakubowicz1 Julio Wainstein1

Zohar Landau1 Itamar Raz2 Bo Ahren3

Nava Chapnik4 Tali Ganz1

Miriam Menaged1 Maayan Barnea5

Yosefa Bar-Dayan1 and Oren Froy4

Diabetes Care Volume 40 November 2017 1573

CARDIOVASCULA

RANDMETA

BOLIC

RISK

The circadian clock controls the activity ofmost enzymes hormones and transportsystems involved in glucose metabolism(12) The central circadian clock locatedin the suprachiasmatic nuclei of the ante-rior hypothalamus generates endoge-nous 24-h rhythms Similar clocks arefound in peripheral tissues such as theliver b-cells muscle and adipose tissue(3ndash5) The core clock mechanism in thebrain and peripheral tissues comprisestwo loops The positive loop consists ofthe CLOCK and BMAL1 heterodimer thatmediates transcription of tissue-specificgenes and those of the negative feedbackloop The negative feedback loop consistsof the period (PER) and cryptochrome(CRY) proteins that inhibit CLOCKBMAL1-mediated transcription (6)Unlike the suprachiasmatic nuclei clock

which responds mainly to the light-darkcycle peripheral clocks respond to mealcontent and timing leading to coordi-nated regulation of digestive and absorp-tive functions and hormone secretionthereby preventing metabolic dysregulation(1ndash4) CLOCKBMAL1 heterodimer plays acritical role in mediating the transcriptionof coactivators that regulate the circadiansynthesis ofmost of the enzymes andhor-mones involved in glucose homeostasisie regulation of hepatic gluconeogene-sis and pancreatic b-cell insulin secretion(7) CRY proteins inhibit gluconeogenicgene expression by regulating CREBP ac-tivity and their hepatic depletion in-creases circulating glucose and CRYoverexpression reduces fasting blood glu-coseand improveswhole-body insulin sen-sitivity in obese mice (7) CLOCKBMAL1heterodimer regulates the expression ofRev-erba andRora (8ndash10) REV-ERBa thenegative regulator of Bmal1 expression(8) is induced during normal adipogenesisand mediates a suppressive effect on he-patic gluconeogenesis and glucose outputby regulating the expression of PEPCKand glucose-6-phosphatase (G6pase)Depletion of REV-ERBa leads to hyper-glycemia (27) In contrast RORa the pos-itive regulator of Bmal1 expression (10)activates the hepatic gluconeogenic en-zyme G6pase and regulates lipogenesisand lipid storage in skeletal muscle (210)AMPK the cellular energy sensor also

plays a key role in the clockmechanismbyenhancing degradation of PER and CRY pro-teins (11) Upregulation of AMPK signalingsignificantly enhances GLUT4 translocationand muscular glucose uptake ensuring

metabolic efficiency and improving post-prandial glucose and insulin responses(12) AMPK exerts a positive effect onSIRT1 associated with beneficial effectson b-cell viability and insulin sensitivitywhich interacts directly with CLOCK anddeacetylates BMAL1 and PER2 (12ndash15)

Reduced glucose-stimulated insulin se-cretion insulin resistance diminishedb-cell proliferation and apoptosis havebeen associated with asynchrony or defi-ciencies in clock genes (16) Moreoverlower transcripts of Bmal1 and Cry2 areinversely correlated with HbA1c levels(1718) Furthermore higher risk of obe-sity metabolic alterations and type 2 di-abetes have been found in shift workersand individuals who underwent acute orchronic forced circadian misalignment(1920) supporting the notion that theclock plays an essential role in the preser-vation of insulin sensitivity and b-cellfunction

It has recently been found that secre-tion of glucagon-like peptide 1 (GLP-1) akey incretin hormone that regulates glu-cose-dependent insulin secretion from in-testinal L cells shows a rhythmic patternin rats and humans in vivo In addition itssecretion is altered by circadian disrup-tors such as constant light exposure con-sumption of a Western diet and feedingduring the inactivity hours The alter-ations in the rhythm of GLP-1 secretoryresponse parallel changes in the patternof insulin responses (5) This would sug-gest that it is important to preserve clockfunctionality in subjects with type 2 dia-betes In fact recent studies suggest thatbreakfast skipping and night eating dis-rupt circadian rhythms and as a resultimpair glucose metabolism and b-cellfunction (162122)

We have recently shown that omissionof breakfast in patients with type 2 diabe-tes led to increasedpostprandial glycemiaand attenuated insulin and GLP-1 re-sponses after lunch and dinner (23) Al-though the mechanism underlying thiseffect is unclear we hypothesize thatbreakfast consumption triggers propercyclic clock gene expression leading toimproved postprandial glycemia and in-sulin and GLP-1 responses Since theacute effect of meals and specificallybreakfast on clock gene expression isnot known in humans we comparedclock and clock-controlled gene expres-sion in association with glucose insu-lin and GLP-1 responses and plasma

dipeptidyl peptidase IV (DPP-IV) activityin healthy individuals and individualswith type 2 diabetes consuming or skip-ping breakfast


