Article

Divergent remodeling of the skeletal muscle

metabolome over 24 h between young, healthy men
and older, metabolically compromised men
Graphical abstract Authors
Jan-Frieder Harmsen,
Michel van Weeghel, Rex Parsons, ...,
Matthijs K.C. Hesselink,
Riekelt H. Houtkooper,
Patrick Schrauwen

Correspondence
r.h.houtkooper@amsterdamumc.nl
(R.H.H.),
p.schrauwen@maastrichtuniversity.nl
(P.S.)

Highlights
d The skeletal muscle metabolome vastly changes over 24 h
In brief
Harmsen et al. examine the skeletal
muscle metabolome over 24 h comparing
serial tissue samples from young, healthy
men versus older, metabolically
compromised men. Different abundance
levels of most metabolites around the
clock, as well as different temporal
dynamics, are evident between groups.
The night period demonstrates the
largest disparity.

d Most muscle metabolites are less abundant in older,

metabolically compromised men

d 24 h rhythms differ between young, healthy men versus older,

metabolically compromised men

d The largest differences between groups are identified during

the night

Harmsen et al., 2022, Cell Reports 41, 111786

December 13, 2022 ª 2022 The Author(s).
https://doi.org/10.1016/j.celrep.2022.111786 ll
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Article
Divergent remodeling of the skeletal muscle
metabolome over 24 h between young, healthy men
and older, metabolically compromised men
Jan-Frieder Harmsen,1 Michel van Weeghel,2,3 Rex Parsons,4 Georges E. Janssens,2 Jakob Wefers,1 Dirk van Moorsel,5
Jan Hansen,1 Joris Hoeks,1 Matthijs K.C. Hesselink,1 Riekelt H. Houtkooper,2,* and Patrick Schrauwen1,6,*
1Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht

University Medical Center, 6200 MD Maastricht, the Netherlands

2Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology, Endocrinology, and Metabolism, Amsterdam Cardiovascular

Sciences, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
3Core Facility Metabolomics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
4Australian Centre for Health Services Innovation and Centre for Healthcare Transformation, School of Public Health and Social Work, Faculty

of Health, Queensland University of Technology, Kelvin Grove, QLD, Australia

5Division of Endocrinology, Department of Internal Medicine, Maastricht University Medical Center, 6200 MD Maastricht, the Netherlands
6Lead contact

*Correspondence: r.h.houtkooper@amsterdamumc.nl (R.H.H.), p.schrauwen@maastrichtuniversity.nl (P.S.)

https://doi.org/10.1016/j.celrep.2022.111786

SUMMARY

24 h whole-body substrate metabolism and the circadian clock within skeletal muscle are both compromised
upon metabolic disease in humans. Here, we assessed the 24 h muscle metabolome by serial muscle sam-
pling performed under 24 h real-life conditions in young, healthy (YH) men versus older, metabolically
compromised (OMC) men. We find that metabolites associated with the initial steps of glycolysis and hexos-
amine biosynthesis are higher in OMC men around the clock, whereas metabolites associated with gluta-
mine-alpha-ketoglutarate, ketone, and redox metabolism are lower in OMC men. The night period shows
the largest number of differently expressed metabolites. Both groups demonstrate 24 h rhythmicity in half
of the metabolome, but rhythmic metabolites only partially overlap. Specific metabolites are only rhythmic
in YH men (adenosine), phase shifted in OMC men (cis-aconitate, flavin adenine dinucleotide [FAD], and uri-
dine diphosphate [UDP]), or have a reduced 24 h amplitude in OMC men (hydroxybutyrate and hippuric acid).
Our data highlight the plasticity of the skeletal muscle metabolome over 24 h and large divergence across the
metabolic health spectrum.

INTRODUCTION hydrocarbon receptor nuclear translocator-like 1 (BMAL1) acting

In lean individuals, skeletal muscle constitutes about 40% of
human body mass and is the largest contributor to whole-body
energy expenditure.1,2 In response to metabolic demand (resting
versus physically active) and nutrient status (fasted versus fed),
healthy skeletal muscle can rapidly modulate its substrate stor-
age and adjust its substrate oxidation to glucose or fat availabil-
ity.3,4 Over the 24 h cycle, skeletal muscle metabolism switches
from predominantly carbohydrate oxidation during the fed and
active period to lipid oxidation during the fasted and inactive
period.5 This predictable fed-fasting cycle in human skeletal
muscle metabolism is reciprocally linked to the endogenous
molecular circadian clock present in muscle cells by providing
systemic time cues to the clock6 but also receiving reinforce-
ment through the clock.5
As for other peripheral clocks, the skeletal muscle clock consists
of a transcriptional-translational feedback loop with circadian lo-
comotor output cycles kaput (CLOCK) and brain and muscle aryl
as transcriptional activators of cryptochrome 1 and 2 (Cry1–2)
and period 1, 2, and 3 (Per1–3) genes, whose encoded proteins
repress CLOCK:BMAL1 with a recurring periodicity of
24 h.7
Accordingly, relevant proportions of the human skeletal muscle
transcriptome (
10% of transcripts8), lipidome (
12%–20% of
lipids9,10), and metabolome (4%–7% of metabolites11) display
24 h rhythmicity. Of note, 24 h rhythms in skeletal muscle meta-
bolism are not only regulated by the endogenous skeletal muscle
clock but are also influenced by the timing of light exposure,
food intake, and physical activity. Thus, unanticipated occurrence
of these factors can alter or blunt 24 h rhythmicity in human meta-
bolism.12,13 Our modern 24 h culture—characterized by eating and
working day and night, reduced sleep, and excessive light expo-
sure at night—is increasingly being recognized for its negative
impact on metabolic health and associated with an increased
risk to develop metabolic diseases such as type 2 diabetes mellitus
(T2DM).12,13 T2DM is often associated with insulin resistance in
skeletal muscle. Interestingly, Gabriel et al.14 recently found

