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SUPPLEMENTAL MATERIALS
Angiotensin II Reduces Mitochondrial Content in Skeletal Muscle and Affects Glycemic
Control
Masanori Mitsuishi, Kazutoshi Miyashita, Ayako Muraki, Hiroshi Itoh
SUPPLEMENTAL RESEARCH DESIGN
AND METHODS
Microarray analysis. Total RNA was
extracted from pooled samples of the
quadriceps from mice infused with AngII for
10 days and from the control (n=3). Isolated
RNAs were processed and analyzed using the
Agilent Whole Mouse Genome Oligo
Microarray (Agilent technologies, Palo Alto,
quantitative PCR
analysis
(ABI7500
Real-Time PCR System; Applied Biosystems,
Foster City, CA), using specific primers for
the mitochondrial DNA encoded 16S
ribosomal RNA gene and the nuclear DNA
encoded hexokinase 2 gene, as described
previously [20]. The level of gene expression
was determined by means of the PCR
reactions (ABI 7500) in the presence of a
CA). Signal intensities were normalized by
per Chip to 50 percentile and analyzed with
the GeneSpring GX (Agilent). The ratios of
the expression levels in the AngII-infused
group and the control were calculated. We
used the Gene Ontology (GO) annotation to
select genes related to the areas of interest,
namely, mitochondrion (GO:0005739) and
lipid metabolism (GO:0006629). The thirty
fluorescent dye (SYBR Premix Ex Taq;
TaKaRa Bio, Otsu, Japan). The relative
quantity of mRNA was calculated after
normalization to that of the internal control,
18S gene.
Quantification of protein levels by western
genes which were most down-regulated or
up-regulated
in
the
quadriceps
of
AngII-infused mice were identified and listed
in order of magnitude of change.
Quantification of mitochondrial DNA copy
number and gene expressions by real time
PCR. Mitochondrial DNA (mtDNA) copy
number was determined by means of
Cruz, CA), mitochondrial transcription factor
A (Tfam or mtTFA) (1:2000; sc-23588, Santa
Cruz), ATP synthase (ATPsyn) (1:2000,
blotting. 10g of total protein was stained
with antibodies against PGC1 (1:500;
sc-13067, Santa Cruz Biotechnology, Santa
subunit , A21350; Molecular Probes),
Cytochrome C Oxidase (COX) (1:1000,
subunit IV, A21348; Molecular Probes),
medium-chain fatty acyl-CoA dehydrogenase
(MCAD) (1:500, 101730-1; Cayman
Chemical, Ann Arbor, MI) and -actin
(1:2000; #4967, Cell Signaling, Danvers,
MA) according to a standard procedure with
the aid of a chemi-luminescence kit (ECL
Plus; GE Healthcare, Pittsburgh, PA) and a
lumino-image analyzer (LAS-4000; FujiFilm,
Tokyo, Japan). The density of the blots for
each protein relative to that for the internal
control, -actin, was estimated by means of
imaging software (MultiGauge; FujiFilm).
Oil-red-O staining. Quadriceps were isolated
from mice that had been infused with AngII
for 10days with or without the AT1R blocker,
CS-866 or the AT2R blocker PD-123319.
change in muscle gene expressions. We could
observe down-regulation of the genes
involved in mitochondrial biogenesis or
function, and fatty acid oxidation; and
up-regulation of genes involved in lipid
synthesis (Supplemental Table 1A-D).
Among the genes categorized in
GO:0005739, mitochondrion, we obtained
compatible changes in expression signals
from 980 gene transcripts. The genes
Tissue sections (10-m thickness) were
stained with Oil-red-O and counterstained
with hematoxylin.
Statistical analysis. All data were expressed
as mean ± standard error. Comparison of
involved in mitochondrial biogenesis or
electron transport, such as, m-mtTFA
precursor (U63712), ATP synthase (Atp5b)
and NADH dehydrogenase (ubiquinone)
flavoprotein 2 (Ndufv2), were among the
genes showing the greatest decrease in
AngII-infused mice (Supplemental Table 1A).
On the other hand, pro-apoptotic genes, such
as, cell death-inducing DNA fragmentation
means between two groups was performed
with Student’s t test. When more than two
groups were compared, analysis of variance
was used to evaluate significant differences
between groups, and if significant differences
were confirmed, each difference was further
examined by means of multiple comparisons.
A p value of less than 0.05 was considered to
be statistically significant.
factor (Cidea), TP53 apoptosis effector (Perp)
and Bcl-2 binding component 3 (Bbc), were
involved in the among up-regulated genes
(Supplemental Table 1B).
We also analyzed genes categorized in
GO:0006629, lipid metabolism (valid
expression signals from 407 gene transcripts).
In this category, genes related to fatty acid
oxidation
(FAO),
such
as
SUPPLEMENTAL RESULTS
Microarray analysis. Gene expression
profiles,
which
are
categorized
in
GO:0005739, mitochondrion or GO:0006629,
lipid metabolism, of the quadriceps of
AngII-infused mice and of the controls were
compared in order to identify AngII-induced
hydroxyacyl-Coenzyme A dehydrogenase
(Hadha) and solute carrier family 27
(Slc27a4, fatty acid transporter), were among
the thirty down-regulated genes in
AngII-infused group (Supplemental Table1C).