ParticipantsThis was a randomized open-labelcrossover-within-subject clinical trial Thestudy population included 18 subjectswith type 2 diabetes and 18 healthy sub-jects All participants habitually ate break-fast did not work shifts within the last5 years and did not cross time zoneswithin the last month prior to the studyParticipants usually woke up at 0600ndash0700 h andwent to sleep at 2200ndash2400 hNone of the participants had impairedthyroid renal or liver function anemiapulmonary disease psychiatric immuno-logical or neoplastic diseases or severediabetic complications or underwent bari-atric surgery All participants were insulinnaıve and only patients takingmetforminwere included Patients taking oral hypo-glycemic agents such as GLP-1 analogsanorectic drugs or steroids were ex-cluded Those patients on metforminwere told to stop taking the medication2 days before test days All participantsgave their informed consent The studywas registered at clinicaltrialsgovNCT01939782 The Helsinki Committee ofthe Wolfson Medical Center where thetrial was performed approved the study

Study DesignThere were two groups of participants agroupwith type 2 diabetes and a group ofhealthy control subjects Both groups un-derwent two separate test days in theclinic with two different experimentalconditions 1) the participants ate break-fast and lunch (YesB) or 2) the partici-pants did not eat breakfast ie continuedthe overnight fast until lunchtime and thenhad lunch (NoB) The two test days wereseparated by 1ndash2 weeks of washout Aperson not involved in the study using acoin flip randomized participants withintheir group to start with the YesB or NoBday Participants ingested their last oraltherapy 18 h before the test days The sub-jects were instructed to eat a standardizedmeal in the evening between 2100 and2200 h prior to test day consisting oftwo whitewheat bread slices and an op-tional drink such as tea or coffee with anartificial sweetener or diet soda Eating astandardizedmeal thenight before the test

1574 Skipping Breakfast Alters Circadian Expression Diabetes Care Volume 40 November 2017

prevents long overnight fasting in the YesBgroup and more so in the NoB group Ad-ditionally participants were instructed toavoid alcohol and excessive physical activ-ity 6 days preceding each test day On theYesB day participants had two identicalstandard meals that were provided in theclinic as breakfast at 0830 h and lunch at1200 h On the NoB day breakfast wasomittedand the individuals continued theirovernight fast until lunch at 1200 h Theenergy and content of each test mealwere identical (572 6 8 kcal 194 fat49 carbohydrates and 32 protein)The primary outcome was to compareclock and clock-controlled gene expres-sion (Clock Bmal1 Per1 Per2 Cry1 Rev-erba Rora Ampk and Sirt1) in whiteblood cells (WBCs) on YesB versus NoBdays in healthy individuals and individualswith type 2 diabetes Gene expressionprofiling in the WBCs has been shown toreflect food-related metabolic changeseven in the postprandial state (24)Gene expression was relative to actin ahousekeeping gene normalizing varia-tions in WBC count The secondary out-come was to compare postprandialplasma glucose insulin intact GLP-1(iGLP-1) and DPP-IV plasma activity

Meal TestsOn the day of the meal tests each partic-ipant reported to the laboratory at 0730 hafter an overnight fast Anthropometricdata were collected in the morning At0800 h a catheter was placed in the veinof the nondominant arm and remainedthere until 1530 h Venous blood samplesfor quantification of plasma glucose insu-lin and iGLP-1were collectedbeforebreak-fast (at 0830 h) at 60 and 120 min afterbreakfast and at 210 min (at 1200 h justbefore lunch) After lunch (at 1200 h) sam-pleswere collected at 60 120 and 210min(420 min including breakfast) On the NoBday blood sampleswere taken at the sametime points as on the YesB day The sam-ples for the assessment of clock and clock-controlled gene expression and DPP-IVactivity were taken after the overnight fast(at 0830 h) before lunch (at 1200 h) and210 min after lunch (at 1530 h)

Biochemical and Hormonal BloodAnalysesPlasma glucose was immediately analyzedwith hexokinase using a Cobas analyzer(Roche Diagnostics Madison WI) Bloodsamples for determining iGLP-1 were

collected into chilled tubes containingEDTA and diprotin A (01 mmolL) Sampleswere centrifuged at 3000 rpm at 4degCfor 10 min and stored at 280degC Insulinwas determined by electrochemilumines-cence immunoassay using a Cobas ana-lyzer (Roche Diagnostics) according tothe manufacturerrsquos instructions iGLP-1was quantified using ELISA (IBL Amer-ica Minneapolis MN) DPP-IV plasmaactivity was analyzed as previouslydescribed (25)

Analysis of Gene Expression inLeukocytesBlood for gene expression was collectedin Tempus tubes (Applied BiosystemsFoster City CA) and total RNA was ex-tractedaccording to themanufacturerrsquosinstructions Total RNAwas DNase I treat-ed using RQ1 DNase (Promega MadisonWI) for 2 h at 37degC as previously de-scribed (26) Two micrograms of DNase