Cell Reports 41, 111786, December 13, 2022 ª 2022 The Author(s). 1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Table 1. Participant characteristics
Young, healthy Older, metabolically
Parameter (n = 12) compromised (n = 12)
Age (years) 22 ± 2 65 ± 9
Height (m) 1.80 ± 0.07 1.78 ± 0.05
Body weight (kg) 73 ± 8 96 ± 12
BMI (kg/m2) 22.4 ± 2.0 30.3 ± 2.7
MEQ-SA score 50 ± 7 57 ± 9
Fasting plasma 5.3 ± 0.6 5.7 ± 0.4
glucose (mmol/L)
Fasting plasma 11.1 ± 4.3 13.8 ± 8.5
insulin (mIU/mL)
Data are presented as mean ± SD. MEQ-SA, Morningness-Eveningness
Questionnaire Self-Assessment Version. Scores of 41 and below indicate
‘‘evening types.’’ Scores of 59 and above indicate ‘‘morning types."
Scores between 42 and 58 indicate ‘‘intermediate types.’’
altered rhythmicity in clock gene expression of cultured primary
myotubes from donors with T2DM compared with age- and
BMI-matched normoglycemic controls. Similarly, we demon-
strated that skeletal muscle clock gene expression was altered
in in older, metabolically compromised men compared with young,
healthy men,15 with Per2 and Cry1 having an altered 24 h rhythm
and Clock lacking 24 h rhythmicity. Furthermore, 24 h rhythmicity
in whole-body substrate oxidation was blunted in older, metabol-
ically compromised men compared with young, healthy men.15
Whereas young, healthy men showed a switch from carbohydrate
oxidation during the day to lipid oxidation during the night, this
fed-to-fasted transition in substrate oxidation was severely atten-
uated in older, metabolically compromised men.
Since skeletal muscle is the major determinant of whole-body
substrate metabolism, we here aimed to explore how the temporal
organization of the skeletal muscle metabolome differs between
the edges of the metabolic health spectrum. We hypothesized
that 24 h rhythmicity in the skeletal muscle metabolome is
disturbed in older, metabolically compromised men compared
with young, healthy men. Such temporal changes of muscle me-
tabolites could provide mechanistic insight into how disrupted
24 h whole-body substrate metabolism and impaired muscle clock
gene expression emerge. Therefore, we performed semi-targeted
metabolomics on muscle biopsy samples taken around the clock
from young, healthy and older, metabolically compromised men.
RESULTS
In two separate cross-sectional cohort studies, our group
previously reported how whole-body substrate metabolism and
skeletal muscle metabolism change across 24 h under real-life
conditions with 3 standardized meals in young, healthy and older,
metabolically compromised men.15,16 Further comparative partic-
ipant characteristics are shown in Table 1. By combining these
previously reported data, diurnal differences between young,
healthy (YH) men and older, metabolically compromised (OMC)
men for the respiratory exchange ratio (RER), core body temper-
ature, plasma metabolites, and skeletal muscle clock gene
expression are summarized in Figure S1. The following semi-tar-
2 Cell Reports 41, 111786, December 13, 2022
Article
geted metabolomics analysis was performed to explore mecha-
nisms associated with these large differences in 24 h whole-
body substrate metabolism and within the circadian clock.
Mean abundance levels of most metabolites differ
substantially between OMC and YH men
To get an initial impression about global differences in the skeletal
muscle metabolome between YH and OMC men, we first looked at
all metabolites with a significant condition effect in the generalized
linear mixed model. This exploratory analysis is equivalent to
comparing the mean abundance levels around the clock between
groups. Of the 115 analyzed metabolites, 64 metabolites (
56%)
displayed altered mean abundance levels between groups, and
the majority of these metabolites had lower levels in OMC men
than YH men. By assigning all 115 metabolites to their respective
superordinate metabolic pathway, we explored overall patterns
in specific pathways with respect to altered mean abundance
levels of the assigned metabolites between groups (Figure 1A).
From the detected metabolites that are associated with the
tricarboxylic acid (TCA) cycle (Figure 2), cis-aconitate, glutamine,
glutamate, and alpha-ketoglutarate demonstrated lower
mean abundance levels in OMC men (condition: p < 0.001). At
the mRNA level, Aco2 encoding mitochondrial aconitase 2, which
catalyzes the interconversion of citrate to isocitrate via cis-aconi-
tate in the second step of the TCA cycle, also demonstrated lower
mean expression levels in OMC men (Figure S2B). In contrast, suc-
cinate, fumarate, malate, acetyl-CoA, and citrate were not different
between groups, suggesting anaplerotic compensation at the level
of succinyl-CoA through amino acids in OMC men. Moreover, 2- or
3-hydroxybutyric acid, associated with ketone metabolism, dis-
played lower levels in OMC men (p < 0.001). Creatine and creati-
nine were also lower in OMC men (p < 0.01). Associated with the
urea cycle, acetylglutamic acid, aspartate, arginine, uric acid,
glutamine, glutamate, and ornithine were lower around the clock
in OMC men (p % 0.001). Several metabolites that are involved
in redox reactions, such as cysteine, glutathione, NADH, NADP+,
NADPH, and pyroglutamic acid, were less abundant in OMC
men (p < 0.05). Nonetheless, the ratios between NAD+/NADH
and ADP/ATP both had higher mean levels in OMC men (p %
0.01; Figure S3).
Strikingly, the glycolysis pathway contrasted the otherwise
consistent global pattern of lower mean metabolite levels in
OMC men (Figure 3). Thus, metabolites associated with the first
steps of glycolysis were higher in OMC men: hexose-phosphate,
dihydroxyacetone-phosphate, D-glyceraldehyde 3-phosphate,
and glyceric acid 1,3-biphosphate (p < 0.05). In contrast, metabo-
lites associated with later steps of glycolysis were again lower in
OMC men: 3-phosphoglyceric acid, 2-phosphoglyceric acid,
and phosphoenolpyruvate (p % 0.003), suggesting a bottleneck
at the enzymatic conversion of glyceric acid 1,3-biphosphate to
3-phosphoglyceric acid involving phosphoglycerate kinase 1
(PGK1). Consistent with this suggestion, mRNA levels of Pgk1
were lower in OMC men (p < 0.001; Figure 3). However, the end
product of glycolysis, pyruvate, was not different between groups.
With respect to glycogenesis, mean abundance levels of uridine
diphosphate (UDP)-hexose and mean mRNA levels of Gys1
encoding glycogen synthase 1 were both lower in OMC men
(Figure S2C), suggesting reduced muscle glycogen synthesis
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capacity in OMC men compared with YH men. A relatively minor
branch of glycolysis, the hexosamine biosynthesis pathway, was
also altered in OMC men with its major end product, UDP
N-acetylglucosamine (UDP-GlcNAc), being higher in OMC men
around the clock (p < 0.001). On the contrary, mRNA levels of
Gfpt1 encoding the rate-limiting enzyme of this pathway, gluta-
mine-fructose-6-phosphate transaminase 1 (GFAT), were lower
in OMC men (p < 0.001; Figure S2A). Lastly, mean mRNA levels
of Slc2a4 encoding GLUT4, Irs1 encoding insulin receptor sub-
strate 1, and Hk2 encoding hexokinase 2 were all lower in OMC
men (Figures 4A–4C), in line with reduced insulin sensitivity in
OMC men.
In summary, the initial comparison of mean abundance levels
around the clock revealed substantial differences in the skeletal
muscle metabolome between groups with mostly lower abun-
dance levels in OMC men, with the exception of those metabo-
lites associated with the initial steps of glycolysis and hexos-
amine biosynthesis metabolism, which were higher in OMC
men. Reduced levels of Pgk1 mRNA in OMC men are consistent
with a bottleneck in the conversion of the early toward later steps
in glycolysis in OMC men.
The greatest differences between OMC and YH men are
present at 4 a.m.
We next explored if the overall differences in metabolite levels
between OMC and YH men could be attributed to specific time
points. For this purpose, we compared differences between
groups over the 5 time points by means of Bonferroni post-hoc
tests. Strikingly, the greatest differences in the muscle metabo-
lome between groups were found during the night at 4 a.m., with
33% of detected metabolites (38 of 115) showing differences be-
tween groups (Figure 1B; Table S1). In the overnight fasted state,
at 8 a.m., 23 metabolites (20%) were different between groups.
In contrast, differences were less pronounced during the post-
prandial daytime, with 16 metabolites (14%) different at 1 p.m.
and 15 metabolites (13%) at 6 p.m. At 11 p.m.—just before par-
ticipants went to bed—22 metabolites (19%) were different be-
tween groups.
Our findings are consistent with the large difference in whole-
body substrate metabolism that we previously observed during
the night—with YH men switching from carbohydrate oxidation
during the day to lipid oxidation during the night, while OMC
men maintained carbohydrate oxidation during the night.15
24 h rhythmicity in the skeletal muscle metabolome is
partly altered in OMC men
Rhythmic versus arrhythmic metabolites
The serial sampling of muscle tissue over 24 h allowed us to
analyze 24 h rhythmicity of metabolites. Seventy metabolites dis-
Figure 1. Global differences within the skeletal muscle metabolome across the metabolic health spectrum
(A) Heatmap depicting abundance levels of muscle metabolites and their associated pathways comparing young healthy (YH) versus older, metabolically
compromised (OMC) men around the clock. The pseudocolor scaling of the metabolite expression is based on the standard deviation (SD) to the mean level
calculated for each metabolite over both groups with lower levels (blue) to higher levels (red) compared with the mean level. Each row represents a single
metabolite, and each column depicts one of the five time points per group.
(B) Overview of the number of metabolites that differed between YH and OMC men at the respective time point (n = 12 subjects per group). The metabolites that
underlie this figure are listed in Table S1.
*Significantly different mean abundance levels between groups (p < 0.05), condition effect in the generalized linear mixed model.
4 Cell Reports 41, 111786, December 13, 2022
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played 24 h rhythmicity in at least one of the two groups, whereas
45 metabolites were arrhythmic in both (Figure 5A). The peak
abundance level occurred at 8 a.m. for most rhythmic metabo-
lites in YH and OMC men, suggesting a superordinate impact
of the overnight fast and/or continuous lack of physical activity
during sleep on the muscle metabolome (Figure S4). Notably,
most metabolites associated with glycolysis (Figure 3) and the
pentose phosphate pathway (Figure 1A) were arrhythmic,
suggesting that these pathways are not strictly controlled by
the molecular clock and/or by environmental rhythmicity such
as human behavior (e.g., feeding/fasting and/or activity/rest cy-
cles). In contrast, detected metabolites involved in the TCA cycle
were consistently rhythmic either in both groups (cis-aconitate,
alpha-ketoglutarate, glutamate, fumarate, malate) or at least
for one group (YH men: acetyl-CoA and succinate; OMC men:
citrate and transcript levels of Aco2). Most TCA metabolites
shared a similar rhythmic pattern, peaking after the longest
period of fasting and rest at 8 a.m., except for acetyl-CoA
(peak at 1 p.m.) and citrate (peak at 6 p.m.) (Figure 2).
Some metabolites demonstrated 24 h rhythmicity either
only in YH men or only in OMC men
Among the rhythmic metabolites, we first zoomed into those me-
tabolites that were only rhythmic in one of the two groups. Based
on a cosinor-based model (see STAR Methods for details), we
found 14 metabolites that were only rhythmic in YH men and
22 metabolites that were only rhythmic in OMC men (see Fig-
ure 5B for the complete list of metabolites). For instance, all three
branched-chain amino acids (BCAAs)—isoleucine, leucine and
valine—were rhythmic in OMC men, characterized by a peak
at 4 or 8 a.m. and a consistent trough at 6 p.m. (Figures S5I
and S5N), suggesting either control by the molecular clock or
by human behavior (e.g., feeding/fasting and/or activity/rest cy-
cles). However, a similar pattern was observed for BCAAs in YH
men that only reached statistical significance for 24 h rhythmicity
for isoleucine. Therefore, to test which of these metabolites
differed most strongly in rhythmicity between groups, we applied
a more stringent analysis using a generalized linear mixed model
to further test for interaction effects. This analysis revealed an
interaction effect only for a fraction of these 36 metabolites:
24 h rhythmicity for adenosine was found in YH men with an in-
crease at night (peak at 4 a.m.) that differed from the stable
pattern in OMC men (Figure 5F). Furthermore, 24 h rhythmicity
for glycerate, kynurenine, and ophthalmic acid was found only
in OMC men. Glycerate had its peak at 8 a.m. and trough at 11
p.m. (Figure 5C), kynurenine had its peak at 11 p.m. and trough
at 1 p.m. (Figure 5D), and ophthalmic acid had its peak at 1 p.m.
and trough at 4 a.m. (Figure 5E). All metabolites that displayed
24 h rhythmicity in only one group without an interaction effect,
and hence no significantly different expression over time, are
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Cytosol Pyruvate
Pyruvate

