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Influences of Breakfast on Clock Gene Expression and Postprandial Glycemia in Healthy Individuals and Individuals With Diabetes: A Randomized Clinical Trial

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Objective: The circadian clock regulates glucose metabolism by mediating the activity of metabolic enzymes, hormones, and transport systems. Breakfast skipping and night eating have been associated with high HbA1c and postprandial hyperglycemia after lunch and dinner. Our aim was to explore the acute effect of breakfast consumption or omission on glucose homeostasis and clock gene expression in healthy individuals and individuals with type 2 diabetes. Research design and methods: In a cross-over design, 18 healthy volunteers and 18 volunteers with 14.5 ± 1.5 years diabetes, BMI 30.7 ± 1.1 kg/m(2), and HbA1c 7.6 ± 0.1% (59.6 ± 0.8 mmol/mol) were randomly assigned to a test day with breakfast and lunch (YesB) and a test day with only lunch (NoB). Postprandial clock and clock-controlled gene expression, plasma glucose, 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, Rorα, and Sirt1 was lower (P < 0.05) but Clock was higher (P < 0.05) after breakfast. In contrast, in individuals with type 2 diabetes, Per1, Per2, and Sirt1 only slightly, but significantly, decreased and Rorα increased (P < 0.05) after breakfast. In healthy individuals, the expression level of Bmal1, Rorα, and Sirt1 was higher (P < 0.05) after lunch on YesB day, whereas the other clock genes remained unchanged. In individuals with type 2 diabetes, Bmal1, Per1, Per2, Rev-erbα, and Ampk increased (P < 0.05) after lunch on the YesB day. Omission of breakfast altered clock and metabolic gene expression in both healthy and individuals with type 2 diabetes. Conclusions: Breakfast consumption acutely affects clock and clock-controlled gene expression leading to normal oscillation. Breakfast skipping adversely affects clock and clock-controlled gene expression and is correlated with increased postprandial glycemic response in both healthy individuals and individuals with diabetes.
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Inuences of Breakfast on Clock
Gene Expression and Postprandial
Glycemia in Healthy Individuals
and Individuals With Diabetes:
A Randomized Clinical Trial
https://doi.org/10.2337/dc16-2753
OBJECTIVE
The circadian clock regulates glucose metabolism by mediating the activity of met-
abolic enzymes, hormones, and transport systems. Breakfast skipping and night
eating have been associated with high HbA
1c
and postprandial hyperglycemia after
lunch and dinner. Our aim was to explore the acute effect of breakfast consumption
or omission on glucose homeostasis and clock gene expression in healthy individuals
and individuals with type 2 diabetes.
RESEARCH DESIGN AND METHODS
In a cross-over design, 18 healthy volunteers and 18 volunteers with 14.5 61.5 years
diabetes, BMI 30.7 61.1 kg/m
2
,andHbA
1c
7.6 60.1% (59.6 60.8 mmol/mol) were
randomly assigned to a test day with breakfast and lunch (YesB) and a test day with
only lunch (NoB). Postprandial clock and clock-controlled gene expression, plasma
glucose, 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,andSirt1 was lower
(P<0.05) but Clock was higher (P<0.05) after breakfast. In contrast, in individuals
with type 2 diabetes, Per1,Per2,andSirt1 only slightly, but signicantly, decreased
and Roraincreased (P<0.05) after breakfast. In healthy individuals, the expression
level of Bmal1,Rora,andSirt1 was higher (P<0.05) after lunch on YesB day, whereas
the other clock genes remained unchanged. In individuals with type 2 diabetes,
Bmal1,Per1,Per2,Rev-erba,andAmpk increased (P<0.05) after lunch on the YesB
day. Omission of breakfast altered clock and metabolic gene expression in both
healthy and individuals with type 2 diabetes.
CONCLUSIONS
Breakfast consumption acutely affects clock and clock-controlled gene expression
leading to normal oscillation. Breakfast skipping adversely affects clock and clock-
controlled gene expression and is correlated with increased postprandial glycemic
response in both healthy individuals and individuals with diabetes.
1
Diabetes Unit, Wolfson Medical Center, Sackler
Faculty of Medicine, Tel Aviv University, Holon,
Israel
2
Diabetes Unit, Hadassah University Hospital,
Ein Kerem Hospital, Hadassah Medical School,
The Hebrew University of Jerusalem, Jerusalem,
Israel
3
Department of Clinical Sciences, Faculty of
Medicine, Lund University, Lund, Sweden
4
Institute of Biochemistry, Food Science and Nu-
trition, TheRobert H. Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of
Jerusalem, Rehovot, Israel
5
Department of Molecular Genetics, Faculty of
Biochemistry, Weizmann Institute of Science,
Rehovot, Israel
Corresponding authors: Oren Froy, oren.froy@
mail.huji.ac.il, and Daniela Jakubowicz, daniela.
jak@gmail.com.
Received 25 December 2016 and accepted 30
July 2017.
Clinical trial reg. no. NCT01939782, clinicaltrials
.gov.
This article contains Supplementary Data online
at http://care.diabetesjournals.org/lookup/
suppl/doi:10.2337/dc16-2753/-/DC1.
D.J. and J.W. contributed equally to this work.
© 2017 by the American Diabetes Association.