IndashtreatedRNAwasreversetranscribedusingM-MuLV reverse transcriptase and randomhexamers (Promega) One-twentieth ofthe reaction was then subjected to quan-titative real-time PCR using primersspanning exon-exon boundaries andthe ABI Prism 7300 Sequence DetectionSystem (Applied Biosystems) The foldchange in target gene expression wascalculated by the 22DDCt relative quanti-fication method (Applied Biosystems)

Sample Size and Power AnalysisAsample sizeofn =16healthyparticipantsand n = 16 participants with type 2 di-abetes in crossover was determinedto have 90 power to detect a truebetween-group difference of 40 6 25relative mRNA expression levels of clockgenes with a P value = 005 The currentstudy provided 80 power to detect 5difference between-group in overall post-prandial plasma area under the curve

Figure 1mdashGeneexpressionofhealthyparticipantsandparticipantswith type2diabeteswithorwithoutbreakfast Blood sampleswere collected after overnight fasting (timepoint 0) afterwhichbreakfastwasgiven (YesB) or fasting continued (NoB) Statistical differences (P 005) between time point 0 andtime point 210min 35 h after breakfast or nobreakfast in the healthy group Statistical differences(P 005) between time point 0 and time point 210min 35 h after breakfast or no breakfast in thegroup with type 2 diabetes Data are means6 SE

carediabetesjournalsorg Jakubowicz and Associates 1575

(AUC) for iGLP-1 insulin and glucose Toallow discontinuation 36 participantswere recruited

Statistical AnalysisThirty-six subjects were enrolled in thestudy and four subjects dropped outThey were excluded from the analysestherefore the results are based on n = 32subjects AUC was calculated by the trap-ezoidal rule For time series a two-wayANOVA (time 3 diet) was performedand a least significant difference pairedStudent t test post hoc analysis wasused for comparison between the YesBand NoB test days at each time point Forclock gene expression fasting (time point0) was determined as baseline for the YesBand NoB groups 35 h after (time point210 min) Time point 210 min was deter-mined as the baseline for the postlunch(time point 420min) gene expression Stu-dent t test analyses were performed toevaluate differences between time pointswithin each group and treatment The re-sults are expressed as mean 6 SEM AP value005was considered statisticallysignificant Statistical analysis was per-formed with JMP software (version 12SAS Institute Inc Cary NC)

RESULTS

ParticipantsThirty-six individuals (18 with type 2 di-abetes and 18 healthy) were enrolled inthe study with 32 participants complet-ing (16 with type 2 diabetes and 16healthy) During the first meal test dayfour dropped out (two healthy and twowith diabetes) because of problems in theinstallation of the intravenous catheterDropouts did not differ significantly fromthose who continued the study The pa-tients with type 2 diabetes were 668 619 years of age and had the diseasefor 145 6 15 years with 596 6 08mmolmol (76 6 01) HbA1c and BMI307 6 11 kgm2 (Supplementary Table1) Ten patients were treated withdiet alone whereas six were treatedwith diet and metformin Seven pa-tients had a history of hypertension andwere treated with ACE inhibitors andorcalcium channel antagonists The partic-ipants in the healthy group were 4436 29 years of age with 49 6 01 (3016 06 mmolmol) HbA1c and BMI 231604 kgm2 (Supplementary Table 1) Theywere not taking any medications

Clock Gene Expression on YesB andNoB Days in Healthy IndividualsVersus Individuals With DiabetesBefore LunchIn healthy individuals the expressionlevel of Per1 Cry1 Rora and Sirt1 waslower (Student t test P 005) but Clockwas higher (Student t test P 005) afterbreakfast (Fig 1) In contrast in individu-als with type 2 diabetes Per1 Per2 andSirt1 only slightly but significantly de-creased and Rora increased (Studentt test P 005) after breakfast Omissionof breakfast altered clock and metabolicgene expression in both healthy peopleand individuals with type 2 diabetes Inhealthy individuals there was an in-crease in the expression level of Bmal1Cry1 Rev-erba Rora and Sirt1 (Studentt test P 005) whereas in individualswith type 2 diabetes Clock expression in-creased (Student t test P 005) (Fig 1)In bothhealthy individuals and individuals

with diabetes Per1 and Per2 expressionwas not affected whether breakfast wasconsumed or not (Fig 1)

Clock Gene Expression on YesB andNoB Days in Healthy IndividualsVersus Individuals With Diabetes AfterLunchIn healthy individuals theexpression levelofBmal1 Rora and Sirt1was higher (Stu-dent t test P 005) after lunch on theYesB day whereas the other clock genesremained unchanged (Fig 2) In individu-als with type 2 diabetes Bmal1 Per1Per2 Rev-erba and Ampk increased (Stu-dent t test P 005) after lunch on theYesB day Omission of breakfast alteredclock andmetabolic gene expression afterlunch in both healthy individuals and in-dividuals with type 2 diabetes In healthyindividuals Per2 and Ampk expression in-creased and Per1 Cry1 and Rev-erba de-creased (Student t test P 005) (Fig 2)