12
Young healthy

Abundance level
8 Older, metabolically compromised

4
time: p = 0.638
condition: p = 0.103
interaction: p = 0.893
0
8:00 13:00 18:00 23:00 4:00

Mitochondrion
Acetyl-CoA
Acetyl-CoA

0.15
Abundance level

0.10

0.05
time: p = 0.009
condition: p = 0.662
interaction: p = 0.210
0.00
8:00 13:00 18:00 23:00 4:00 Citrate
Citric acid

0.4
Abundance level

0.3
Oxaloacetate
0.2
Malate
Malate (n.d.)
0.1 time: p = 0.011
0.5 time: p < 0.001
condition: p = 0.137
interaction: p = 0.601
condition: p = 0.098
Abundance level

0.4 interaction: p = 0.610 0.0

8:00 13:00 18:00 23:00 4:00
0.3
0.2
0.1
0.0
Cis-aconitate
Cis-Aconitate

8:00 13:00 18:00 23:00 4:00 1.5 *

*
Abundance level

1.0

Fumarate
Fumarate

0.6
TCA 0.5
time: p = 0.013
condition: p = 0.002
Abundance level

interaction: p = 0.030
0.0
0.4
cycle 8:00 13:00 18:00 23:00 4:00

0.2
time: p < 0.001

0.0
condition: p = 0.232
interaction: p = 0.545 Isocitrate (n.d.)
8:00 13:00 18:00 23:00 4:00

D-ketoglutarate
Alpha-Ketoglutarate
Succinate
Succinate
15 time: p < 0.001
0.12
Abundance level

condition: p < 0.001

* interaction: p = 0.225
Abundance level

10 *
0.08
5
0.04
time: p = 0.039
condition: p = 0.851 0
interaction: p = 0.164
0.00 8:00 13:00 18:00 23:00 4:00
8:00 13:00 18:00 23:00 4:00
Glutamate
Glutamate Glutamine
Glutamine

20 250
*
*
succinyl-CoA
Abundance level

Abundance level

15 200

(n.d.) 10
150
100
5 time: p = 0.037
50 time: p = 0.188
condition: p < 0.001 condition: p < 0.001
interaction: p = 0.547 interaction: p = 0.953
0 0
8:00 13:00 18:00 23:00 4:00 8:00 13:00 18:00 23:00 4:00