Readers may use this article as long as the work
is properly cited, the use is educational and not
for prot, and the work is not altered. More infor-
mation is available at http://www.diabetesjournals
.org/content/license.
Daniela Jakubowicz,
1
Julio Wainstein,
1
Zohar Landau,
1
Itamar Raz,
2
Bo Ahren,
3
Nava Chapnik,
4
Tali Ganz,
1
Miriam Menaged,
1
Maayan Barnea,
5
Yosefa Bar-Dayan,
1
and Oren Froy
4
Diabetes Care 1
CARDIOVASCULAR AND METABOLIC RISK
Diabetes Care Publish Ahead of Print, published online August 22, 2017
The circadian clock controls the activity of
most enzymes, hormones, and transport
systems involved in glucose metabolism
(1,2). The central circadian clock, located
in the suprachiasmatic nuclei of the ante-
rior hypothalamus, generates endoge-
nous ;24-h rhythms. Similar clocks are
found in peripheral tissues, such as the
liver, b-cells, muscle, and adipose tissue
(35). The core clock mechanism in the
brain and peripheral tissues comprises
two loops. The positive loop consists of
the CLOCK and BMAL1 heterodimer that
mediates transcription of tissue-specic
genes and those of the negative feedback
loop. The negative feedback loop consists
of the period (PERs) and cryptochrome
(CRYs) proteins that inhibit CLOCK:BMAL1-
mediated transcription (6).
Unlike the suprachiasmatic nuclei clock,
which responds mainly to the light-dark
cycle, peripheral clocks respond to meal
content and timing, leading to coordi-
nated regulation of digestive and absorp-
tive functions, hormone secretion, thereby
preventing metabolic dysregulation (14).
BMAL1:CLOCK heterodimer plays a criti-
cal role in mediating the transcription of
coactivators that regulate the circadian
synthesis of most of the enzymesand hor-
mones involved in glucose homeostasis,
i.e., regulation of hepatic gluconeogene-
sis and pancreatic b-cell insulin secretion
(7). CRYs inhibit gluconeogenic gene ex-
pression by regulating CREBP activity and
their hepatic depletion increases circulat-
ing glucose, and CRY overexpression re-
duces fasting blood glucose and improves
whole-body insulin sensitivity in obese
mice (7). CLOCK:BMAL1 heterodimer reg-
ulates the expression of Rev-erbaand
Rora(810). REV-ERBa, the negative reg-
ulator of Bmal1 expression (8), is induced
during normal adipogenesis and mediates
a suppressive effect on hepatic gluconeo-
genesis and glucose output by regulating
the expression of PEPCK and glucose-
6-phosphatase (G6pase). Depletion of
REV-ERBaleads to hyperglycemia (2,7).
In contrast, RORa, the positive regulator
of Bmal1 expression (10), activates the
hepatic gluconeogenic enzyme G6pase
and regulates lipogenesis and lipid stor-
age in skeletal muscle (2,10).
AMPK, the cellular energy sensor, also
plays a key role in the clock mechanism by
enhancing degradation of PERs and CRYs
(11). Upregulation of AMPK signaling sig-
nicantly enhances GLUT4 translocation
and muscular glucose uptake, ensuring
metabolic efciency and improving post-
prandial glucose and insulin responses
(12). AMPK exerts a positive effect on
SIRT1, associated with benecial effects
on b-cell viability and insulin sensitivity,
which interacts directly with CLOCK and
deacetylates BMAL1 and PER2 (1215).
Reduced glucose-stimulated insulin se-
cretion, insulin resistance, diminished
b-cell proliferation, and apoptosis have
been associated with asynchrony or de-
ciencies in clock genes (16). Moreover,
lower transcripts of Bmal1 and Cry2 are
inversely correlated with HbA
1c
levels
(17,18). Furthermore, higher risk of obe-
sity, metabolic alterations, and type 2 di-
abetes have been found in shift workers
and individuals who underwent acute or
chronic forced circadian misalignment
(19,20), supporting the notion that the
clock plays an essential role in the preser-
vation of insulin sensitivity and b-cell
function.
It has recently been found that secre-
tion of glucagon-like peptide-1 (GLP-1), a
key incretin hormone that regulates glu-
cose-dependent insulin secretion from in-
testinal L cells, shows a rhythmic pattern
in rats and humans in vivo. In addition, its
secretion is altered by circadian disrup-
tors, such as constant light exposure, con-
sumption of a Western diet, and feeding
during the inactivity hours. The alter-
ations in the rhythm of GLP-1 secretory
response parallel changes in the pattern
of insulin responses (5). This would sug-
gest that it is important to preserve clock
functionality in subjects with type 2 dia-
betes. In fact, recent studies suggest that
breakfast skipping and night eating dis-
rupt circadian rhythms and, as a result,
impair glucose metabolism and b-cell
function (16,21,22).
We have recently shown that omission
of breakfast in patients with type 2 diabe-
tes led to increased postprandial glycemia
and attenuated insulin and GLP-1 re-
sponses after lunch and dinner (23). Al-
though the mechanism underlying this
effect is unclear, we hypothesize that
breakfast consumption triggers proper
cyclic clock gene expression, leading to
improved postprandial glycemia and in-
sulin and GLP-1 responses. Since the
acute effect of meals and specically
breakfast on clock gene expression is
not known in humans, we compared
clock and clock-controlled gene expres-
sion in association with glucose, insu-
lin, and GLP-1 responses and plasma
dipeptidyl peptidase IV (DPP-IV) activity
in healthy individuals and individuals
with type 2 diabetes consuming or skip-
ping breakfast.