Figure 2mdashGene expression of healthy participants and participants with type 2 diabetes afterlunch Blood samples were collected 35 h after breakfast or no breakfast (time point 210 min)and 35 h after lunch (time point 420 min) Statistical differences (P 005) between timepoint 210 min and time point 420 min in the healthy group Statistical differences (P 005)between time point 210 min and time point 420 min in the group with type 2 diabetes Data aremeans6 SE


whereas in individuals with type 2 diabe-tes Bmal1 Per1 and Ampk expressiondecreased and Rora expression increased(Student t test P 005) after lunch onthe NoB day (Fig 2) Taken togetherbreakfast and lunch led to increased ex-pression of the positive loop and down-regulation of the negative feedback loopwhereas breakfast skipping altered thisexpression pattern Lunch as the firstmeal of the day could not rectify the al-tered clock expression In addition indi-viduals with type 2 diabetes who skipbreakfast have a greater disruption oftheir circadian rhythms compared withthose who consume breakfast

Glucose Insulin and iGLP-1 Levels andDPP-IV Activity on YesB and NoB DaysIn the healthy group and group with di-abetes the AUCglucose after lunchwas 15ndash18 higher during the NoB day versusthe YesB day (P 0001) (Fig 3 andSupplementary Table 2) In healthy par-ticipants AUCinsulin after lunch was notsignificantly different between the tests(P = 05) In contrast in the group withdiabetes the AUCinsulin after lunch was25 lower on the NoB day comparedwith the YesB day (P 0004) (Fig 3and Supplementary Table 2) In both thehealthy group and group with diabetestheAUCiGLP-1 after lunchwas35 loweron the NoB day compared with theYesB day (P 00001) (Fig 3 andSupplementary Table 2) No significantchange in plasma DPP-IV activity wasfound after lunch between the groupson YesB or NoB day

CONCLUSIONS

In this study we report for the first timean acute postprandial effect on clock andclock-controlled gene expression in bothhealthy individuals and individuals withtype 2 diabetes We showed that break-fast skipping acutely disrupts circadianrhythms in both healthy individuals andpeople with type 2 diabetes Further-more clock gene expression after lunchwas different and depended on whetherbreakfast was consumedor not Thus thestudy results suggest that breakfast con-sumption is of high relevance for preserv-ing clock gene activity and this is true forboth healthy subjects and subjects withdiabetesSeveral reports have suggested that

meal timing exerts a pivotal influenceon peripheral clocks and clock output

systems involved in the regulation ofmet-abolic pathways (126ndash29) Breakfastskipping (2230) andor eating at hoursdesigned to sleep (21) for the long runlead to disruption of clock gene expres-sion and are associated with weight gainand diabetes (3132) In contrast acclima-tion to timed feeding high-energy break-fast and fasting during the hoursdesigned for sleep reversed impairedclock gene expression and enhanced

AMPK expression resulting in a moreefficient weight loss insulin sensitivityand reduction of lipid accumulation(212230ndash32)

In healthy people breakfast and lunchacutely led to an overall increased expres-sion of the positive loop of the core clockmechanism whereas the expression ofthe negative feedback loop was downre-gulated (Fig 4) This pattern maintainsthe normal cyclic expression of output

Figure 3mdashLine charts of healthy participants (A) and participants with type 2 diabetes (B) on YesBand NoB days for glucose insulin iGLP-1 and DPP-IV Breakfast was given to the YesB group at timepoint 0 Lunch was given to both groups at time point 210min Statistical difference between YesBand NoB at a specific time point Data are means6 SE


systems (Fig 4) In contrast breakfastskipping in healthy people led to an al-tered clock gene expression pattern Afterlunch on the NoB day Sirt1 Clock Bmal1and Rorawere upregulated and Per1wasdownregulated similarly to the effect onthe YesB day (Fig 4) However unlike onthe YesB day after lunch Per2 and Ampkwere upregulated and Cry1 did notchange disrupting the normal cyclic ex-pression (Fig 4)In patients with type 2 diabetes break-

fast and lunch eventually led to an overallaltered clock gene expression (Fig 4)However Ampk expression was upregu-lated after lunch As AMPK activationleads to glucose uptake this upregulationmay indicate improved glycemic controlas indeed was found herein In contrastbreakfast skipping in peoplewith diabetesfurther imparted a greater disruption as italso led to reduced Ampk levels accentu-ating blood glucose dysregulation (Fig 4)Our results show that the effect of

