Lipotoxicity and muscular mitochondrial
function in insulin resistance
Matthijs Hesselink
Nutrition and Toxicology Research Institute Maastricht (NUTRIM)
Department of Human Movement Sciences
Maastricht University
The Netherlands
www.hb.unimaas.nl/muscle
Q: Why muscle?
Muscle & insulin sensitivity
·
·
~ 80% of post-prandial glucose uptake occurs in
muscle
Lipid overflow to non-adipose tissue results in
muscular fat storage, which associates with insulin
resistance
Q: Why mitochondria?
Mitochondria & insulin sensitivity
·
·
·
·
Oxidative degradation of nutrients occurs in
mitochondria
Mitochondria are the major site of production of
reactive oxygen species
Mitochondria are vital organelles for (myo)cellular
function and are regulators of apoptosis
Mitochondrial dysfunction may result in muscular
fat storage and insulin resistance
Lipotoxicity, mitochondrial function and
insulin resistance
Lipid overflow
Low (fat) oxidative capacity
Muscular FA (derivatives) ↑
-
Impeded insulin signalling
Mitochondrial FA accumulation ↑
FA export
Uncoupling
(UCP3, FA, ANT)
Main sites of intervention
ROS
Lipid peroxidation
Lipidperoxide export
Lipotoxicity
Mitochondrial damage/dysfunction
Main ouput parameters
Tissues examined & Models used
IMCL is negatively associated with
insulin sensitivity
Krssak et al., Diabetologia 2002
Sinha et al., Diabetes 2002
Fourouhi et al., Diabetologia 1999
Jacob et al., Diabetes 1999
High FFA levels acutely induce muscle TG
accumulation and insulin resistance
Hoeks et al, Diabetologia 2006
Paradox: endurance training
also increases IMCL content
14 days endurance training
1.5
1
+54%
0.5
0
before
after
Schrauwen-Hinderling et al., Obesity 2005
IMCL as % of water signal
IMCL as % of water signal
7 days high-fat diet
0.8
0.6
+42%
0.4
0.2
0
before
after
Schrauwen-Hinderling et al., JCEM 2003
Training, not high-fat diet downregulates ACC2
AcetylCoA
ACC-2
MalonylCoA
ACC2 (% of baseline)
250%
P=0.06
fattyacylcarnitine
CPT1
fattyacylCoA
200%
150%
100%
P<0.05
50%
0%
LF
before training
HF
after training
Schrauwen et al., Diabetes 2002
Q: is IMCL only ‘harmful’ in combination
with a low oxidative capacity?
Muscular mitochondrial function in T2DM
and controls, matched for BMI
Diabetic patients
(n=12, all males)
Controls
(n=9, all males)
p-value
BMI (kg/m2)
29.4 ± 3.3
29.3 ± 2.7
n.s.
Age (years)
61.8 ± 4.4
56.1 ± 6.8
P < 0.05
VO2max
(ml*min-1*kg-1)
30.6 ± 5.8
34.6 ± 4.8
n.s.
plasma glucose (mM)
9.56 ± 2.12
5.73 ± 0.37
P < 0.01
insulin sensitivity
(GIR, μmol*min-1*kg-1 )
18.9 ± 8.1
26.0± 6.7
P < 0.05
(mean ± stdev)
Schrauwen-Hinderling et al., Diabetologia 2007
Muscle mitochondrial function in vivo
by
31P
NMR Spectroscopy
PCr
ATP
Pi
7.5
0
-7.5
-15
relative resonance frequency (ppm)
Muscle mitochondrial function in vivo
by
31P
NMR Spectroscopy
recovery
exercise
Meyer et al., 1988
Muscle mitochondrial function in vivo
31P
NMR Spectroscopy
PCr
by
PCr resynthesis is
almost purely aerobic
time
PCr recovery half-time
reflects oxidative capacity
Muscular fat content in T2DM and BMI
matched normoglycemic controls is similar
percentage of water signal
2
1,5
1
0,5
0
controls
T2DM
Schrauwen-Hinderling et al., Diabetologia 2007
Q: How about mitochondrial function?
In vivo mitochondrial function reduced in
T2DM, correlates with glucose and HbA1C
35
*
R2 =0.49; P<0.01
60
25
PCr recovery (s)
P C r rec overy (s )
30
20
15
10
50
40
30
20
5
10
0
0
CON
DM
6
8
10
12
14
Plasma glucose
Schrauwen-Hinderling et al., Diabetologia 2007
Q1:is mitochondrial dysfunction already present
in the pre-diabetic state?
