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Underlying mechanisms & causal links: high fat feeding & circadian rhythms
Fondation Merieux Conference, April 6-8, 2016
Better Foods for Better Health Microbiota & Health: The Challenges of a Promising Approach
Eugene B. Chang, M.D.Knapp Center for Biomedical Discovery
University of Chicago
Type 2 Diabetes
Obesity (BMI ≥ 30kg/m2)
The obesity and diabetes epidemic
1994 2000 2010
No Data <4.5% 4.5%–5.9% 6.0%–7.4% 7.5%–8.9% >9.0%
1994 2000 20101985
No Data <10% 10-13.9% <14% 14–17.9% 18–21.9% 22.0%–25.9% 26.0%
High fat diets affect the murine enteric microbiome differently
16S rRNA gene sequences determined by Sanger-based clone library sequencing
Devkota et al. Nature (2012)
Suzanne Devkota
Western-diet induced changes in humanized mouse gut microbiota promote adipose tissue
Turnbaugh, et al. Sci. Transl. Med. 2010
Microbiota from Human subjects on different diets transferred to GF mice
LF/PP – Low fat, plant polysaccharide-richWestern – High fat, high simple sugar
FMT from lean donors reduces insulin resistance in obese subjects
Lean male
donorsObese male recipients
BMI ≥ 30
6 weeks
*Improved
symptoms of
Type II Diabetes
and
Metabolic
syndrome
Vrieze et al., Gastro.
(2012) 143:913-916
Hepatic genes upregulated in GF versus conventionalized mice
0 3 6 9
Androgen and Estrogen Metabolism
PXR/RXR Activation
Methionine Metabolism
LXR/RXR Activation
Fatty Acid Metabolism
Linoleic Acid Metabolism
Circadian Rhythm Signaling
Xenobiotic Metabolism Signaling
Metabolism of Xenobiotics by Cytochrome P450
Biosynthesis of Steroids
LPS/IL-1 Mediated Inhibition of RXR Function
p<0.05
Vanessa Leone
Leone, et al. Cell Host & Microbe (2015)
Circadian clock networks regulate daily metabolic functions
Bass, J. Nature 2012
Germ free (GF) mice are resistant to obesogenic diets
Body weight Caloric consumption
n = 17 or 18 age-matched individually housed male mice/trt group
Leone, et al. Cell Host & Microbe (2015)
Germ-free mice have altered circadian
mediobasal hypothalamic and liver circadian
clock gene expression profiles
(Leone, et al., 2015)
0 4 8 12 16 20 240.00
0.01
0.02
0.03
0.04
SPF - LFSPF - HF
Zeitgeber Time (ZT)
Bm
al1
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 24
0.000
0.004
0.008
0.012
0.016
0.020
0.024
0.028
SPF - LF
SPF - HF
Zeitgeber Time (ZT)
Bm
al1
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 240.00
0.01
0.02
0.03
0.04
GF - LFGF - HF
Zeitgeber Time (ZT)
Bm
al1
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 24
0.000
0.004
0.008
0.012
0.016
0.020
0.024
0.028
GF - LFGF - HF
Zeitgeber Time (ZT)
Bm
al1
exp
ressio
n (
rela
tive G
AP
DH
)Mediobasal hypothalamus
Liver
Mediobasal hypothalamus
Liver
0 4 8 12 16 20 24
0.02
0.04
0.06
0.08
SPF - LF
SPF - HF
Zeitgeber Time (ZT)
Per2
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 24
0.00
0.08
0.16
0.24
0.32SPF - LFSPF - HF
Zeitgeber Time (ZT)
Per2
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 24
0.00
0.02
0.04
0.06
0.08
GF - LFGF - HF
Zeitgeber Time (ZT)
Per2
exp
ressio
n (
rela
tive G
AP
DH
)
0 4 8 12 16 20 24
0.00
0.08
0.16
0.24
0.32
GF - LFGF - HF
Zeitgeber Time (ZT)
Per2
exp
ressio
n (
rela
tive G
AP
DH
)
Bmal1 (Inducer) Per2 (repressor)
High fat dietary intake shifts cecal microbial
diversity and taxonomic structure
Highfat Lowfat
PCoA – PC1 vs. PC2
Highfat Lowfat3.5
4.0
4.5
5.0
5.5
6.0
6.5***
Sh
an
no
n D
iversit
y In
dex
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Highfat Lowfat