breakfast is acute as only 1 dayof skippingbreakfast led to such a deleterious effecton clock and clock-controlled geneexpres-sion The rapid change in clock gene ex-pression on YesB versus NoB days isconsistent with reports in animal modelsin which a 30-min feeding stimulus al-tered clock gene expression within 2ndash4 h(3334) As feeding poses such an acuteeffect it is clear why long-term abnormalpatterns of clock gene expression aremanifested in obese individuals and indi-viduals with diabetesA change in clock and clock-controlled

gene expression due to breakfast omis-sion is expected to yield altered metabo-lism Impaired circadian rhythms havebeen closely associated with the patho-physiology of type2diabetes (4) Reducedglucose-stimulated insulin secretion insu-lin resistance diminished b-cell prolifera-tion and apoptosis have been associatedwith asynchrony or deficiencies in clockgenes (16) Indeed in people with diabe-tes omission of breakfast led to a signif-icant increase in postprandial glucoseexcursions with reduced insulin andiGLP-1 responses after lunch comparedwith YesB day Furthermore this delete-rious effect of breakfast skipping was alsoobserved in healthy participants althoughwith a lower magnitudeThe reduction of hyperglycemia and

enhanced insulin response after lunchwith prior consumption of breakfast hasbeen described as the second meal

phenomenon and has been previously re-ported in several studies in healthy in-dividuals and individuals with type 2diabetes (2335) This has been explainedby enhanced b-cell responsiveness at thesecond meal induced by the first meal(3637) This explanation is based on thefindings that both the first and the secondphase of insulin release are influenced byb-cell memory and the magnitude of in-sulin release is enhanced significantly byprevious glucose exposure (37) The ab-sence of glucose elevation due to fastinguntil noon may diminish b-cell respon-siveness and memory leading to a re-duced and delayed insulin responseafter lunch on the NoB day Indeed itwas recently reported that lower insulinrelease by b-cells in response to nutrientdepletion or starvation induces lysosomaldegradation of nascent secretory insulingranules and this is controlled by proteinkinase D (PKD) a key player in secretorygranule biogenesis (38) The impaired in-sulin secretion at lunch on the NoB daymay also be due to perturbed incretinregulation since both b-cell memoryand sensitivity to glucose are enhancedby GLP-1 Therefore the higher levels ofGLP-1 on the YesB day may explain boththe enhanced insulin secretion and the

reduced glycemic response after lunchAt the cellular level the reduction of post-prandial glycemia after lunch on the YesBday can be explained by the fact thatbreakfast triggers correct cyclic expres-sion of clock geneswhich in turn assuresmetabolic efficiency leading to normalglucose insulin and GLP-1 responses af-ter lunch Ampk upregulation in partici-pants with type 2 diabetes may enhanceGLUT4 translocation and muscular glu-cose uptake leading to improved post-prandial glycemia

At baseline clock gene levels were dif-ferent between the healthy group andthe group with type 2 diabetes presum-ably due todifferences related to age bodyweight andor HbA1c levels Although theage and body weight of the group withtype 2 diabetes was significantly differentfrom the healthy group and may be alimitation in our study changes in clockgene expression as a result of breakfastskipping cannot be attributed to theseparameters as each group was normal-ized to its own baseline In addition thecrossover study assured that the sameperson had both YesB and NoB treat-ments reiterating the effect of breakfastskipping Another limitation of our studyis that it was performed until after lunch

Figure 4mdashEffect of lunch with or without prior breakfast on the interrelations between the clockmechanism and metabolic proteins CLOCK and BMAL1 mediate the expression of clock and clock-controlled genes regulating circadian hormone secretion (insulin and glucagon) metabolism (glu-coneogenesis and glycolysis) and glucose homeostasis PER1 PER2 and CRY1 serve as the negativefeedback loop that inhibits CLOCKBMAL1-mediated expression SIRT1 and AMPK when activatedunder low cellular energy levels relieve the inhibition mediated by the negative feedback loopBMAL1 expression is positively regulated by RORa and negatively regulated by REV-ERBa Breakfastconsumption followed by lunch assures this overall cyclic regulation is maintained Omissionof breakfast disrupts this cyclic regulation in both healthy people and patients with type 2 diabetes


Analyses of subsequent dinner and othertime points during the nighttime are re-quired in order to determine overall dailyrhythms and how each meal affects thisoscillatory pattern In summary our studydemonstrates that breakfast consump-tion exerts a rapid influence on clockand clock-controlled genes involved inglucose regulation such as Ampk andthis effect is associated with a significantreduction in postprandial glycemia andenhanced insulin and GLP-1 responsesafter subsequent lunch In contrast break-fast skipping adversely affects the expres-sion of clock and clock-controlled genesinvolved in glucose metabolism in bothhealthy individuals and individuals with di-abetes This study emphasizes the con-sumption of breakfast as an importantstrategy when targeting glycemic controlin type 2 diabetes As the circadian clockalso regulates blood pressure heart rateand cardiovascular activity as well as adi-pose tissue and other metabolic organs(39) meal timing may affect overall me-tabolism and influence chronic complica-tions of obesity and type 2 diabetes