(legend on next page)

Cell Reports 41, 111786, December 13, 2022 5

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shown in Figures S5 and S6. On the transcript level, Gfpt1 mRNA
was only rhythmic in OMC men, peaking at 8 a.m., following the
rhythmic pattern in glucose abundance levels (Figure S2A), but
no interaction effect was found compared with YH men.
By focusing on those metabolites that showed 24 h rhythmicity
in only one of the two groups, a lack of rhythmicity for adenosine
and presence of rhythmicity for glycerate, kynurenine and
ophthalmic acid was identified in OMC men compared with
YH men.
Many metabolites had 24 h rhythmicity in both groups
but displayed differences in mesor, amplitude, or
acrophase
Although the overall presence of 24 h rhythmicity in the
skeletal muscle metabolome seemed to be comparable be-
tween OMC and YH men, we hypothesized that specific proper-
ties of 24 h rhythmicity are different between groups. Using the
CircaCompare model,17 we estimated differences in mesor
(rhythm-adjusted mean abundance level of the metabolite),
amplitude, and acrophase (time at which the peak of the 24 h
rhythm occurs) in those 38 metabolites with 24 h rhythmicity in
both groups (Figure 5B). Most prominently, 21 out of 38 metab-
olites displayed a lower mesor level in OMC men (Figure 5B), in
line with the global pattern of lower mean abundance levels in
OMC men as described earlier, whereas only ADP-ribose dis-
played a higher mesor level in OMC men (p < 0.001). In OMC
men, a phase shift was detected for cis-aconitate (p = 0.013),
flavin adenine dinucleotide (FAD; p = 0.001), and UDP (p =
0.036) (Figures 5I–5K) when compared with YH men, with FAD
showing the largest phase shift. The only two metabolites with
an altered amplitude were 2- or 3-hydroxybutyric acid (p =
0.022) and hippuric acid (p < 0.001), which both exhibited a
smaller amplitude in OMC versus YH men (Figures 5G and 5H).
For nine metabolites (asparagine, CDP, fumarate, histidine,
itaconic acid, malate, proline, serine, and taurine), no differences
in mesor, amplitude, or phase were detected between
groups, suggesting either control by the molecular clock or
by diurnal human behavior independent of metabolic health
status.
For those pathways that showed the most differences
between groups, we performed complementary analyses of
transcript levels. Associated with glucose metabolism, Slc2a4,
Irs1, Hk2 (Figures 4A–4C), and Gys1 (Figure S2C) were all rhyth-
mic in both groups with lower mesor levels in OMC men
(p < 0.001), except for Hk2. With regard to substrate oxidation,
mRNA levels of Ppard encoding peroxisome proliferator-acti-
vated receptor delta and Pdk4 encoding pyruvate dehydroge-
nase kinase 4, both known to promote mitochondrial fatty acid
oxidation over glucose oxidation,18,19 demonstrated 24 h rhyth-
micity, with Ppard peaking at 6 p.m. and Pdk4 strongly peaking
at 4 a.m., but no differences were found between groups
(Figures 4D and 4E). We also analyzed mRNA of additional clock
genes that were not analyzed in our initial study (i.e., Per1, Per3,
and Rora; Figure S7). Per1 was unchanged between groups,
Figure 2. Differences in metabolites associated with the tricarboxylic acid (TCA) cycle
Abundance levels of metabolites are shown for YH (blue dots and line) and OMC men (red dots and line). Data are presented as mean ± SEM; n = 12 subjects per
group. *Significantly different between groups (p < 0.05), condition effect in the generalized linear mixed model followed up by Bonferroni post-hoc tests at each
time point; n.d. refers to metabolites that were not detected.
6 Cell Reports 41, 111786, December 13, 2022
Article
while Per3 had a greater amplitude in OMC men (p = 0.017).
Rora was phase shifted between groups (p < 0.001), in line
with Per2 and Cry1 (Figure S1).
Collectively, by comparing rhythmic features within the pro-
portion of the skeletal muscle metabolome that demonstrated
24 h rhythmicity in both groups, we identified remarkable differ-
ences between OMC and YH men with respect to mesor, acrop-
hase, and amplitude. The lower mesor levels in OMC men were in
line with the global pattern of mostly lower mean abundance
levels in most detected metabolites irrespective of 24 h
rhythmicity.
2-Hydroxybutyric acid may contribute to shifting
substrate metabolism in skeletal muscle between
carbohydrate and lipid oxidation around the clock
Sato et al.20 recently identified 2-hydroxybutyric acid to play
an important and time-of-day-dependent signaling role in
systemic and local tissue metabolism. In sedentary mice,
2-hydroxybutyric acid displayed synchronized 24 h rhythmicity
in serum, liver, and skeletal muscle that peaked at the beginning
of the active phase.21 In line with these pre-clinical findings, 2- or
3-hydroxybutyric acid peaked at the beginning of the active
phase after the overnight fast in YH and OMC men (Figure 5F).
Since injecting 2-hydroxybutyric acid into mice has been shown
to induce a sudden and persistent shift from carbohydrate to lipid
oxidation,20 we also correlated abundance levels of 2- or
3-hydroxybutyric acid to the RER measured at 4 a.m. in both
groups, when the RER showed the largest difference between
groups.15 Interestingly, 2- or 3-hydroxybutyric acid levels and
the RER were negatively correlated (Spearman r = 0.637, p =
0.001; Figure S8), and this significant correlation persisted
even when all five time points were considered (Spearman r =
0.274, p = 0.003). A phase shift and reduced 24 h rhythm
amplitude was also found for both (2- or 3-hydroxybutyric acid:
p = 0.078 and p = 0.022; RER: p = 0.043 and p = 0.072).
DISCUSSION
Diurnal regulation of whole-body metabolism in response to
nutrient status and metabolic demand can be reinforced by
circadian clocks located in peripheral tissues. In the metaboli-
cally compromised state, the endogenous clock within skeletal
muscle is also compromised,14,15 accompanied by the blunted
ability of whole-body substrate metabolism to switch from car-
bohydrate oxidation during the fed, active daytime period to
lipid oxidation during the fasted, inactive night-time period.15
Here, we explored how the temporal organization of the skeletal
muscle metabolome differs between the edges of the metabolic
health spectrum. By serial sampling of human skeletal muscle
tissue over a 24 h cycle in two distinct cohorts, we provide a
detailed comparative analysis of the muscle metabolome
between YH and OMC men. We demonstrate that the temporal
organization of the muscle metabolome in OMC men diverges
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Figure 3. Differences in metabolites involved in glycolysis

Abundance levels of metabolites are shown for YH (blue dots and line) and OMC men (red dots and line). Pgk1 mRNA expression, encoding for phosphoglycerate
kinase 1, which converts glyceric acid 1,3-biphosphate to 3-phosphoglyceric acid, is shown to complement the metabolomics data.
Data are presented as mean ± SEM; n = 12 subjects per group. *Significantly different between groups (p < 0.05), condition effect in the generalized linear mixed
model followed up by Bonferroni post-hoc tests at each time point.