Verrucomicrobia
Spirochaetes
Cyanobacteria
Proteobacteria
Actinobacteria
Tenericutes
Bacteroidetes
Firmicutes
Bacteria;Other
V4-V5 16s rRNA amplicon sequencing
performed using Illumina Miseq
PC1 – 56.20%
PC
2 –
13
.95
%
Leone, et al. Cell Host & Microbe (2015)
Shifts in microbial diversity exhibit diurnal
oscillations that are influenced by diet
Leone, et al. Cell Host & Microbe (2015)
– 56.20%
-1
3.9
5%
Enteral feeding is not the only driver of
diurnal variations in gut microbes
Leone, et al. Cell Host & Microbe (2015)
OTU #306066 - Lachnospiraceae
0 4 8 12 16 20 240
20
40
60
80
100
120
140
*
SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nce
OTU #265793 - Oscillospira
0 4 8 12 16 20 240
1
2
100
150
200
250SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve a
bu
nd
an
ce
OTU #2374993 - Lachnospiraceae
0 4 8 12 16 20 240
25
50
75
100
125
SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nce
OTU #263272 - Ruminococcus
0 4 8 12 16 20 240
25
50
75
100
125 SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive
ab
un
da
nc
e
OTU #267411 - Lachnospiraceae
0 4 8 12 16 20 240
10
20
30
100
150
200
250 SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive
ab
un
dan
ce
OTU #167034 - Oscillospira
0 4 8 12 16 20 240
25
50
75
100
125
150SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive a
bu
nd
an
ce
OTU #261365 - Ruminococcus
0 4 8 12 16 20 240
1
2
3
4
255075
100125150
SPF - HFSPF - RC
Zeitgeber Time (ZT)
Re
lati
ve a
bu
nd
an
ce
OTU #180535 - Oscillospira
0 4 8 12 16 20 240
25
50
75
100
125
150
175SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive a
bu
nd
an
ce
RC oscillating OTUs
HF oscillating OTUs
OTU #306066 - Lachnospiraceae
0 4 8 12 16 20 240
25
50
75
100
125
150
175SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nc
e
OTU #176691 - Clostridiales
0 4 8 12 16 20 240
100
200
300
400
500
600
700SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nc
e
Oscillatory cecal OTUs found in mice fed regular, low fat chow (RC) diets
Leone, et al. Cell Host & Microbe (2015)
OTU #306066 - Lachnospiraceae
0 4 8 12 16 20 240
20
40
60
80
100
120
140
*
SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nce
OTU #265793 - Oscillospira
0 4 8 12 16 20 240
1
2
100
150
200
250SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve a
bu
nd
an
ce
OTU #2374993 - Lachnospiraceae
0 4 8 12 16 20 240
25
50
75
100
125
SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nce
OTU #263272 - Ruminococcus
0 4 8 12 16 20 240
25
50
75
100
125 SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive
ab
un
da
nc
e
OTU #267411 - Lachnospiraceae
0 4 8 12 16 20 240
10
20
30
100
150
200
250 SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive
ab
un
dan
ce
OTU #167034 - Oscillospira
0 4 8 12 16 20 240
25
50
75
100
125
150SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive a
bu
nd
an
ce
OTU #261365 - Ruminococcus
0 4 8 12 16 20 240
1
2
3
4
255075
100125150
SPF - HFSPF - RC
Zeitgeber Time (ZT)
Re
lati
ve a
bu
nd
an
ce
OTU #180535 - Oscillospira
0 4 8 12 16 20 240
25
50
75
100
125
150
175SPF - RCSPF - HF
Zeitgeber Time (ZT)
Rela
tive a
bu
nd
an
ce
RC oscillating OTUs
HF oscillating OTUs
OTU #306066 - Lachnospiraceae
0 4 8 12 16 20 240
25
50
75
100
125
150
175SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nc
e
OTU #176691 - Clostridiales
0 4 8 12 16 20 240
100
200
300
400
500
600
700SPF - RCSPF - HF
Zeitgeber Time (ZT)
Re
lati
ve
ab
un
da
nc
e