Q2:does intrinsic mitochondrial dysfunction
underlie reduced in vivo mitochondrial
function?
Intrinsic mitochondrial function ex vivo
using high-resolution respirometry
250
State u
120
150
State 3
80
100
40
50
0
0
00:00:00
00:07:30
00:15:00
vezels
R1
00:22:30
00:30:00
Range [hh:mm:ss]: 00:45:00
mal
glut
ADP
succ
00:19:0600:21:30
00:26:07
00:37:30
FCCP+1+1
00:33:39 00:36:34
00:45:00
O 2 F lu x p e r V ( A ) [p m o l/(s *m l) ]
O 2 C o n c e n tra tio n ( A ) [n m o l/m l]
160
200
Pre-diabetic subject Characteristics
Controls
FDR
T2DM
N=16
N=12
N=10
Age (y)
59.2 ± 0.7
60.1 ± 0.9
61.4 ± 1.6
n.s.
BMI (kg/m2)
29.1 ± 0.7
30.1 ± 1.2
28.9 ± 0.7
n.s.
31.2. ± 1.6
32.6 ± 9.6
28.2 ± 2.3
n.s.
28.9 ± 3.7
22.1 ± 3.4
11.2 ± 2.8
* p<0.05
VO2max
(ml/kg/min)
Ins-stim Rd
(umol/kg/min)
Phielix et al., Diabetes 2008
Lower ex vivo mitochondrial function in T2DM
irrespective of mitochondrial density (mtDNA)
*
state 3/ mtDNA copy number
70
60
50
40
30
Control
FDR
T2DM
20
10
0
Phielix et al., Diabetes 2008
Lower in vivo mitochondrial function in
T2DM and first-degree relatives
*
30
PCr half-time (s)
25
P=0.08
20
15
Control
FDR
T2DM
10
5
0
Phielix et al., Diabetes 2008
Q: Is compromised mitochondrial function in the prediabetic state required to develop insulin resistance?
Transition of pre-diabetes to T2D during
maturation of ZDF rats
Lenaers et al., submitted
O2 consumption (nmol/min/mg mito)
No mitochondrial dysfunction during the
development of T2D in ZDF rats
250
* p < 0.05 : compared to 6 Weeks
# p < 0.05 : compared to 12 Weeks
Oxidation of a lipid substrate
ZDF
control
200
*
150
*
100
#
* #
50
0
6 weeks
12 weeks
Age
19 weeks
Lenaers et al., submitted
Increased UCP3 protein blunted in ZDF rats
ZDF
control
250
#
*
UCP 3 (AU)
200
150
100
50
0
6 Weeks
12 Weeks
19 Weeks
Age
Lenaers et al., submitted
Uncoupling protein 3 (UCP3)
● ~55% homology to UCP1 which is responsible for the
thermogenesis in of brown adipose tissue
● Primarily expressed in skeletal muscle and heart
● UCP3 is able to lower the mitochondrial proton gradient upon activation by fatty acids - by transporting H+ or FA-
Intermembrane
space
H+
H+
H+
FA-
H+
Cyt c
Inner
mitochondrial
membrane
H+
Q
NADH
NAD+
UCP
?
2H+ + 1/2 O2 H2O
ADP
Mitochondrial
matrix
ATP
H+
FA-
H+
Electron transport chain
ATP
synthase
UCP leak
UCP3 is reduced in (pre)diabetic patients
Schrauwen et al., JCEM 2006
Q: Is UCP3 involved in the development of
mitochondrial dysfunction in T2D?
Lipotoxicity, mitochondrial function and insulin
resistance
Lipid overflow
Low (fat) oxidative capacity
Muscular FA (derivatives) ↑
-
Impeded insulin signalling
Mitochondrial FA accumulation ↑
FA export
Uncoupling
(UCP3, FA, ANT)
ROS
Lipid peroxidation
Lipidperoxide export
Lipotoxicity
Mitochondrial damage/dysfunction
Q: Can insulin sensitizing by exercise training restore
mitochondrial aberrations observed in T2D?
Insulin sensitizing by Rosiglitazone
restores UCP3 protein content in T2D…
Schrauwen et al, JCEM 2006
Mensink et al, Diab Obes Met 2006
Exercise training in T2D and controls
improves insulin sensitivity
Glucose infusion rate
35
umol/min/kg BM
30
*
25
*
20
15
10
5
0
Control subjects
Data are means ± SE
before training
diabetic subjects
after training
Meex et al., in progress
UCP3 protein expression (AU)
Exercise training in T2D restores UCP3 content
45
40
35
30
25
20
15
10
5
0
Before
Data are means ± SE
After
Controls
T2D
Meex et al., in progress
Q: Can mitochondrial uncoupling reduce
superoxide production in diabetogenic conditions?