from YH men, with the greatest differences present at 4 a.m.
We found that some metabolites associated with glycolysis
and hexosamine biosynthesis metabolism demonstrated
higher levels in OMC men around the clock, whereas metabo-
lites associated with the urea cycle and glutamine-alpha-
ketoglutarate, ketone, and redox metabolism were lower in
OMC men.
Beyond classifying differences between cohorts in a dichoto-
mous manner (i.e., lower versus higher levels), the great advan-
tage of serial muscle sampling around the clock is that temporal
organization patterns evolve. Thereby, we could classify muscle
metabolites as being rhythmic over 24 h and do so separately in
each group. Based on this analysis, we identified several metab-
olites that were only rhythmic in YH men (14 metabolites). These
are of particular interest since their lack of rhythmicity in OMC
men may contribute to the impaired 24 h rhythmicity in substrate
metabolism and/or skeletal muscle clock gene expression, as we
have previously shown15 (Figure S1). Interestingly, 24 h rhyth-
micity of adenosine was markedly different in YH men compared
with the stable pattern in OMC men. Adenosine can modify circa-
dian clockwork through adenosine A1/A2A receptor signaling and
downstream Ca2+ release regulating the clock genes Per1 and
Per2.22 We here demonstrate that adenosine follows a 24 h
rhythm in skeletal muscle from YH donors with highest levels at
the beginning of the inactive period (11 p.m. and 4 a.m.). Since
interstitial adenosine levels within skeletal muscle rise in response
to contractions/exercise,23 potential reductions in physical activity
in OMC men could explain the lack of 24 h rhythmicity in adeno-
sine, although in the current study, YH and OMC men were on
the same fixed activity schedule during the day of muscle sam-
pling. It remains to be investigated why glycerate, kynurenine,
and ophthalmic acid were the only metabolites with the presence
of a 24 h rhythm in OMC men that strongly differed from the stable
pattern in YH men.

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A B C

D E

Figure 4. Transcript levels of key regulators of glucose and lipid metabolism show 24 h rhythms and differ across the metabolic health
spectrum
Additional mRNA expression analysis between YH (blue dots and line) and OMC men (red dots and line): (A) Slc2a4 encoding GLUT4; (B) Irs1 encoding insulin
receptor substrate 1; (C) Hk2 encoding hexokinase 2; (D) Ppard encoding peroxisome proliferator-activated receptor delta; and (E) Pdk4 encoding pyruvate
dehydrogenase kinase 4. Note that all five genes showed 24 h rhythmicity in their expression pattern; no differences in rhythmicity were found except for Slc2a4
and Irs1, which had a lower mesor in OMC men.
Data are presented as mean ± SEM; n = 12 subjects per group. *Significantly different between groups (p < 0.05), p values are derived from the generalized linear
mixed model followed up by Bonferroni post-hoc tests at each time point.

From the 38 metabolites that displayed 24 h rhythmicity in both
groups, only 9 metabolites did not differ in their rhythmic proper-
ties, whereas the vast majority (21 metabolites) displayed lower
mesor levels in OMC men, in line with the global pattern of
lower mean abundance levels of most metabolites independent
of 24 h rhythmicity. Moreover, we found phase shifts for cis-aco-
nitate, FAD, and UDP. In particular, the identification of FAD
being phase shifted is intriguing since the connection between
redox homeostasis, involving FAD, FADH2, NAD+, NADH,
NADP+, and NADPH, and the circadian clock is established.13
Previously, FAD has been shown to display circadian rhythmicity
in mouse liver.24 Furthermore, through an FAD binding pocket,
FAD can stabilize cryptochrome proteins CRY1 and CRY2,
with FAD levels usually increasing right before the accumulation
of CRY proteins. FAD treatment can even lengthen the circadian
period of cell cultures in a dose-dependent manner.24 As CRY
proteins inhibit glucagon-inducible genes and thereby gluconeo-
genesis,25 FAD can indirectly influence glycolytic pathways.
Thus, our finding of a phase shift in FAD, paralleled by the
phase shift in Cry1 gene expression for OMC muscle, suggests
a changed temporal organization of glycolytic pathways
compared with YH muscle, which could contribute to the greater
abundance in metabolites of the first steps of glycolysis
(Figure 3).
In this regard, we found contrasting differences between
groups with respect to the initial versus later steps of the glycol-
ysis pathway. While OMC men demonstrated higher mean abun-
dance levels of most glycolytic metabolites from glucose until
glyceric acid 1,3-biphosphate, lower levels were found for
3-phosphoglyceric acid until phosphoenolpyruvate. In line with
this, we found that transcript levels of Pgk1, which encodes
the enzyme PGK1 converting glyceric acid 1,3-biphosphate to
3-phosphoglyceric acid, were lower in OMC men. Acute sleep
loss has been shown to reduce levels of PGK1 and other glyco-
lytic enzymes and increase muscle protein breakdown.26 The
lower levels of most amino acids and the changes within the
glycolysis pathway in OMC men may therefore mimic the skel-
etal muscle metabolic profile after acute sleep loss, suggesting
that circadian misalignment plays a role in both metabolic
phenotypes. Despite the differences in glycolytic intermediates,
pyruvate levels did not differ between OMC and YH men. How-
ever, it should be noted that pyruvate is not solely dependent
on glycolysis but can also be formed by the degradation of amino
acids such as alanine, serine, cysteine, glycine, threonine, and
tryptophan. Consistently, we found lower levels of cysteine
and tryptophan in OMC men, suggesting that a lower glycolytic
output of pyruvate could be compensated by a greater degree
of amino acid degradation to maintain pyruvate supply to the
TCA cycle.
Next to changes in the glycolysis and TCA pathway, upregula-
tion of the hexosamine synthesis pathway (HSP)27 and defi-
ciency in scavengers of reactive oxygen species (e.g., gluta-
thione)28 have both been previously suggested as contributing
factors to reduced metabolic health. In line with this notion, our
findings in human skeletal muscle highlight that higher hexos-
amine and lower glutathione levels persist throughout the day
in OMC men. Enhanced flux through HSP requires sustained
high glucose concentrations, which is why this pathway has
also been proposed to function as a cellular nutrient sensor.27
Originating out of the conversion of glucose and glutamine,

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A B

C D E

F G H

I J K

(legend on next page)