High fat diet-induced gut microbes exhibit altered diurnal oscillations of known microbial metabolites
0 4 8 12 16 20 240
25
50
75
100
125
150
175
200
225
250
275SPF - RCSPF - HF
***
** *
*
Zeitgeber Time (ZT)
Bu
tyra
te (
mm
ol/g
of
co
nte
nts
)
0 4 8 12 16 20 240.0
2.0
4.0
6.0
SPF - RCSPF - HF
** *
Zeitgeber Time (ZT)
H2S
(m
mo
l/g
of
co
nte
nts
)
Leone, et al. Cell Host & Microbe (2015)
Effects of SCFAs and H2S
(NaHS) on hepatocyte
circadian clock functionHepatic organoid
Butyrate
NaHS
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
5mM Butyrate
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
1mM NaHS
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
5mM Butyrate
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
1mM NaHS
Pe
r2 e
xp
ress
ion
(r
ela
tive
GA
PD
H)
Pe
r2 e
xp
ress
ion
(r
ela
tive
GA
PD
H)
Bm
al1
ex
pre
ss
ion
(r
ela
tive G
AP
DH
)
Time after serum-shock (hours)
*** *
**
* *
Time after serum-shock (hours)
Bm
al1
exp
ressio
n(r
ela
tive G
AP
DH
)
Leone, et al. Cell Host & Microbe (2015)
Figure 4
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
5mM Butyrate
0 8 16 24 32 40 48
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
1mM NaHS
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
5mM Butyrate
0 8 16 24 32 40 48
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
1mM NaHS
Pe
r2 e
xp
ressio
n
(re
lati
ve G
AP
DH
)
Pe
r2 e
xp
res
sio
n
(rela
tive
GA
PD
H)
Per2
ex
pre
ss
ion
(r
ela
tive
GA
PD
H)
Bm
al1
exp
res
sio
n
(re
lati
ve
GA
PD
H)
Bm
al1
exp
res
sio
n
(re
lati
ve G
AP
DH
)
Bm
al1
exp
ressio
n
(re
lati
ve G
AP
DH
)
Time after serum-shock (hours)
*** *
*
**
* *
*
Time after serum-shock (hours)
Diet
Absent microbial Zeitgeber Time
Diet
Effects of diet on host CC function and metabolism in absence of gut microbiota
Normal CC function
Lean Normal CC rhythm
Normal
microbe-
derived
metabolic
signals
MBH Regulation by central
clock
CC dysfunction
Obese Disturbed CC rhythm
Altered microbe-
derived metabolic
signals
MBH
MBH
Lean Disturbed CC rhythm
CC dysfunction
MBH
CC dysfunction
Differential regulation by central clock
Lean
Disturbed CC rhythm
Diet
Normal CCfunction
LeanNormal N lCC rhythm
Normal
microbe-m
derived
metabolic m
signals
MBH Regulationn by centrall
clock
CC
Obese Disturbed CC rhythm
Altered microbe-
derived metabolic
signals
MBH
Zeitgeber Time Ab
Diet
Effects of diet on host CC function and metabolism in absence of gut microbiota
MBH
Lean Disturbed CC rhythm CC h th
CC dysfunction
MBH
CC dysfunction
Differential regulation bycentral clock
LeanDisturbed CC rhythm CC rhythm
Eff t f di t h t CC f ti d t b li i b f t i bi t
Absent microbe-
derived metabolic
signals
Absent microbe-
derived metabolic
signals
Effects of diet on host CC function and metabolism with gut microbiota present
Differential regulation by central clock
A B
high fat
diet
high fat
diet
low fat
diet
low fat
diet
Working model
Saline But - ZT2 But - ZT142.5
3.0
3.5
4.0
4.5
5.0 NS,p = 0.053
NS,p = 0.135
Treatment group
per2
:bm
al1
mR
NA
rati
o
Saline But - ZT2 But - ZT140
2
4
6
8
10
12
14
16
18
20
22 *
NS,
p = 0.018
p = 0.281
Treatment group
pe
r2:b
mal1
mR
NA
ra
tio
Mediobasal hypothalamus
Liver
C D
Figure 4
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
5mM Butyrate
0 8 16 24 32 40 48
0
0.002
0.004
0.006
0.008
0.01
0 8 16 24 32 40 48
No trt
1mM NaHS
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
5mM Butyrate
0 8 16 24 32 40 48
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 8 16 24 32 40 48