RESEARCH DESIGN AND METHODS
Participants
This was a randomized, open-label,
crossover-within-subject clinical trial. The
study population included 18 subjects
with type 2 diabetes and 18 healthy sub-
jects. All participants habitually ate break-
fast, did not work shifts within the last
5 years, and did not cross time zones
within the last month prior to the study.
Participants usually woke up at 0600
0700 h and went to sleep at 22002400 h.
None of the participants had impaired
thyroid, renal, or liver function; anemia;
pulmonary disease; psychiatric, immuno-
logical, or neoplastic diseases; or severe
diabetic complications or underwent bari-
atric surgery. All participants were insulin
na¨
ıve, and only patients taking metformin
were included. Patients taking oral hypo-
glycemic agents, such as GLP-1 analogs,
anorectic drugs, or steroids, were ex-
cluded. Those patients on metformin
were told to stop taking the medication
2 days before test days. All participants
gave their informed consent. The study
was registered at clinicaltrials.gov,
NCT01939782. The Helsinki Committee of
the Wolfson Medical Center, where the
trial was performed, approved the study.
Study Design
There were two groups of participants: a
group with type 2 diabetes and a group of
healthy control subjects. Both groups un-
derwent two separate test days in the
clinic, with two different experimental
conditions: 1) the participants ate break-
fast and lunch (YesB), or 2) the partici-
pants did not eat breakfast, i.e., continued
the overnight fast until lunchtime and then
hadlunch(NoB).Thetwotestdayswere
separated by 12 weeks of washout. A
person not involved in the study, using a
coin ip, randomized participants within
their group to start with the YesB or NoB
day. Participants ingested their last oral
therapy 18 h before the test days. The sub-
jects were instructed to eat a standardized
meal in the evening, between 2100 and
2200 h, prior to test day, consisting of
twowhite/wheatbreadslicesandanop-
tional drink, such as tea or coffee with an
articial sweetener or diet soda. Eating a
standardized meal the night before the test
2 Breakfast Skipping Alters Circadian Expression Diabetes Care
prevents long overnight fasting in the YesB
group and more so in the NoB group. Ad-
ditionally, participants were instructed to
avoid alcohol and excessive physical activ-
ity 6 days preceding each test day. On the
YesB day, participants had two identical
standard meals that were provided in the
clinic as breakfast at 0830 h and lunch at
1200 h. On the NoB day, breakfast was
omitted and the individuals continued their
overnight fast until lunch at 1200 h. The
energy and content of each test meal
were identical (572 68 kcal; 19.4% fat,
49% carbohydrates, and 32% protein).
The primary outcome was to compare
clock and clock-controlled gene expres-
sion (Clock,Bmal1, Per1, Per2, Cry1,Rev-
erba,Rora,Ampk,andSirt1)inwhite
blood cells (WBCs) on YesB versus NoB
days in healthy individuals and individuals
with type 2 diabetes. Gene expression
proling in the WBCs has been shown to
reect food-related metabolic changes,
even in the postprandial state (24).
Gene expression was relative to actin, a
housekeeping gene, normalizing varia-
tions in WBC count. The secondary out-
come was to compare postprandial
plasma glucose, insulin, intact GLP-1
(iGLP-1), and DPP-IV plasma activity.
Meal Tests
On the day of the meal tests, each partic-
ipant reported to the laboratory at 0730 h
after an overnight fast. Anthropometric
data were collected in the morning. At
0800 h, a catheter was placed in the vein
of the nondominant arm and remained
there until 1530 h. Venous blood samples
for quantication of plasma glucose, insu-
lin, and iGLP-1 were collected before break-
fast (at 0830 h), at 60 and 120 min after
breakfast, and at 210 min (at 1200 h, just
before lunch). After lunch (at 1200 h), sam-
ples were collected at 60, 120, and 210 min
(420 min including breakfast). On the NoB
day, blood samples were taken at the same
time points as on the YesB day. The sam-
ples for the assessment of clock and clock-
controlled gene expression and DPP-IV
activity were taken after the overnight fast
(at 0830 h), before lunch (at 1200 h), and
210 min after lunch (at 1530 h).
Biochemical and Hormonal Blood
Analyses
Plasma glucose was immediately analyzed
with hexokinase using a Cobas analyzer
(Roche Diagnostics, Madison, WI). Blood
samples for determining iGLP-1 were
collected into chilled tubes containing
EDTA and diprotin A (0.1 mmol/L). Samples
were centrifuged at 3,000 rpm at 4°C
for 10 min and stored at 280°C. Insulin
was determined by electrochemilumines-
cence immunoassay using a Cobas ana-
lyzer (Roche Diagnostics) according to
the manufacturers instructions. iGLP-1
was quantied using ELISA (IBL Amer-
ica, Minneapolis, MN). DPP-IV plasma
activity was analyzed as previously
described (25).
