structural basis for lipopolysaccharide recognition by peptidoglycan ...
The Impact of Lipopolysaccharide on Mitochondrial …...Poster presented 18 at: The Impact of...
1
Poster presented at: BES2018 The Impact of Lipopolysaccharide on Mitochondrial Efficiency in Brown Adipocytes Farah Omran 1 , Philip G McTernan 2 , Mark Christian 1 . 1Warwick Medical School, University of Warwick, Coventry, West Midland, UK. 2School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS, UK. The presence of brown adipose tissue (BAT) in adult humans opens new avenues for research to ameliorate obesity consequences including metabolic and cardiovascular disease because of its special feature to expend rather than store energy. Lipopolysaccharide (LPS) is known to be elevated in obesity and initiate an inflammatory state, but its effect has not been fully explored in BAT. β3 adrenergic receptor ligands such as CL 316,243 (CL) induce BAT activity through stimulation of UCP1. The interaction between the LPS receptor TLR4 and β 3 adrenergic receptors has not been well studied in BAT. Therefore, the objective of this study is to investigate the effect of LPS on the CL response and examine how LPS may alter mitochondrial function in BAT. BACKGROUND METHODS 1. Immortalized brown adipocytes were differentiated with or without LPS (100ng/ml or 1000ng/ml) and treated with or without CL. 2. key brown fat genes and mitochondrial genes were measured with qRT-PCR 3. UCP1, the thermogenic respiration protein levels were measured with WB 4. Mitochondrial respiration was assessed using Seahorse Bioscience XF24 5. Reactive oxygen species (ROS) was quantified using DCF assay. RESULTS LPS significantly decreased key brown fat genes (CIDEA, UCP1, PGC-1a). Furthermore, LPS-treated cells showed significantly lower UCP1, PGC1a gene induction in response to CL. P-value compared to control or CL. 0 1 2 3 4 5 CL LPS 100+CL LPS 1000+CL mRNA expresion (fold change) UCP1 UCP1 protein also decreased in LPS-treated cells and was less induced in response to CL. P-value compared to control or CL. key mitochondrial genes (ATPase8, CPT1B, CytC, ND1) were reduced by LPS treatment. P-value compared to control. This implicates LPS in acting to impair mitochondrial function and is supported by a reduced O 2 consumption rate in LPS treated cells as well as loss of mitochondrial membrane potential ΔΨm. Also, LPS increased susceptibility to hydrogen peroxide (oxidative agent) which was accompanied by reduced catalase gene expression levels (CAT). CONCLUSIONS These findings suggest that systemic LPS poses a risk to damaging mitochondrial function in BAT. As such, this current data indicates that blocking LPS-TLR4 signalling has potential to enhance BAT activity and mitigate inflammation to counteract obesity and type 2 diabetes 0 1 2 3 4 5 6 7 8 9 CL LPS 100 +CL LPS 1000+CL mRNA expresion (fold change) PGC1a 0 2 4 6 8 10 12 14 Day 0 Control LPS100 UCP1/ß-actin ratio (fold change) UCP1 Protein UCP1 ß-actin Day 0 Control LPS 100 0 0.5 1 1.5 2 2.5 3 3.5 4 Control CL LPS 100 LPS 100+CL UCP1/ß-actin ratio (fold change) UCP1 Protein Control LPS 100 LPS 100+CL CL UCP1 ß-actin ACKNOWLEGMENT The Graduate School, University of Warwick CARA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 mRNA expression (fold change) ATP synthase 0.0 0.2 0.4 0.6 0.8 1.0 1.2 mRNA expresion (fold change) NDI 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 mRNA expresion (fold change) CPT1B 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 mRNA expresion (fold change) CYTc Assay of mitochondrial DNA copy number revealed a large reduction in mitochondrial number. P-value compared to control. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 control LPS100 mtDNA copy number mtDNA copy number 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 20 40 60 80 100 120 140 OCR (pMoles/min) Time (min) Control LPS 100 CL LPS 100+CL Oxygen Consumption Rate oligo FCCP Rot+ Antimycin CL CL 0 0.5 1 1.5 2 2.5 3 None H2O2 Control LPS 100 LPS1000 ROS assay Florescence intensity (fold change) 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 mRNA expresion (fold change) UCP1 0.00 0.20 0.40 0.60 0.80 1.00 1.20 mRNA expresion (fold change) CIDEA 0.00 0.20 0.40 0.60 0.80 1.00 1.20 mRNA expresion (fold change) PGC1a ** ** *** *** *** *** *** ** ** ** *** *** *** *** ** *** * ** * *** ** * *** * * * *** *** 6. Confocal images were taken to assess mitochondrial membrane potential ΔΨm Control LPS 100 0 50 100 150 control lps florescence MTR/MTG (%of basal) MMP ΔΨm *** 0.0 0.2 0.4 0.6 0.8 1.0 1.2 control LPS 100 LPS 1000 mRNA expresion (fold change) CAT * 157--P Farah Omran Obesity and metabolism
Transcript of The Impact of Lipopolysaccharide on Mitochondrial …...Poster presented 18 at: The Impact of...
Poster presented
at:
BES2018
The Impact of Lipopolysaccharide on Mitochondrial Efficiency in Brown Adipocytes
Farah Omran1, Philip G McTernan2, Mark Christian1.