Funding This study was funded by D-Cure andthe Ministry of Health Israel (grant 3-0000-11406)Duality of Interest No potential conflicts of in-terest relevant to this article were reportedAuthor Contributions DJ and OF contrib-uted to the conception and design of the studyacquired analyzed and interpreted data anddrafted and revised the manuscript JW con-tributed to the conception and design of thestudy acquired and interpreted data anddrafted the manuscript ZL NC TG MMand YB-D contributed to the conception anddesignof thestudyacquiredand interpreteddataorganized the randomization and drafted themanuscript IR and BA researched data con-tributed to the interpretation of the data anddraftedand revised themanuscriptMB analyzedand interpreted data DJ is the guarantor of thiswork and as such had full access to all the data inthe study and takes responsibility for the integrityof the data and the accuracy of the data analysisPrior Presentation This study was presentedorally at the 77th Scientific Sessions of the Amer-ican Diabetes Association San Diego CA 9ndash13June 2017

References1 Froy O Metabolism and circadian rhythmsndashimplications for obesity Endocr Rev 2010311ndash242 Jordan SD Lamia KA AMPK at the crossroadsof circadian clocks and metabolism Mol Cell En-docrinol 2013366163ndash1693 Zvonic S Ptitsyn AA Conrad SA et al Charac-terization of peripheral circadian clocks in adiposetissues Diabetes 200655962ndash970

4 Ando H Ushijima K Yanagihara H et al Clockgene expression in the liver and adipose tissues ofnon-obese type 2 diabetic Goto-Kakizaki rats ClinExp Hypertens 200931201ndash2075 Brubaker PL Gil-Lozano M Glucagon-likepeptide-1 themissing link in themetabolic clockJ Diabetes Investig 20167(Suppl 1)70ndash756 Reppert SM Weaver DR Coordination of cir-cadian timing inmammals Nature 2002418935ndash9417 Feng D LazarMA Clocks metabolism and theepigenome Mol Cell 201247158ndash1678 Preitner N Damiola F Lopez-Molina L et alThe orphan nuclear receptor REV-ERBalpha con-trols circadian transcription within the positivelimb of the mammalian circadian oscillator Cell2002110251ndash2609 Ueda HR ChenW Adachi A et al A transcrip-tion factor response element for gene expressionduring circadian night Nature 2002418534ndash53910 Sato TK Panda S Miraglia LJ et al A func-tional genomics strategy reveals Rora as a com-ponent of themammalian circadian clock Neuron200443527ndash53711 Lamia KA Sachdeva UM DiTacchio L et alAMPK regulates the circadian clock by crypto-chrome phosphorylation and degradation Sci-ence 2009326437ndash44012 Hardie DG Ross FA Hawley SA AMPK a nu-trient and energy sensor that maintains energyhomeostasis Nat Rev Mol Cell Biol 201213251ndash26213 Pinho AV Bensellam M Wauters E et alPancreas-specific Sirt1-deficiency inmice compro-mises beta-cell function without development ofhyperglycemia PLoS One 201510e012801214 Asher G Gatfield D StratmannM et al SIRT1regulates circadian clock gene expression throughPER2 deacetylation Cell 2008134317ndash32815 Nakahata Y Sahar S Astarita G Kaluzova MSassone-Corsi P Circadian control of the NAD+salvage pathway by CLOCK-SIRT1 Science 2009324654ndash65716 Vieira E Burris TP Quesada I Clock genespancreatic function and diabetes Trends MolMed 201420685ndash69317 Pappa KI Gazouli M Anastasiou E IliodromitiZ Antsaklis A Anagnou NP Circadian clock geneexpression is impaired in gestational diabetesmellitus Gynecol Endocrinol 201329331ndash33518 Ando H Takamura T Matsuzawa-Nagata Net al Clock gene expression in peripheral leuco-cytes of patients with type 2 diabetes Diabetolo-gia 200952329ndash33519 Vetter C Devore EE Ramin CA Speizer FEWillettWC Schernhammer ESMismatch of sleepand work timing and risk of type 2 diabetes Di-abetes Care 2015381707ndash171320 Scheer FA HiltonMFMantzoros CS Shea SAAdverse metabolic and cardiovascular conse-quences of circadian misalignment Proc NatlAcad Sci U S A 20091064453ndash445821 Arble DM Bass J Laposky AD Vitaterna MHTurek FW Circadian timing of food intake contrib-utes to weight gain Obesity (Silver Spring) 2009172100ndash210222 Yoshida C Shikata N Seki S Koyama NNoguchi Y Early nocturnal meal skipping altersthe peripheral clock and increases lipogenesis inmice Nutr Metab (Lond) 201297823 Jakubowicz DWainstein J Ahren B Landau ZBar-Dayan Y Froy O Fasting until noon triggers