Mitochondrial proton gradient and ROS
production
Skulachev et al., BBA 1997
Measuring superoxide production by
electron spin resonance in isolated mitos
muscle mito 0.2 mg/ml
5 min 37° C
100 mM DMPO
muscle mito 0.2 mg/ml
5 min 37° C
100 mM DMPO
10 mM glutamate
10 mM succinaat
3 mM malate
idem 2
500 U/ml SOD
Hoeks et al., FEBS 2008
Aging related increased superoxide
production is blunted in UCP3 tg mice
+ 4HNE
WT
Overexpressor
Nabben et al., FEBS Lett 2008
Type 2 diabetes, fatty acid-induced uncoupling
and superoxide production
● Increased supply of fatty acids to the muscle
● Decreased fat oxidative capacity in skeletal muscle
● Increased storage of lipid (intermediates) in muscle
● Increased production of reactive oxygen species (ROS)
● Increased levels of lipid peroxidation (FA + ROS)
FA-induced uncoupling and ROS
Mouse skeletal muscle mitochondria, n=3
Hoeks et al., in progress
Hypotheses
●
FA-induced uncoupling controls mitochondrial ROS
production when facing high intracellular fatty acid levels
●
Reduced or defective FA-induced uncoupling contributes to the
progression of type 2 diabetes
Q: Is FA-induced uncoupling lower in
diabetic ZDF Rats?
FA-induced uncoupling
Mito
Pyr
Fatty acid
Oligo
Hoeks et al., in progress
Oxygen consumption (nmol O2/min/mg)
FA-induced uncoupling
140
Vmax
120
100
80
60
40
EC50
20
0
0.1
1
10
100
1000
10000
[Palmitate] nM
Hoeks et al., in progress
140
Diabetic ZDF rats less sensitive to FAinduced uncoupling
Vmax (nmol O2/min/mg)
120
100
control
ZDF
80
60
40
500
20
*
0
+/+
fa/fa
EC50 (nM free palmitate)
400
300
200
100
0
+/+
n=4
fa/fa
Hoeks et al., in progress
Q: What mechanisms could be responsible
for mitochondrial (FFA) uncoupling?
Mitochondrial uncoupling
Uncoupling Protein 3 (UCP3)
● Exact physiological function not yet established
● Hypothesis: FA (peroxides) activate UCP3 protein → proton leak
Adenine Nucleotide Translocator (ANT)
● Primary function: mitochondrial exchange of ADP and ATP
● Hypothesis: ANT (partly) mediates FA-induced uncoupling and
contributes to basal proton leak in some tissues
Lower UCP3 but comparable ANT levels in
diabetic ZDF rat
Control
Diabetic ZDF
n=6
Hoeks et al., in progress
ANT inhibition: CarboxyAtractyloside
Hoeks et al., in progress
After ANT inhibition similar sensitivity to FAinduced uncoupling; ANT defect?
Control
Diabetic ZDF
Hoeks et al., in progress
Conclusions
·
·
·
·
·
·
·
·
·
Intrinsic mitochondrial abberations underlie mitochondrial dysfunction
in T2D
First-degree relatives (pre-diabetics) already tend to have lower
mitochondrial function
Failure to maintain adaptive improvements in mitochondrial function
parallel the transition of the prediabetic state towards full blown type 2
diabetes (rats)
Mitochondrial dysfunction in T2D does not augment muscular fat
storage compared to (obese) controls
Insulin sensitizing interventions restore UCP3 content in T2D
Overexpression of UCP3 blunts age related superoxide production
FA-induced uncoupling reduces superoxide production
Diabetic
ZDF
rats± SE
are less sensitive to FA-induced uncoupling (need
Data are
means
more FA for the same level of uncoupling)
The difference in sensitivity to FA-induced uncoupling is mediated via
ANT
Thanks to…
Patrick Schrauwen
Ruth Meex
Vera Schrauwen-Hinderling
Gert Schaart
Esther Kornips
Johan de Vogel
Noud van Herpen
Miranda Nabben
Joris Hoeks
Esther Phielix
Ellen Lenaers
Ronnie Minnaard
Denis van Beurden
Katarina Fredriksson
Johanna Jörgensen
Silvie Timmers