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the end product of HSP, UDP-GlcNAc, is the donor of
N-acetylglucosamine to the hydroxyl group of serine and threo-
nine residues in specific proteins, initiating the process known as
O-GlcNAcylation. Muscle-specific overexpression of the rate-
limiting enzyme of the HSP, glutamine-fructose-6-phosphate
aminotransferase (GFPT1), results in insulin resistance in
mice.29 On the transcript level, Gfpt1 mRNA levels were lower
in OMC men despite higher levels of UDP-GlcNAc. While such
protein O-GlcNAcylation is thus known to induce changes in
gene expression in a way that facilitates the development of
T2DM,27 it can also directly regulate the transcriptional activity
and stability of BMAL1, CLOCK, and PER2 with consequences
for entrainment and the period length of the circadian clock.30–
32
Concurrently, depleted levels of glutathione throughout the
day render the skeletal muscle susceptible to mitochondrial
damage from oxidative stress.28 Taken together, the upregu-
lated HSP and affected glutathione metabolism, evident in
OMC men around the clock, may contribute to the disturbed
24 h rhythmicity in the muscle molecular clock and mitochondrial
function as observed in OMC men.15 However, our observational
study does not allow us to determine cause and effect. There-
fore, future pre-clinical studies are needed to test these
hypotheses.
The skeletal muscle metabolome of OMC men differed the
most from YH men during the night at 4 a.m., coinciding with
the time of the designated metabolic switch from carbohydrate
to lipid oxidation that was present in YH men but blunted in
OMC men.15 At this time, the largely disrupted muscle metabo-
lome might fail to create the necessary conditions to facilitate
the metabolic switch. Complementing mechanistic animal
work,20 we provide some evidence for a link between 2- or
3-hydroxybutyric acid and human substrate metabolism. The
reduced levels of hydroxybutyric acid in OMC men upon over-
night fasting could be the result of attenuated ketogenesis in
the liver and hence a reduced systemic supply of ketone bodies,
further illustrating that OMC men do not enter a fully fasted state.
In support of this, the increase of plasma free fatty acid levels in
response to the overnight fast was also markedly attenuated in
OMC men compared with YH men (Figure S1E).15 Taken
together, the metabolic impact of the overnight fast on whole-
body substrate oxidation and on the skeletal muscle metabolome
is blunted in metabolically compromised individuals. Therefore,
the fasted night/sleep period is a time window of opportunity
for targeted intervention. In this context, early time-restricted
eating (TRE) and afternoon to evening exercise could be prom-
ising interventional tools to influence nocturnal skeletal muscle
metabolism. Both early TRE33,34 and afternoon to evening
exercise35–37 have been shown to improve metabolic outcomes
in metabolically compromised volunteers and may have a stron-
ger impact on nocturnal metabolism by inducing a more pro-
nounced fasting state during the night. More studies are needed
to investigate if such interventions exert their beneficial metabolic
effects by influencing the nocturnal skeletal muscle metabolome.
In an attempt to determine the confounding effect of aging, we
compared our data with recently published human skeletal mus-
cle metabolomics data from our group,38 for which we used the
same metabolomics approach and platform ensuring compara-
bility to our current data in terms of identification of metabolites.
In that study, we compared the skeletal muscle metabolome in
the overnight fasted state (i.e., one single time point) between
YH men (23 ± 2 years, BMI: 22.7 ± 3.4 kg/m2) and healthy, older
adults (71 ± 4 years, BMI: 25.8 ± 3.3 kg/m2). By comparing the
log10 p value of the difference in metabolites between those
healthy, older and healthy, young men with the difference in me-
tabolites between our OMC and YH men at the 8 a.m. time point,
we were able to identify muscle metabolites that change due to
aging, while others seem to change rather due to the metabolic
impairment (Figure S9). Overall, linear regression analysis on
these data was indicative of a negative correlation between the
two comparisons (Pearson r = 0.33, p < 0.001; Figure S9A),
suggesting rather opposite changes within the fasted muscle
metabolome between metabolically healthy versus metaboli-
cally compromised aging. On the one hand, higher levels in
glycerate, taurine, dihydoxyacetone-phosphate, and ophthalmic
acid and lower levels in hydroxyphenyllactic acid, NADH,
NADP+, 2- or 3-hydroxybutyric acid, UDP-hexose, creatinine,
tryptophan, beta-alanine, and aspartate were consistent in
both older groups, suggesting modulation with aging (Fig-
ure S9B). However, most metabolites demonstrated opposite
directional changes between the two comparisons. In particular,
metabolites associated with glycolysis, like, e.g., glucose, ADP-
ribose, and UDP-GlcNAc, tended to be lower in healthy, older
adults while higher in OMC men (Figure S9B). In addition,
oxidized glutathione, ornithine, alpha-ketoglutarate, and lysine
(Figure S9B) were higher in healthy, older adults while being
lower in OMC men. Therefore, the direction of change in these
metabolites is likely to be linked to the metabolically compro-
mised state.
An important finding of our study, with practical implications, is
that a single time point (i.e., overnight fasted), as is routinely used
in metabolic cross-sectional studies,39 would have revealed only
23 metabolites as different between groups (i.e., at 8 a.m.),
whereas serial muscle sampling around the clock revealed 33
additional metabolites to be different, underscoring the gain in
information through around-the-clock sampling. In particular,
none of the glycolytic metabolites would have been identified

Figure 5. 24 h rhythms differ across the metabolic health spectrum

The comparison of 24 h rhythms in skeletal muscle metabolites between YH (blue dots and line) and OMC men (red dots and line). Out of 115 detected me-
tabolites, 70 metabolites displayed 24 h rhythmicity in at least one of the two groups, whereas 52 were arrhythmic in both groups (A). Out of these rhythmic
metabolites, 38 metabolites were rhythmic in both groups, 15 were only rhythmic in YH men, and 17 were only rhythmic in OMC men (B). For the metabolites that
are underscored in (B), an interaction effect was found between groups: glycerate (C); kynurenine (D); ophthalmic acid (E); adenosine (F); 2- or 3-hydroxybutyric
acid (G); hippuric acid (H); cis-aconitate (I); FAD (J); and UDP (K). The solid line represents the fitted cosinor-based model and is only displayed for metabolites and
groups where the data proved to be rhythmic. p values in (G)–(K) are derived from the nonlinear mixed-effects regression model CircaCompare;17 see STAR
Methods for more information. To better visualize 24 h rhythmicity, the x axis indicating clock time has been extended beyond the 5 time points of muscle
sampling (8–4 a.m.) to allow double plotting. Data are presented as mean ± SEM; n = 12 subjects per group.