Analysis of Gene Expression in
Leukocytes
Blood for gene expression was collected
in Tempus tubes (Applied Biosystems,
Foster City, CA) and total RNA extracted
according to the manufacturers instruc-
tions. Total RNA was DNase I treated us-
ing RQ1 DNase (Promega, Madison, WI)
for 2 h at 37°C, as previously described
(26). Two micrograms of DNase Itreated
RNA was reverse transcribed using
MMuLV reverse transcriptase and ran-
dom hexamers (Promega). One-twentieth
of the reaction was then subjected to
quantitative real-time PCR using primers
spanning exon-exon boundaries and the
ABI Prism 7300 Sequence Detection Sys-
tem (Applied Biosystems). The fold
change in target gene expression was
calculated by the 2
2DDCt
relative quanti-
cation method (Applied Biosystems).
Sample Size and Power Analysis
Asamplesizeofn= 16 healthyparticipants
and n= 16 participants with type 2 di-
abetes in crossover was determined
to have .90% power to detect a true,
between-group difference of 40 625%
relative mRNA expression levels of clock
genes with a Pvalue = 0.05. The current
study provided 80% power to detect 5%
difference between-group in overall post-
prandial plasma area under the curve
Figure 1Gene expression of healthy participants and participants with type 2 diabetes with or without
breakfast. Blood samples were collected after overnight fasting (time point 0), after which breakfast was
given (YesB) or fasting continued (NoB). Hashes denote statistical differences (P,0.05) between time
point 0 and time point 210 min, 3.5 h after breakfast or no breakfast in the healthy group. Asterisks
denote statistical differences (P,0.05) between time point 0 and time point 210 min, 3.5 h after
breakfast or no breakfast in the group with type 2 diabetes. Data are means 6SE.
care.diabetesjournals.org Jakubowicz and Associates 3
(AUC) for iGLP-1, insulin, and glucose. To
allow discontinuation, 36 participants
were recruited.
Statistical Analysis
Thirty-six subjects were enrolled in the
study and four subjects dropped out.
They were excluded from the analyses;
therefore, the results are based on n=32
subjects. AUC was calculated by the trap-
ezoidal rule. For time series, a two-way
ANOVA (time 3diet) was performed
and a least signicant difference paired
Student ttest post hoc analysis was
used for comparison between the YesB
and NoB test days at each time point. For
clock gene expression, fasting (time point
0)wasdeterminedasbaselinefortheYesB
and NoB groups 3.5 h after (time point
210 min). Time point 210 min was deter-
mined as the baseline for the postlunch
(time point 420 min) gene expression. Stu-
dent ttest analyses were performed to
evaluate differences between time points
within each group and treatment. The re-
sults are expressed as mean 6SEM. A
Pvalue #0.05 was considered statistically
signicant. Statistical analysis was per-
formed with JMP software (version 12;
SAS Institute Inc., Cary, NC).
RESULTS
Participants
Thirty-six individuals (18 with type 2 di-
abetes and 18 healthy) were enrolled in
the study, with 32 participants complet-
ing (16 with type 2 diabetes and 16
healthy). During the rst meal test day,
four dropped out (two healthy and two
with diabetes) because of problems in the
installation of the intravenous catheter.
Dropouts did not differ signicantly from
those who continued the study. The pa-
tients with type 2 diabetes were 66.8 6
1.9 years of age and had the disease
for 14.5 61.5 years with 59.6 60.8
mmol/mol (7.6 60.1%) HbA
1c
and BMI
30.7 61.1 kg/m
2
(Supplementary Table
1). Ten patients were treated with
diet alone, whereas six were treated
with diet and metformin. Seven pa-
tients had a history of hypertension and
were treated with ACE inhibitors and/or
calcium channel antagonists. The partic-
ipants in the healthy group were 44.3
62.9 years of age with 4.9 60.1%
(30.1 60.6 mmol/mol) HbA
1c
and BMI
23.1 60.4 kg/m
2
(Supplementary Table
1). They were not taking any medications.
Clock Gene Expression on YesB and
NoB Days in Healthy Individuals
Versus Individuals With Diabetes
Before Lunch
In healthy individuals, the expression
level of Per1,Cry1,Rora,andSirt1 was
lower (Student ttest, P,0.05), but Clock
was higher (Student ttest, P,0.05) after
breakfast (Fig. 1). In contrast, in individu-
als with type 2 diabetes, Per1,Per2,and
Sirt1 only slightly, but signicantly, de-
creased and Roraincreased (Student
ttest, P,0.05) after breakfast. Omission
of breakfast altered clock and metabolic
gene expression in both healthy people
and individuals with type 2 diabetes. In
healthy individuals, there was an in-
crease in the expression level of Bmal1,
Cry1,Rev-erba,Roraand Sirt1 (Student
ttest, P,0.05), whereas in individuals
with type 2 diabetes, Clock expression
increased (Student ttest, P,0.05) (Fig.
1). In both healthy individuals and
individuals with diabetes, Per1 and Per2
expression was not affected whether
breakfast was consumed or not (Fig. 1).