Genes related to positive regulation of FAO,
such as AMP-activated protein kinase
(Prkag1, AMPK) and adiponectin receptor 1
(Adipor1), and genes reflecting the degree of
FAO, such as crystallin lambda 1 (Cryl1) and
C1q and tumor necrosis factor related protein
2 (C1qtnf2) were also among the
down-regulated genes. Superoxide dismutase
1 (SOD1) which is known to play a role in
scavenging ROS was also decreased in the
muscle as a result of AngII infusion. On the
other hand, genes involved in fatty acid
synthesis such as (Fasn), stearoyl-Coenzyme
shown). Furthermore, many important genes
that are related to carbohydrate and lipid
anabolism were up-regulated, including
apo-lipoproteins, perilipin and gluconeogenic
genes, phosphoenolpyruvate carboxykinase 1
(Pck1, PEPCK) and pyruvate carboxylase
(Pcx). The changes in the expression levels of
the representative genes observed in
microarray analysis were also confirmed by
quantitative PCR (Supplemental Figure 1).
A desaturase 1 (Scd1) and Scd2 were among
the thirty most up-regulated genes in
AngII-infused group (Supplemental Table1D),
associated with a 1.56-fold increase in the
expression of the master regulator of lipid
synthesis, sterol regulatory element binding
transcription factor 1 (SREBP1, data not
These results indicate that the AngII
infusion in mice affects glycemic control at
least partly by regulating the expression of
the genes involved in mitochondrial
biogenesis, their function and lipid
metabolism in the skeletal muscle.
Supplemental Table 1 Microarray analysis of the skeletal muscle of AngII-infused mice.
A: Thirty genes included in GO:0005739 (mitochondrion) that were down-regulated in
the skeletal muscle of AngII-infused mice
Genbank
Gene Symbol
Product
AII/
Vehicle
NM_008294 Hsd3b4
NM_009648 Akap1
AK028953
Apool
NM_031877 Wasf1
AK050856
Hadha
U63712
U63712 *
NM_019880
NM_027570
NM_026542
NM_008906
AK054185
NM_010438
NR_001460
NM_145494
AK085055
NM_030225
Mtch1
Ldhd
Slc25a39
Ctsa
Mrpl3
Hk1
Rmrp
Me2
Clic4
Dlst
NM_173011 Idh2
AK081256
9030617O03R
ik
NM_025500 Mrpl37
NM_013543 H2-Ke6
NM_011591 Timm17b
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and
steroid delta-isomerase 4
A kinase (PRKA) anchor protein 1
0.50
apolipoprotein O-like
WASP family 1
hydroxyacyl-Coenzyme A
dehydrogenase/3-ketoacyl-Coenzyme A
thiolase/enoyl-Coenzyme A hydratase (trifunctional
protein), alpha subunit
Mus musculus testis-specific HMG-box protein
m-tsHMG precursor mRNA, partial cds, and
mitochondrial transcription factor m-mtTFA
precursor mRNA
0.53
0.55
0.58
mitochondrial carrier homolog 1 (C. elegans)
lactate dehydrogenase D
solute carrier family 25, member 39
cathepsin A
mitochondrial ribosomal protein L3
hexokinase 1
RNA component of mitochondrial RNAase P
malic enzyme 2, NAD(+)-dependent, mitochondrial
Chloride intracellular channel 4 (mitochondrial)
dihydrolipoamide S-succinyltransferase (E2
0.60
0.60
0.61
0.62
0.63
0.64
0.65
0.65
0.68
0.69
component of 2-oxo-glutarate complex)
isocitrate dehydrogenase 2 (NADP+), mitochondrial
RIKEN cDNA 9030617O03 gene
0.69
0.70
mitochondrial ribosomal protein L37
H2-K region expressed gene 6
translocase of inner mitochondrial membrane 17b
0.70
0.71
0.71
0.52
0.59
AK004470
NM_138600
NM_019826
NM_024188
NM_181407
1190003J15Ri
k
Aldh7a1
Ivd
Oxct1
Me3
XM_132976 Caprin2
NM_016774 Atp5b *
BC030946
Ndufv2 *
NM_138601 D10Jhu81e
RIKEN cDNA 1190003J15 gene
0.71
aldehyde dehydrogenase family 7, member A1
isovaleryl coenzyme A dehydrogenase
3-oxoacid CoA transferase 1
malic enzyme 3, NADP(+)-dependent,
mitochondrial
caprin family member 2
ATP synthase, H+ transporting mitochondrial F1
0.72
0.73
0.73
0.73
0.74
0.74
complex, beta subunit
NADH dehydrogenase (ubiquinone) flavoprotein 2
DNA segment, Chr 10, Johns Hopkins University
81 expressed
0.74
0.74
* genes involved in mitochondrial biogenesis or electron transport.