10 Cell Reports 41, 111786, December 13, 2022

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to be different between groups if only the overnight fasted time
point had been taken into account. Moreover, for skeletal muscle
metabolites with 24 h rhythmicity, past and future reports about
decreased or increased levels in metabolomics studies with sin-
gle time point analyses should be interpreted with caution since
these changes could also be explained by phase shifts in the
respective metabolites. Our analysis of the skeletal muscle me-
tabolome around the clock in two distinct cohorts with respect
to metabolic health status can be used as an informative
resource for future studies to estimate if 24 h rhythmicity has
to be taken into account for sampling and analyzing the metab-
olite of interest in humans.
Limitations of the study
The following limitations of the current study should be taken into
account for the interpretation of findings and for designing
follow-up trials: first, differences between the groups at both
tions in metabolites associated with the initial steps of glycolysis
as well as with HSP that demonstrated higher levels in OMC
men. Our findings underscore the relevance of taking timing for
targeted intervention into account. Changes within the temporal
organization of the skeletal muscle metabolome could contribute
to the disturbed molecular clock in metabolically compromised
individuals and their blunted ability for metabolic switching be-
tween carbohydrate and lipid oxidation over the 24 h cycle.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact

sides of the metabolic health spectrum can be attributed to dif- B Materials availability

ferences in metabolic state but also to differences in age, which B Data and code availability

by itself is also known to affect metabolic health. Since no 24 h d EXPERIMENTAL MODEL AND SUBJECT DETAILS
data are available in age-matched controls, confounding due B Participants

to aging cannot be ruled out. Secondly, our metabolomics B Study protocol overview

analysis was performed on skeletal muscle biopsies, which is a d METHOD DETAILS

naturally diverse sample type, in particular in terms of muscle B Metabolomics analysis

fiber type and intracellular heterogeneity (e.g., cytosolic versus B Gene transcript quantification

mitochondrial subcellular location cannot be distinguished), d QUANTIFICATION AND STATISTICAL ANALYSIS

both between and within individuals over time. In particular, it re-
mains to be investigated if differences in metabolite abundance
levels or rhythmicity properties were due to changes in subcellu-
lar metabolite pools. Thirdly, our study was restricted to male
participants; how temporal dynamics within the skeletal muscle
metabolome are influenced by sex, and how the metabolome
changes across the metabolic health spectrum in females, re-
quires future investigation. In general, it should also be high-
lighted that our human trial was designed to mimic real-life con-
ditions with three typical meals and some physical activity
throughout daytime. As a consequence, we suspect that the
high proportion of rhythmic metabolites evident irrespective of
the metabolic health status is likely linked to the superordinate
impact of the overnight fast and/or continuous lack of physical
activity during sleep. To better isolate the role of the endogenous
circadian clock on 24 h rhythms within the muscle metabolome,
future trials should make use of ‘‘constant routine’’ protocols that
keep confounding factors such as light, food intake, physical ac-
tivity, and wakefulness constant over 24 h, as previously done by
Perrin et al.8 when studying the 24 h muscle transcriptome with
participants in a continuous semi-recumbent position. Lastly,
our findings are observational; hence, cause and effect relation-
ships cannot be determined.
Conclusions
In conclusion, through serial sampling of skeletal muscle tissue
around the clock, we found large divergence in the skeletal mus-
cle metabolome between YH and OMC men that would have
been mostly missed by a single time point analysis. Indeed,
the muscle metabolome was most different at night. With the
global pattern of lower mean abundance levels in most metabo-
lites throughout the 24 h cycle in OMC men, we identified excep-
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2022.111786.
ACKNOWLEDGMENTS
This work is partly financed by the Netherlands Organisation for Scientific
Research (TOP 40-00812-98-14047 to P.S.). We acknowledge support from
the Netherlands Cardiovascular Research Initiative: an initiative with the sup-
port of the Dutch Heart Foundation (CVON2014-02 ENERGISE). Work in the
Houtkooper group is financially supported by a grant from the Velux Stiftung
(no. 1063).
AUTHOR CONTRIBUTIONS
J.-F.H., J.W., D.v.M., J. Hansen, J. Hoeks, R.H.H., and P.S. designed the
research; J.W., D.v.M., and J. Hansen collected data; J.-F.H., M.v.W., and
R.P. analyzed data; M.v.W. and R.H.H. provided the analytic tools; R.P. devel-
oped software necessary to analysis data; and all authors wrote the
manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: March 25, 2022
Revised: July 28, 2022
Accepted: November 15, 2022
Published: December 13, 2022
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Biological samples
Human muscle samples Van Moorsel et al.16 and Wefers et al.15 N/A
Critical commercial assays
RNeasy tissue kit Qiagen 74104
High capacity RNA -to- cDNA kit ThermoFischer 4387406
HOT FIREPol Probe qPCR mix Bioconnect 08-14-00001
plus (ROX)
Sensimix sybr Hi-ROX Bioline QT 605-05
Deposited data
Raw and analyzed metabolomics https://www.ebi.ac.uk/metabolights/ MTBLS6049
data in MetaboLights MTBLS6049/descriptors
Oligonucleotides
Primers for gene expression This paper N/A
analysis, see Table S2
Software and algorithms
R (v4.1.2) CRAN https://cran.r-project.org/
circacompare (0.1.1) CRAN https://cran.r-project.org/web/
packages/circacompare/index.html
ComplexHeatmap (2.13.1) CRAN https://cran.r-project.org/
ggplot2 (3.3.6) CRAN https://cran.r-project.org/
Biorad CFX manager 3.1 for Biorad https://www.bio-rad.com/
gene expression analysis

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Patrick Schrauwen (p.
schrauwen@maastrichtuniversity.nl).

Materials availability
This study did not generate new unique reagents.

Data and code availability

d The raw metabolomics data is available via MetaboLights under the identifier MTBLS6049.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Participants
Skeletal muscle tissue was obtained around the clock in two previously published studies.15,16 The design of these studies was
essentially identical, apart from the study population, i.e. young healthy men16 vs. older metabolically-compromised men.15 To
establish volunteers with compromised metabolic health, we selected participants with reduced glucose tolerance and insulin sensi-
tivity based on an oral glucose tolerance test and a glucose clearance rate <360 mL/kg/min established insulin resistance.40 The
participant characteristics of the two study populations are listed in Table 1. Both studies were approved by the Ethics Committee
of the Maastricht University Medical Center. All participants provided written informed consent. Both studies were registered at
https://clinicaltrials.gov with identifiers NCT02261168 and NCT03733743, respectively.

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Study protocol overview

Detailed descriptions of the study procedures can be found elsewhere (see15,16). In short, after seven days of adherence to a
standardized lifestyle with respect to eating and sleeping times at home, YH and OMC men were admitted to our research unit
for 44 h in total, under standardized conditions mimicking real-life conditions with scheduled physical activity, meals and normal
room lighting conditions. After spending the first day (last meal at 6PM) and night in a respiration chamber, the main test day began
with participants waking up at 7AM. At
8AM,
1PM,
6PM,
11PM and
4AM, indirect calorimetry was performed in a recumbent
position and directly after each measurement, a muscle biopsy was taken from the vastus lateralis muscle. Three standardized meals
were provided immediately after the first three muscle biopsies, i.e. at
9AM,
2PM and
7PM. Energy requirements were calcu-
lated by multiplying the sleeping metabolic rate (assessed during the first night spent in a respiration chamber) with an activity factor
of 1.5. Breakfast accounted for 21 energy% (E%), lunch for 30 E%, and dinner for 49 E%. Daily macronutrient composition was 55 E
% as carbohydrates (3 E% fibers), 31 E% as fat (9 E% saturated), and 14 E% as protein. 2 days preceding the admission to the
research unit, meals were provided in a standardized manner at similar clock times for consumption at home.