Clock Gene Expression on YesB and
NoB Days in Healthy Individuals
Versus Individuals With Diabetes After
Lunch
In healthyindividuals, the expression level
of Bmal1,Rora,andSirt1 was higher (Stu-
dent ttest, P,0.05) after lunch on the
YesB day, whereas the other clock genes
remained unchanged (Fig. 2). In individu-
als with type 2 diabetes, Bmal1,Per1,
Per2,Rev-erba,andAmpk increased (Stu-
dent ttest, P,0.05) after lunch on the
YesB day. Omission of breakfast altered
clock and metabolic gene expression after
lunch in both healthy individuals and in-
dividuals with type 2 diabetes. In healthy
individuals, Per2 and Ampk expression in-
creased and Per1,Cry1,andRev-erbade-
creased (Student ttest, P,0.05) (Fig. 2),
Figure 2Gene expression of healthy participants and participants with type 2 diabetes after lunch.
Blood samples were collected 3.5 h after breakfast or no breakfast (time point 210 min) and 3.5 h
after lunch (time point 420 min). Hashes denote statistical differences (P,0.05) between time
point 210 min and time point 420 min in the healthy group. Asterisks denote statistical differences
(P,0.05) between time point 210 min and time point 420 min in the group with type 2 diabetes.
Data are means 6SE.
4 Breakfast Skipping Alters Circadian Expression Diabetes Care
whereas in individuals with type 2 diabe-
tes, Bmal1,Per1, and Ampk expression
decreased and Roraexpression increased
(Student ttest, P,0.05) after lunch on
the NoB day (Fig. 2). Taken together,
breakfast and lunch led to increased ex-
pression of the positive loop and down-
regulation of the negative feedback loop,
whereas breakfast skipping altered this
expression pattern. Lunch, as the rst
meal of the day, could not rectify the al-
tered clock expression. In addition, indi-
viduals with type 2 diabetes who skip
breakfast have a greater disruption of
their circadian rhythms compared with
those who consume breakfast.
Glucose, Insulin, and iGLP-1 Levels and
DPP-IV Activity on YesB and NoB Days
In the healthy group and group with di-
abetes, the AUC
glucose
after lunch was 15
18% higher during the NoB day versus
the YesB day (P,0.001) (Fig. 3 and
Supplementary Table 2). In healthy par-
ticipants, AUC
insulin
after lunch was not
signicantly different between the tests
(P= 0.5). In contrast, in the group with
diabetes, the AUC
insulin
after lunch was
25% lower on the NoB day compared
with the YesB day (P,0.004) (Fig. 3
and Supplementary Table 2). In both the
healthy group and group with diabetes,
the AUC
iGLP-1
after lunch was ;35% lower
on the NoB day compared with the
YesB day (P,0.0001) (Fig. 3 and
Supplementary Table 2). No signicant
change in plasma DPP-IV activity was
found after lunch between the groups
on YesB or NoB day.
CONCLUSIONS
In this study, we report for the rst time
an acute postprandial effect on clock and
clock-controlled gene expression in both
healthy individuals and individuals with
type 2 diabetes. We showed that break-
fast skipping acutely disrupts circadian
rhythms in both healthy individuals and
people with type 2 diabetes. Further-
more, clock gene expression after lunch
was different and depended on whether
breakfast was consumed or not. Thus, the
study results suggest that breakfast con-
sumption is of high relevance for preserv-
ing clock gene activity, and this is true for
both healthy subjects and subjects with
diabetes.
Several reports have suggested that
meal timing exerts a pivotal inuence
on peripheral clocks and clock output
systems involved in the regulation of met-
abolic pathways (1,2629). Breakfast
skipping (22,30) and/or eating at hours
designed to sleep (21), for the long run,
lead to disruption of clock gene expres-
sion and are associated with weight gain
and diabetes (31,32). In contrast, acclima-
tion to timed feeding, high-energy break-
fast, and fasting during the hours
designed for sleep reversed impaired
clock gene expression and enhanced
AMPK expression, resulting in a more
efcient weight loss, insulin sensitivity,
and reduction of lipid accumulation
(21,22,3032).
In healthy people, breakfast and lunch
acutely led to an overall increased expres-
sion of the positive loop of the core clock
mechanism, whereas the expression of
the negative feedback loop was downre-
gulated (Fig. 4). This pattern maintains
the normal cyclic expression of output
Figure 3Line charts of healthy participants (A) and participants with type 2 diabetes (B) on YesB
and NoB daysfor glucose, insulin, iGLP-1, and DPP-IV.Breakfast was given to the YesB group at time
point 0. Lunchwas given to both groups at timepoint 210 min. Asterisks denote statisticaldifference
between YesB and NoB at a specic time point. Data are means 6SE.
care.diabetesjournals.org Jakubowicz and Associates 5
systems (Fig. 4). In contrast, breakfast
skipping in healthy people led to an al-
tered clock gene expression pattern. After
lunchontheNoBday,Sirt1,Clock,Bmal1,
and Rorawere upregulated and Per1 was
downregulated, similarly to the effect on
the YesB day (Fig. 4). However, unlike on
the YesB day, after lunch, Per2 and Ampk
were upregulated and Cry1 did not
change, disrupting the normal cyclic ex-
pression (Fig. 4).
In patients with type 2 diabetes, break-
fast and lunch eventually led to an overall
altered clock gene expression (Fig. 4).