No trt
1mM NaHS
Pe
r2 e
xp
ressio
n
(re
lati
ve G
AP
DH
)
Pe
r2 e
xp
res
sio
n
(rela
tive
GA
PD
H)
Per2
ex
pre
ss
ion
(r
ela
tive
GA
PD
H)
Bm
al1
exp
res
sio
n
(re
lati
ve
GA
PD
H)
Bm
al1
exp
res
sio
n
(re
lati
ve G
AP
DH
)
Bm
al1
exp
ressio
n
(re
lati
ve G
AP
DH
)
Time after serum-shock (hours)
*** *
*
**
* *
*
Time after serum-shock (hours)
Diet
Absent microbial Zeitgeber Time
Diet
Effects of diet on host CC function and metabolism in absence of gut microbiota
Normal CC function
Lean Normal CC rhythm
Normal
microbe-
derived
metabolic
signals
MBH Regulation by central
clock
CC dysfunction
Obese Disturbed CC rhythm
Altered microbe-
derived metabolic
signals
MBH
MBH
Lean Disturbed CC rhythm
CC dysfunction
MBH
CC dysfunction
Differential regulation by central clock
Lean
Disturbed CC rhythm
Diet
Normal CCfunction
LeanNormal N lCC rhythm
Normal
microbe-m
derived
metabolic m
signals
MBH Regulationn by centrall
clock
CC
Obese Disturbed CC rhythm
Altered microbe-
derived metabolic
signals
MBH
Zeitgeber Time Ab
Diet
Effects of diet on host CC function and metabolism in absence of gut microbiota
MBH
Lean Disturbed CC rhythm CC h th
CC dysfunction
MBH
CC dysfunction
Differential regulation bycentral clock
LeanDisturbed CC rhythm CC rhythm
Eff t f di t h t CC f ti d t b li i b f t i bi t
Absent microbe-
derived metabolic
signals
Absent microbe-
derived metabolic
signals
Effects of diet on host CC function and metabolism with gut microbiota present
Differential regulation by central clock
A B
high fat
diet
high fat
diet
low fat
diet
low fat
diet
Working model
Saline But - ZT2 But - ZT142.5
3.0
3.5
4.0
4.5
5.0 NS,p = 0.053
NS,p = 0.135
Treatment group
per2
:bm
al1
mR
NA
rati
o
Saline But - ZT2 But - ZT140
2
4
6
8
10
12
14
16
18
20
22 *
NS,
p = 0.018
p = 0.281
Treatment group
pe
r2:b
mal1
mR
NA
ra
tio
Mediobasal hypothalamus
Liver
C D
Effects of intraperitoneal administration of butyrate on CC networks of GF mice
control Sal Sal
But ZT2 But Sal
But ZT14 Sal But
ZT 0 ZT2
6 12 ZT14
18 24
Gut microbiota sense dietary cues and translating into an output that needed for Circadian networks
Microbial
Oscillations
Gut microbiota
Microbial
signals
Metabolome
Circadian NetworksDietary intake• When• How much• What
Aberrant Microbial
Oscillations
Conventionally-
raised
High fat diet
Obese
Aberrant
microbial
signals
Circadian disruption
Normal Microbial
Oscillations
Conventionally-
raised
Lean
Normal
microbial
signals
Normal
circadian function
Low-fat diet
Absent Microbial
Oscillations
Germ-free
High fat diet
Lean
Absent
microbial
signals
Circadian disruption
Low-fat diet
or
Impact of gut microbes on host metabolism
Body
Weight
Age
The challenge to treating obesity –
changing energy balance
2015
50-100 yr ago
Challenge
AcknowledgementsUniversity of ChicagoVanessa Leone
Mark Musch
A. Murat Eren
Brian Prendergast
Betty Theriault
Joseph Pierre
Candace Cham
Aaron Dinner
Kristina Martinez
Edmond Huang
Yong Huang
Sean Gibbons
Alan Hutchison
Nathaniel Hubert, M.S.
Anuradha Nadimpalli
Elizabeth Zale
UW-MadisonKenneth Kudsk
Aaron Heneghan
Argonne National Lab Jack Gilbert
Rick Stevens
Chris Henry
Dionysios Antonopoulos
Marine Biological LabMitch Sogin
Joe Vineis
Hilary Morisson
Biofortis - Merieux
NutriScienceChad M. Cook
DeAnn Liska
Pam Coleman
Funding
NIDDK P30 DDRCC
Sunrise from the Knapp
Center for Biomedical
Discovery after a long
circadian study
Funding: NIDDK, Gastrointestinal Research Foundation of Chicago