increased postprandial hyperglycemia and im-paired insulin response after lunch and dinner inindividuals with type 2 diabetes a randomizedclinical trial Diabetes Care 2015381820ndash182624 Kawakami Y Yamanaka-Okumura H SakumaM et al Gene expression profiling in peripheralwhite blood cells in response to the intake of foodwith different glycemic index using a DNA micro-array J Nutrigenet Nutrigenomics 20136154ndash16825 Gunnarsson PT Winzell MS Deacon CF et alGlucose-induced incretin hormone release and in-activation are differently modulated by oral fatand protein in mice Endocrinology 20061473173ndash318026 Sherman H Frumin I Gutman R et al Long-term restricted feeding alters circadian expressionand reduces the level of inflammatory anddiseasemarkers J Cell Mol Med 2011152745ndash275927 Hatori M Vollmers C Zarrinpar A et al Time-restricted feeding without reducing caloric intakepreventsmetabolic diseases inmice fed a high-fatdiet Cell Metab 201215848ndash86028 Chaix A Zarrinpar A Miu P Panda S Time-restricted feeding is a preventative and therapeuticintervention against diverse nutritional challengesCell Metab 201420991ndash100529 Yasumoto Y Hashimoto C Nakao R et alShort-term feeding at the wrong time is sufficienttodesynchronizeperipheralclocksand induceobesitywith hyperphagia physical inactivity and metabolicdisorders in mice Metabolism 201665714ndash72730 Wu T Sun L ZhuGe F et al Differential rolesof breakfast and supper in rats of a daily three-meal schedule upon circadian regulation andphysiology Chronobiol Int 201128890ndash90331 Kudo T Akiyama M Kuriyama K Sudo MMoriya T Shibata S Night-time restricted feedingnormalises clock genes and Pai-1 gene expressionin the dbdb mouse liver Diabetologia 2004471425ndash143632 FuseYHiraoA KurodaHOtsukaM Tahara YShibata S Differential roles of breakfast only (onemeal per day) and a bigger breakfast with a smalldinner (two meals per day) in mice fed a high-fatdiet with regard to induced obesity and lipid me-tabolism J Circadian Rhythms 201210433 Wu T Ni Y Kato H Fu Z Feeding-inducedrapid resetting of the hepatic circadian clock is as-sociated with acute induction of Per2 and Dec1transcription in rats Chronobiol Int 2010271ndash1834 Wu T Fu O Yao L Sun L Zhuge F Fu Z Dif-ferential responses of peripheral circadian clocksto a short-term feeding stimulus Mol Biol Rep2012399783ndash978935 Jovanovic A Gerrard J Taylor R The second-meal phenomenon in type 2 diabetes DiabetesCare 2009321199ndash120136 Nesher R Cerasi E Modeling phasic insulinrelease immediate and time-dependent effectsof glucose Diabetes 200251(Suppl 1)S53ndashS5937 Vagn Korsgaard T Colding-Joslashrgensen MTime-dependent mechanisms in beta-cell glucosesensing J Biol Phys 200632289ndash30638 Goginashvili A Zhang Z Erbs E et al Insulingranules Insulin secretory granules control auto-phagy in pancreatic b cells Science 2015347878ndash88239 Richards J Diaz AN Gumz ML Clock genes inhypertension novel insights from rodent modelsBlood Press Monit 201419249ndash254


The circadian clock controls the activity ofmost enzymes hormones and transportsystems involved in glucose metabolism(12) The central circadian clock locatedin the suprachiasmatic nuclei of the ante-rior hypothalamus generates endoge-nous 24-h rhythms Similar clocks arefound in peripheral tissues such as theliver b-cells muscle and adipose tissue(3ndash5) The core clock mechanism in thebrain and peripheral tissues comprisestwo loops The positive loop consists ofthe CLOCK and BMAL1 heterodimer thatmediates transcription of tissue-specificgenes and those of the negative feedbackloop The negative feedback loop consistsof the period (PER) and cryptochrome(CRY) proteins that inhibit CLOCKBMAL1-mediated transcription (6)Unlike the suprachiasmatic nuclei clock

which responds mainly to the light-darkcycle peripheral clocks respond to mealcontent and timing leading to coordi-nated regulation of digestive and absorp-tive functions and hormone secretionthereby preventing metabolic dysregulation(1ndash4) CLOCKBMAL1 heterodimer plays acritical role in mediating the transcriptionof coactivators that regulate the circadiansynthesis ofmost of the enzymes andhor-mones involved in glucose homeostasisie regulation of hepatic gluconeogene-sis and pancreatic b-cell insulin secretion(7) CRY proteins inhibit gluconeogenicgene expression by regulating CREBP ac-tivity and their hepatic depletion in-creases circulating glucose and CRYoverexpression reduces fasting blood glu-coseand improveswhole-body insulin sen-sitivity in obese mice (7) CLOCKBMAL1heterodimer regulates the expression ofRev-erba andRora (8ndash10) REV-ERBa thenegative regulator of Bmal1 expression(8) is induced during normal adipogenesisand mediates a suppressive effect on he-patic gluconeogenesis and glucose outputby regulating the expression of PEPCKand glucose-6-phosphatase (G6pase)Depletion of REV-ERBa leads to hyper-glycemia (27) In contrast RORa the pos-itive regulator of Bmal1 expression (10)activates the hepatic gluconeogenic en-zyme G6pase and regulates lipogenesisand lipid storage in skeletal muscle (210)AMPK the cellular energy sensor also