However, Ampk expression was upregu-
lated after lunch. As AMPK activation
leads to glucose uptake, this upregulation
may indicate improved glycemic control,
as indeed was found herein. In contrast,
breakfast skipping in people with diabetes
further imparted a greater disru ption, as it
also 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 day of skipping
breakfast led to such a deleterious effect
on clock and clock-controlled gene expres-
sion. The rapid change in clock gene ex-
pression on YesB versus NoB days is
consistent with reports in animal models,
in which a 30-min feeding stimulus al-
tered clock gene expression within 24h
(33,34). As feeding poses such an acute
effect, it is clear why long-term abnormal
patterns of clock gene expression are
manifested in obese individuals and indi-
viduals with diabetes.
A change in clock and clock-controlled
gene expression, due to breakfast omis-
sion, is expected to yield altered metabo-
lism. Impaired circadian rhythms have
been closely associated with the patho-
physiology of type 2 diabetes(4). Reduced
glucose-stimulated insulin secretion, insu-
lin resistance, diminished b-cell prolifera-
tion, and apoptosis have been associated
with asynchrony or deciencies in clock
genes (16). Indeed, in people with diabe-
tes, omission of breakfast led to a signif-
icant increase in postprandial glucose
excursions with reduced insulin and
iGLP-1 responses after lunch compared
with YesB day. Furthermore, this delete-
rious effect of breakfast skipping was also
observed in healthy participants, although
with a lower magnitude.
The reduction of hyperglycemia and
enhanced insulin response after lunch
with prior consumption of breakfast has
been described as the second meal
phenomenon and has been previously re-
ported in several studies in healthy in-
dividuals and individuals with type 2
diabetes (23,35). This has been explained
by enhanced b-cell responsiveness at the
second meal induced by the rst meal
(36,37). This explanation is based on the
ndings that both the rst and the second
phase of insulin release are inuenced by
b-cell memory, and the magnitude of in-
sulin release is enhanced signicantly by
previous glucose exposure (37). The ab-
sence of glucose elevation due to fasting
until noon may diminish b-cell respon-
siveness and memory, leading to a re-
duced and delayed insulin response
after lunch on the NoB day. Indeed, it
was recently reported that lower insulin
release by b-cells in response to nutrient
depletion or starvation induces lysosomal
degradation of nascent secretory insulin
granules, and this is controlled by protein
kinase D (PKD), a key player in secretory
granule biogenesis (38). The impaired in-
sulin secretion at lunch on the NoB day
may also be due to perturbed incretin
regulation, since both b-cell memory
and sensitivity to glucose are enhanced
by GLP-1. Therefore, the higher levels of
GLP-1 on the YesB day may explain both
the enhanced insulin secretion and the
reduced glycemic response after lunch.
At the cellular level, the reduction of post-
prandial glycemia after lunch on the YesB
day can be explained by the fact that
breakfast triggers correct cyclic expres-
sion of clockgenes, which, in turn, assures
metabolic efciency, leading to normal
glucose, insulin, and GLP-1 responses af-
ter lunch. Ampk upregulation in partici-
pants with type 2 diabetes may enhance
GLUT4 translocation and muscular glu-
cose uptake, leading to improved post-
prandial glycemia.
At baseline, clock gene levels were dif-
ferent between the healthy group and
the group with type 2 diabetes, presum-
ably due to differences related to age, body
weight, and/or HbA
1c
levels. Although the
age and body weight of the group with
type 2 diabetes was signicantly different
from the healthy group, and may be a
limitation in our study, changes in clock
gene expression as a result of breakfast
skipping cannot be attributed to these
parameters, as each group was normal-
ized to its own baseline. In addition, the
crossover study assured that the same
person had both YesB and NoB treat-
ments, reiterating the effect of breakfast
skipping. Another limitation of our study
is that it was performed until after lunch.
Figure 4Effect of lunch with or without prior breakfast on the interrelations between the clock
mechanism 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 glucosehomeostasis. PER1, PER2, and CRY1 serve as the negative
feedback loop that inhibits CLOCK:BMAL1-mediated expression. SIRT1 and AMPK, when activated
under low cellular energy levels, relieve the inhibition mediated by the negative feedback loop.
BMAL1 expression is positively regulated by RORaand negatively regulated by REV-ERBa. Breakfast
consumption followed by lunch assures this overall cyclic regulation is maintained. Omission
of breakfast disruptsthis cyclic regulation in both healthy people and patientswith type 2 diabetes.
(A high-quality color representation of this gure is available in the online issue.)
6 Breakfast Skipping Alters Circadian Expression Diabetes Care
Analyses of subsequent dinner and other
time points during the nighttime are re-
quired in order to determine overall daily
rhythms and how each meal affects this
oscillatory pattern. In summary, our study
demonstrates that breakfast consump-
tion exerts a rapid inuence on clock
and clock-controlled genes involved in
glucose regulation, such as Ampk,and
this effect is associated with a signicant
reduction in postprandial glycemia and
enhanced insulin and GLP-1 responses
after subsequent lunch. In contrast, break-
fast skipping adversely affects the expres-
sion of clock and clock-controlled genes
involved in glucose metabolism in both
healthy individuals and individuals with di-
abetes. This study emphasizes the con-
sumption of breakfast as an important
strategy when targeting glycemic control
in type 2 diabetes. As the circadian clock
also regulates blood pressure, heart rate,
and cardiovascular activity, as well as adi-
pose tissue and other metabolic organs
(39), meal timing may affect overall me-
tabolism and inuence chronic complica-
tions of obesity and type 2 diabetes.