1Warwick Medical School, University of Warwick, Coventry, West Midland, UK. 2School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS, UK.
The presence of brown adipose tissue (BAT) in adult humans opens new avenues for research to ameliorate obesity consequences includingmetabolic and cardiovascular disease because of its special feature to expend rather than store energy.
Lipopolysaccharide (LPS) is known to be elevated in obesity and initiate an inflammatory state, but its effect has not been fully explored inBAT. β3 adrenergic receptor ligands such as CL 316,243 (CL) induce BAT activity through stimulation of UCP1. The interaction between the LPSreceptor TLR4 and β 3 adrenergic receptors has not been well studied in BAT.
Therefore, the objective of this study is to investigate the effect of LPS on the CL response and examine how LPS may alter mitochondrialfunction in BAT.
BACKGROUND
METHODS
1. Immortalized brown adipocytes were differentiated with or without LPS (100ng/ml or 1000ng/ml) and treated with or without CL.
2. key brown fat genes and
mitochondrial genes were
measured with qRT-PCR
3. UCP1, the thermogenic
respiration protein levels were
measured with WB
4. Mitochondrial respiration was assessed using
Seahorse Bioscience XF24
5. Reactive oxygen species
(ROS) was quantified using
DCF assay.
RESULTS
LPS significantly decreased key brown fat genes (CIDEA, UCP1, PGC-1a). Furthermore, LPS-treated cells showed significantly lower UCP1, PGC1a gene induction in response to CL. P-value compared to control or CL.
0
1
2
3
4
5
CL LPS 100+CL LPS 1000+CL
mR
NA
exp
resi
on
(fo
ld c
han
ge) UCP1
UCP1 protein also decreased in LPS-treated cells and was less induced in response to CL. P-value compared to control or CL.
key mitochondrial genes (ATPase8, CPT1B, CytC, ND1) were reduced by LPS treatment. P-value compared to control.
This implicates LPS in acting to impair mitochondrial function and issupported by a reduced O2 consumption rate in LPS treated cells as well asloss of mitochondrial membrane potential ΔΨm. Also, LPS increasedsusceptibility to hydrogen peroxide (oxidative agent) which wasaccompanied by reduced catalase gene expression levels (CAT).
CONCLUSIONSThese findings suggest that systemic LPS poses a risk to damaging mitochondrial function in BAT. As such, this current data indicates
that blocking LPS-TLR4 signalling has potential to enhance BAT activity and mitigate inflammation to counteract obesity and type 2
diabetes
0
1
2
3
4
5
6
7
8
9
CL LPS 100 +CL LPS 1000+CL
mR
NA
exp
resi
on
(fo
ld c
han
ge) PGC1a
0
2
4
6
8
10
12
14
Day 0 Control LPS100
UCP
1/ß
-act
in r
atio
(f
old
ch
ange
)
UCP1 Protein
UCP1
ß-actin
Day 0 Control LPS 100
0
0.5
1
1.5
2
2.5
3
3.5
4
Control CL LPS 100 LPS 100+CL
UCP
1/ß
-act
in r
atio
(f
old
ch
ange
)
UCP1 Protein
Control LPS 100 LPS 100+CLCL
UCP1
ß-actin
ACKNOWLEGMENT The Graduate School, University of Warwick
CARA
0.00.20.40.60.81.01.21.41.6
mR
NA
exp
ress
ion
(fo
ld c
han
ge) ATP synthase
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mR
NA
exp
resi
on
(fo
ld c
han
ge) NDI
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
mR
NA
exp
resi
on
(fo
ld c
han
ge) CPT1B
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
mR
NA
exp
resi
on
(fo
ld c
han
ge)
CYTc
Assay of mitochondrial DNA copy number revealed a large reduction in mitochondrial number. P-value compared to control.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
control LPS100
mtD
NA
co
py
nu
mb
er
mtDNA copy number
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 20 40 60 80 100 120 140
OCR
(p
Mo
les/
min
)
Time (min)
Control
LPS 100
CL
LPS 100+CL
Oxygen Consumption Rate
oligo FCCPRot+Antimycin
CL
CL
0
0.5
1
1.5
2
2.5
3
None H2O2
Control LPS 100 LPS1000
ROS assay
Flo
resc
ence
inte
nsi
ty (
fold
ch
ange
)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
mR
NA
exp
resi
on
(fo
ld c
han
ge) UCP1
0.00
0.20
0.40
0.60
0.80
1.00
1.20
mR
NA
exp
resi
on
(fo
ld c
han
ge) CIDEA
0.00
0.20
0.40
0.60
0.80
1.00
1.20
mR
NA
exp
resi
on
(fo
ld c
han
ge) PGC1a
** *****
*** ***
******
******
***
***
***
*** *****
*
**
*
*** **
*
***
*
*
*
******
6. Confocal images were taken to assess
mitochondrial membrane potential
ΔΨm
Control LPS 100
0
50
100
150
control lpsflo
resc
ence
MTR
/MTG
(%o
f b
asal
)
MMP ΔΨm
***
0.0
0.2
0.4
0.6
0.8
1.0
1.2
control LPS 100 LPS 1000
mR
NA
exp
resi
on
(fol
d ch
ange
)
CAT
*
157--PFarah Omran
Obesity and metabolism