plays a key role in the clockmechanismbyenhancing degradation of PER and CRY pro-teins (11) Upregulation of AMPK signalingsignificantly enhances GLUT4 translocationand muscular glucose uptake ensuring

metabolic efficiency and improving post-prandial glucose and insulin responses(12) AMPK exerts a positive effect onSIRT1 associated with beneficial effectson b-cell viability and insulin sensitivitywhich interacts directly with CLOCK anddeacetylates BMAL1 and PER2 (12ndash15)

Reduced glucose-stimulated insulin se-cretion insulin resistance diminishedb-cell proliferation and apoptosis havebeen associated with asynchrony or defi-ciencies in clock genes (16) Moreoverlower transcripts of Bmal1 and Cry2 areinversely correlated with HbA1c levels(1718) Furthermore higher risk of obe-sity metabolic alterations and type 2 di-abetes have been found in shift workersand individuals who underwent acute orchronic forced circadian misalignment(1920) supporting the notion that theclock plays an essential role in the preser-vation of insulin sensitivity and b-cellfunction

It has recently been found that secre-tion of glucagon-like peptide 1 (GLP-1) akey incretin hormone that regulates glu-cose-dependent insulin secretion from in-testinal L cells shows a rhythmic patternin rats and humans in vivo In addition itssecretion is altered by circadian disrup-tors such as constant light exposure con-sumption of a Western diet and feedingduring the inactivity hours The alter-ations in the rhythm of GLP-1 secretoryresponse parallel changes in the patternof insulin responses (5) This would sug-gest that it is important to preserve clockfunctionality in subjects with type 2 dia-betes In fact recent studies suggest thatbreakfast skipping and night eating dis-rupt circadian rhythms and as a resultimpair glucose metabolism and b-cellfunction (162122)

We have recently shown that omissionof breakfast in patients with type 2 diabe-tes led to increasedpostprandial glycemiaand attenuated insulin and GLP-1 re-sponses after lunch and dinner (23) Al-though the mechanism underlying thiseffect is unclear we hypothesize thatbreakfast consumption triggers propercyclic clock gene expression leading toimproved postprandial glycemia and in-sulin and GLP-1 responses Since theacute effect of meals and specificallybreakfast on clock gene expression isnot known in humans we comparedclock and clock-controlled gene expres-sion in association with glucose insu-lin and GLP-1 responses and plasma

dipeptidyl peptidase IV (DPP-IV) activityin healthy individuals and individualswith type 2 diabetes consuming or skip-ping breakfast


ParticipantsThis was a randomized open-labelcrossover-within-subject clinical trial Thestudy population included 18 subjectswith type 2 diabetes and 18 healthy sub-jects All participants habitually ate break-fast did not work shifts within the last5 years and did not cross time zoneswithin the last month prior to the studyParticipants usually woke up at 0600ndash0700 h andwent to sleep at 2200ndash2400 hNone of the participants had impairedthyroid renal or liver function anemiapulmonary disease psychiatric immuno-logical or neoplastic diseases or severediabetic complications or underwent bari-atric surgery All participants were insulinnaıve and only patients takingmetforminwere included Patients taking oral hypo-glycemic agents such as GLP-1 analogsanorectic drugs or steroids were ex-cluded Those patients on metforminwere told to stop taking the medication2 days before test days All participantsgave their informed consent The studywas registered at clinicaltrialsgovNCT01939782 The Helsinki Committee ofthe Wolfson Medical Center where thetrial was performed approved the study

Study DesignThere were two groups of participants agroupwith type 2 diabetes and a group ofhealthy control subjects Both groups un-derwent two separate test days in theclinic with two different experimentalconditions 1) the participants ate break-fast and lunch (YesB) or 2) the partici-pants did not eat breakfast ie continuedthe overnight fast until lunchtime and thenhad lunch (NoB) The two test days wereseparated by 1ndash2 weeks of washout Aperson not involved in the study using acoin flip randomized participants withintheir group to start with the YesB or NoBday Participants ingested their last oraltherapy 18 h before the test days The sub-jects were instructed to eat a standardizedmeal in the evening between 2100 and2200 h prior to test day consisting oftwo whitewheat bread slices and an op-tional drink such as tea or coffee with anartificial sweetener or diet soda Eating astandardizedmeal thenight before the test













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influences of breakfast on clock - diabetes care · breakfast consumption acutely affects clock and...

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