Funding. This study was funded by D-Cure and
the Ministry of Health, Israel (grant 3-0000-
11406).
Duality of Interest. No potential conicts of in-
terest relevant to this article were reported.
Author Contributions. D.J. and O.F. contrib-
uted to the conception and design of the study,
acquired, analyzed, and interpreted data, and
drafted and revised the manuscript. J.W. con-
tributed to the conception and design of the
study, acquired and interpreted data, and
drafted the manuscript. Z.L., N.C., T.G., M.M.,
and Y.B.-D. contributed to the conception and
designof the study,acquired andinterpreteddata,
organized the randomization, and drafted the
manuscript. I.R. and B.A. researched data, con-
tributed to the interpretation of the data, and
draftedand revisedthe manuscript.M.B. analyzed
and interpreted data. D.J. is the guarantor of this
work and, as such, had full access to all the data in
the study and takes responsibility forthe integrity
of the data and the accuracy of the data analysis.
Prior Presentation. This study was presented
orally at the 77th Scientic Sessions of the Amer-
ican Diabetes Association, San Diego, CA, 913
June 2017.
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care.diabetesjournals.org Jakubowicz and Associates 7
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Skipping breakfast has been consistently associated with high HbA1c and postprandial hyperglycemia (PPHG) in patients with type 2 diabetes. Our aim was to explore the effect of skipping breakfast on glycemia after a subsequent isocaloric (700 kcal) lunch and dinner. In a crossover design, 22 patients with diabetes with a mean diabetes duration of 8.4 ± 0.7 years, aged 56.9 ± 1.0 years, BMI 28.2 ± 0.6 kg/m(2), and HbA1c 7.7 ± 0.1% (61 ± 0.8 mmol/mol) were randomly assigned to two test days: one day with breakfast, lunch, and dinner (YesB) and another with lunch and dinner but no breakfast (NoB). Postprandial plasma glucose, insulin, C-peptide, free fatty acids (FFA), glucagon, and intact glucagon-like peptide-1 (iGLP-1) were assessed. Compared with YesB, lunch area under the curves for 0-180 min (AUC0-180) for plasma glucose, FFA, and glucagon were 36.8, 41.1, and 14.8% higher, respectively, whereas the AUC0-180 for insulin and iGLP-1 were 17% and 19% lower, respectively, on the NoB day (P < 0.0001). Similarly, dinner AUC0-180 for glucose, FFA, and glucagon were 26.6, 29.6, and 11.5% higher, respectively, and AUC0-180 for insulin and iGLP-1 were 7.9% and 16.5% lower on the NoB day compared with the YesB day (P < 0.0001). Furthermore, insulin peak was delayed 30 min after lunch and dinner on the NoB day compared with the YesB day. Skipping breakfast increases PPHG after lunch and dinner in association with lower iGLP-1 and impaired insulin response. This study shows a long-term influence of breakfast on glucose regulation that persists throughout the day. Breakfast consumption could be a successful strategy for reduction of PPHG in type 2 diabetes. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
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Aims/hypothesis: Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. Methods: This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. Results: We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. Conclusions/interpretation: This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.
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Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function. Copyright © 2015, American Association for the Advancement of Science.
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Because current therapeutics for obesity are limited and only offer modest improvements, novel interventions are needed. Preventing obesity with time-restricted feeding (TRF; 8-9 hr food access in the active phase) is promising, yet its therapeutic applicability against preexisting obesity, diverse dietary conditions, and less stringent eating patterns is unknown. Here we tested TRF in mice under diverse nutritional challenges. We show that TRF attenuated metabolic diseases arising from a variety of obesogenic diets, and that benefits were proportional to the fasting duration. Furthermore, protective effects were maintained even when TRF was temporarily interrupted by ad libitum access to food during weekends, a regimen particularly relevant to human lifestyle. Finally, TRF stabilized and reversed the progression of metabolic diseases in mice with preexisting obesity and type II diabetes. We establish clinically relevant parameters of TRF for preventing and treating obesity and metabolic disorders, including type II diabetes, hepatic steatosis, and hypercholesterolemia. Copyright © 2014 Elsevier Inc. All rights reserved.
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Background/aims: Transcriptomics technology in human nutrition intervention studies would allow for genome-wide screening of the effects of nutrients. We observed the time course of gene expression changes in peripheral white blood cells (WBC) to elucidate the metabolic changes in the postprandial state that are a reflection and a marker of whole body metabolic changes. Methods: In a randomized crossover study, 7 healthy subjects consumed test meals of glucose (GL), white rice (WR) and rolled barley (BAR), each containing 75 g of available carbohydrate, and water (WAT). Blood glucose, insulin and nonesterified fatty acid concentrations, as well as the subjective levels of fullness and hunger were measured. Microarray analysis of the WBC and the real-time PCR were examined during 360 min after the intake of the test meals. Results: The number of genes that changed more than 1.5-fold and the expression patterns in the time course were different between the GL, the WR and the BAR groups. Several genes involved in glycolysis and fatty acid β-oxidation were markedly changed after the intake of the GL, the WR and the BAR; however, these genes did not change at any time point in the WAT. Conclusions: Gene expression profiling in the WBC can reflect food-related metabolic changes, even in the postprandial state.