METHOD DETAILS

Metabolomics analysis
Corresponding to Janssens et al. (2022), the following internal standards diluted in water were added to approximately 5 mg
of freeze-dried muscle tissue: adenosine-15N5-monophosphate (5 nmol), adenosine-15N5-triphosphate (5 nmol), D4-alanine
(0.5 nmol), D7-arginine (0.5 nmol), D3-aspartic acid (0.5 nmol), D3-carnitine (0.5 nmol), D4-citric acid (0.5 nmol), 13C1-citrulline
(0.5 nmol), 13C6-fructose-1,6-diphosphate (1 nmol), guanosine-15N5-monophosphate (5 nmol), guanosine-15N5-triphosphate
(5 nmol), 13C6-glucose (10 nmol), 13C6-glucose-6-phosphate (1 nmol), D3-glutamic acid (0.5 nmol), D5-glutamine (0.5 nmol), D5-gluta-
thione (1 nmol), 13C6-isoleucine (0.5 nmol), D3-lactic acid (1 nmol), D3-leucine (0.5 nmol), D4-lysine (0.5 nmol), D3-methionine
(0.5 nmol), D6-ornithine (0.5 nmol), D5-phenylalanine (0.5 nmol), D7-proline (0.5 nmol), 13C3-pyruvate (0.5 nmol), D3-serine
(0.5 nmol), D6-succinic acid (0.5 nmol), D5-tryptophan (0.5 nmol), D4-tyrosine (0.5 nmol), D8-valine (0.5 nmol). Subsequently, 1:1
(v/v) methanol:water (to a total volume of 1000 mL) and a 5 mm stainless-steel bead were added to the samples and homogenized
for 5 min at a frequency of 30 times/sec using a TissueLyser II (Qiagen, Hilden, Germany). To each sample 1000 mL of chloroform was
added, thoroughly mixed and centrifuged for 10 min at 18.000g. The top ‘‘polar’’ layer was transferred to a new 1.5 mL tube and dried
using a vacuum concentrator at 60 C. The residues were reconstituted in 100 mL 3:2 (v/v) methanol:water. Metabolites were analyzed
using a Waters Acquity ultra-high performance liquid chromatography system coupled to a Bruker Impact II Ultra-High Resolution
Qq-Time-Of-Flight mass spectrometer. Samples were kept at 12 C during analysis and 5 mL of each sample was injected. Chromato-
graphic separation was achieved using a Merck Millipore SeQuant ZIC-cHILIC column (PEEK 100 3 2.1 mm, 3 mm particle size). Col-
umn temperature was held at 30 C. Mobile phase consisted of (A) 1:9 (v/v) acetonitrile:water and (B) 9:1 (v/v) acetonitrile:water, both
containing 5 mmol/L ammonium acetate. Using a flow rate of 0.25 mL/min, the LC gradient consisted of: 100% B for 0-2 min, reach
0% B at 28 min, 0% B for 28-30 min, reach 100% B at 31 min, 100% B for 31-32 min. Column re-equilibration is achieved at a flow rate
of 0.4 mL/min at 100% B for 32-35 min. MS data were acquired using full scan negative and positive ionization mode over the range of
m/z 50-1200. Data were analyzed using Bruker TASQ software version 2.1.22.3. All reported metabolite intensities were normalized
to their corresponding internal standards based on comparable retention time and responses in the MS as well as dry tissue weight.
Metabolite identification has been based on a combination of accurate mass, (relative) retention times and fragmentation spectra,
compared to the analysis of a library of standards (Sigma-Aldrich MSMLS). General repeatability of metabolite analysis was assessed
for each metabolite using repeated measurements of a pooled sample. Additionally, all peak integrations were manually checked for
quality in each sample, as large natural variance may skew pooled sample results.

Gene transcript quantification

RNA was previously isolated from 10 mg of muscle material by TRIzol lysis (Qiagen, Hilden, Germany). RNA was further purified by the
RNeasy kit from Qiagen (Hilden, Germany). RNA yield was measured using a NanoDrop spectrophotometer (Thermo Fisher Scien-
tific, Waltham, USA). The high-capacity RNA-to-cDNA kit from Applied Biosystems (Foster City, USA) was used for transcribing
100 ng of RNA to cDNA. Transcript abundance was determined using a CFX384 Real-Time System (Biorad-Laboratories, Veenen-
daal, NL). To minimize the variability in reference gene normalization, the geometric mean of two reference genes (RPL26 and RPL0),
which were both stably expressed over time, was used. This geometric mean was used as the internal reference for comparative
gene expression analysis over time and between groups.41 Primers used in this study are listed in Table S2.

QUANTIFICATION AND STATISTICAL ANALYSIS

In total, 115 metabolites were subjected to statistical analyses over time between groups. If certain metabolites were below the
detection limit at single time points for individual subjects, these were treated as missing values. To assess differences in rhythmic
parameters – mesor, amplitude and acrophase – between groups, we applied nonlinear mixed-effects regression using the
R-package for CircaCompare17 with a random-effect for the mesor (k) and a pre-defined period of 24-h. Before the model is fit to

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compare the two groups, each group is independently assessed for the presence of rhythmicity, defined as non-zero parameter es-
timate (p < 0.05) for amplitude (⍺) when fitting the following model:

Yb = k + a cosðt  4Þ
In this equation, Y represents the response or outcome variable, k represents the mesor, a rhythm-adjusted mean, a represents
amplitude, t refers to the time in hours, and 4 is the phase. If rhythmicity was observed in both groups for a given metabolite, differ-
ences in mesor, amplitude and acrophase for that metabolite between OMC and YH were modeled.
To determine whether there was an effect of time on metabolite expression and whether this was different between groups, we
conducted a generalized linear mixed model with time (categorical), condition (YH vs. OMC) and their interaction (time x condition)
as fixed effects with random intercepts for subjects. In this model, the interaction effect represents different expression of the respec-
tive metabolite over time between groups (e.g. an increasing vs. a decreasing abundance pattern over time between groups or
consistently different peak time points between groups). This model takes missing values into account, which occurred due to
missing samples or poor muscle tissue quality at a few time points (out of 60 muscle samples per group 5 time points for 12 sub-
jects– 5 samples were missing for OMC and 2 samples for YH). In the absence of interaction, we interpreted the main effects for con-
dition and time, and in the case of statistical significance, we followed up the respective main effects with pairwise comparisons per
time point using Bonferroni post-hoc tests. To better visualize 24h-rhythmicity in figures, the x axis indicating clock time has been
extended beyond the 5 time points of muscle sampling (8AM–4AM) to allow double-plotting. For metabolites that did not demon-
strate 24h-rhythmicity in both groups, the generalized linear mixed model as described above was conducted to compare changes
in metabolites over time between the two groups. mRNA levels were analyzed in the same manner as metabolites. The Spearman
rank correlation was used to establish the relationship between specific metabolites and the RER.
Data are presented as mean ± SEM (standard error of the mean). The level of significance was set to <0.05 for all analyses. Sta-
tistical analyses were performed using the R programming language (version 4.0.4; http://www.r-project.org) and further processed
with the GraphPad Prism 8 software package (GraphPad Software, San Diego, CA, USA). The heatmap (Figure 1) was generated
using the ComplexHeatmap R-package.42

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