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Background The circadian clock regulates various physiological and behavioral rhythms such as feeding and locomotor activity. Feeding at unusual times of the day (inactive phase) is thought to be associated with obesity and metabolic disorders in experimental animals and in humans. Objective The present study aimed to determine the underlying mechanisms through which time-of-day-dependent feeding influences metabolic homeostasis. Methods We compared food consumption, wheel-running activity, core body temperature, hormonal and metabolic variables in blood, lipid accumulation in the liver, circadian expression of clock and metabolic genes in peripheral tissues, and body weight gain between mice fed only during the sleep phase (DF, daytime feeding) and those fed only during the active phase (NF, nighttime feeding). All mice were fed with the same high-fat high-sucrose diet throughout the experiment. To the best of our knowledge, this is the first study to examine the metabolic effects of time-imposed restricted feeding (RF) in mice with free access to a running wheel. Results After one week of RF, DF mice gained more weight and developed hyperphagia, higher feed efficiency and more adiposity than NF mice. The daily amount of running on the wheel was rapidly and obviously reduced by DF, which might have been the result of time-of-day-dependent hypothermia. The amount of daily food consumption and hypothalamic mRNA expression of orexigenic neuropeptide Y and agouti-related protein were significantly higher in DF, than in NF mice, although levels of plasma leptin that fluctuate in an RF-dependent circadian manner, were significantly higher in DF mice. These findings suggested that the DF induced leptin resistance. The circadian phases of plasma insulin and ghrelin were synchronized to RF, although the corticosterone phase was unaffected. Peak levels of plasma insulin were remarkably higher in DF mice, although HOMA-IR was identical between the two groups. Significantly more free fatty acids, triglycerides and cholesterol accumulated in the livers of DF, than NF mice, which resulted from the increased expression of lipogenic genes such as Scd1, Acaca, and Fasn. Temporal expression of circadian clock genes became synchronized to RF in the liver but not in skeletal muscle, suggesting that uncoupling metabolic rhythms between the liver and skeletal muscle also contribute to DF-induced adiposity. Conclusion Feeding at an unusual time of day (inactive phase) desynchronizes peripheral clocks and causes obesity and metabolic disorders by inducing leptin resistance, hyperphagia, physical inactivity, hepatic fat accumulation and adiposity.
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To examine whether a mismatch between chronotype (i.e., preferred sleep timing) and work schedule is associated with type 2 diabetes risk. In the Nurses' Health Study 2, we followed 64,615 women from 2005 to 2011. Newly developed type 2 diabetes was the outcome measure (n = 1,452). A question on diurnal preference ascertained chronotype in 2009; rotating night shift work exposure was assessed regularly since 1989. Compared with intermediate chronotypes, early chronotypes had a slightly decreased diabetes risk after multivariable adjustment (odds ratio 0.87 [95% CI 0.77-0.98]), whereas no significant association was observed for late chronotypes (1.04 [0.89-1.21]). Among early chronotypes, risk of type 2 diabetes was modestly reduced when working daytime schedules (0.81 [0.63-1.04]) and remained similarly reduced in women working <10 years of rotating night shifts (0.84 [0.72-0.98]). After ≥10 years of shift work exposure, early chronotypes had a nonsignificant elevated diabetes risk (1.15 [0.81-1.63], Ptrend = 0.014). By contrast, among late chronotypes, the significantly increased diabetes risk observed among day workers (1.51 [1.13-2.02]) appeared largely attenuated if their work schedules included night shifts (<10 years: 0.93 [0.76-1.13]; ≥10 years: 0.87 [0.56-1.34]; Ptrend = 0.14). The interaction between chronotype and shift work exposure was significant (Pinteraction = 0.0004). Analyses restricting to incident cases revealed similar patterns. In early chronotypes, type 2 diabetes risk increased with increasing duration of shift work exposure, whereas late types had the highest diabetes risk working daytime schedules. These data add to the growing body of evidence that workers could benefit from shift schedules minimizing interference with chronotype-dependent sleep timing. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
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Circadian physiology is responsible for the temporal regulation of metabolism to optimize energy homeostasis throughout the day. Disturbances in the light/dark cycle, sleep/wake schedule, or feeding/activity behavior can affect the circadian function of the clocks located in the brain and peripheral tissues. These alterations have been associated with impaired glucose tolerance and type 2 diabetes. Animal models with molecular manipulation of clock genes and genetic studies in humans also support these links. It has been demonstrated that the endocrine pancreas has an intrinsic self-sustained clock, and recent studies have revealed an important role of clock genes in pancreatic β cells, glucose homeostasis, and diabetes. Copyright © 2014 Elsevier Ltd. All rights reserved.
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The circadian clock plays an integral role in the regulation of physiological processes, including the regulation of blood pressure. However, deregulation of the clock can lead to pathophysiological states including hypertension. Recent work has implicated the circadian clock genes in the regulation of processes in the heart, kidney, vasculature, and the metabolic organs, which are all critical in the regulation of the blood pressure. The goal of this review is to provide an introduction and general overview into the role of circadian clock genes in the regulation of blood pressure with a focus on their deregulation in the etiology of hypertension. This review will focus on the core circadian clock genes CLOCK, BMAL1, Per, and Cry.