B: Thirty genes included in GO:0005739 (mitochondrion) that were up-regulated in the
skeletal muscle of AngII-infused mice
Genbank
Gene Symbol
Description
AII/
Vehicle
NM_153150 Slc25a1
6.68
NM_181315 Car5b
NM_146214 Tat
solute carrier family 25 (mitochondrial carrier,
citrate transporter), member 1
cell death-inducing DNA fragmentation factor,
alpha subunit-like effector A
solute carrier family 25 (mitochondrial carrier,
dicarboxylate transporter), member 10
solute carrier family 25 (mitochondrial carrier,
phosphate carrier), member 25
carbonic anhydrase 5b, mitochondrial
tyrosine aminotransferase
NM_019946
NM_027324
NM_134154
NM_026405
NM_009653
NM_008220
NM_008513
microsomal glutathione S-transferase 1
sideroflexin 1
solute carrier family 25, member 45
RAB32, member RAS oncogene family
aminolevulinic acid synthase 2, erythroid
hemoglobin, beta adult major chain
Low density lipoprotein receptor-related protein 5
3.90
3.53
3.04
3.02
2.88
2.76
2.48
NM_007702 Cidea *
NM_013770 Slc25a10
NM_146118 Slc25a25
Mgst1
Sfxn1
Slc25a45
Rab32
Alas2
Hbb-b1
Lrp5
6.50
4.91
4.89
4.49
4.38
NM_013546
NM_008218
NM_008797
NM_175177
AK003929
Hebp1
Hba-a1
Pcx
Bdh1
Auh
heme binding protein 1
hemoglobin alpha, adult chain 1
pyruvate carboxylase
3-hydroxybutyrate dehydrogenase, type 1
AU RNA binding protein/enoyl-coenzyme A
hydratase
sarcosine dehydrogenase
PERP, TP53 apoptosis effector
solute carrier family 25, member 35
2.30
2.23
2.09
2.03
1.99
1.83
1.80
1.75
NM_133234 Bbc3 *
NM_007981 Acsl1
glucokinase
neurolysin (metallopeptidase M3 family)
glutathione transferase zeta 1 (maleylacetoacetate
isomerase)
solute carrier family 22 (organic cation transporter),
member 4
ATP-binding cassette, sub-family D (ALD),
member 3
Bcl-2 binding component 3
acyl-CoA synthetase long-chain family member 1
NM_007830 Dbi
NM_028048 Slc25a35
diazepam binding inhibitor
solute carrier family 25, member 35
1.63
1.60
NM_138665 Sardh
NM_022032 Perp *
AK035739
Slc25a35
NM_010292 Gck
NM_029447 Nln
NM_010363 Gstz1
NM_019687 Slc22a4
NM_008991 Abcd3
1.96
1.88
1.84
1.73
1.72
1.67
1.67
* genes involved in pro-apoptotic processes
C: Thirty genes included in GO:0006629 (lipid metabolism) that were down-regulated
in the skeletal muscle of AngII-infused mice
Genbank
Gene Symbol
Product
AII/
Vehicle
AK020717
Cryl1
NM_013473 Anxa8
crystallin, lambda 1
annexin A8
0.36
0.41
AK046660
Recql
NM_011110 Pla2g5
AK050856
Hadha *
RecQ protein-like
phospholipase A2, group V
hydroxyacyl-Coenzyme A
dehydrogenase/3-ketoacyl-Coenzyme A
thiolase/enoyl-Coenzyme A hydratase (trifunctional
protein), alpha subunit
lactate dehydrogenase D
0.45
0.54
0.58
NM_027570 Ldhd
0.60
NM_011365
NM_010184
NM_010173
AK080908
NM_007460
NM_011989
Itsn2
Fcer1a
Faah
Sod1
Ap3d1
Slc27a4 *
NM_023538 Agk
NM_018743 Agpat6
NM_013782 Ptdss2
NM_016781 Prkag1 #
NM_026792
NM_178882
NM_009384
NM_026979
NM_148927
Agpat5
D2hgdh
Tiam1
C1qtnf2
Plekha4
NM_018858 Pebp1
NM_153502 Ankrd23
AK053879
Plekha1
AK080941
Ankhd1
NM_008094 Gba
NM_008816 Pecam1
NM_011256 Pitpnm2
NM_148937 Plcd4
NM_028320 Adipor1 #
SH3 domain protein 1B
Fc receptor, IgE, high affinity I, alpha polypeptide
fatty acid amide hydrolase
superoxide dismutase 1, soluble
adaptor-related protein complex 3, delta 1 subunit
solute carrier family 27 (fatty acid transporter),
member 4
multi-substrate lipid kinase
lysophosphatidic acid acyltransferase zeta
0.60
0.66
0.67
0.68
0.69
0.70
phosphatidylserine synthase 2
AMP-activated protein kinase, noncatalytic
gamma-1 subunit
1-acylglycerolphosphate acyltransferase-epsilon
D-2-hydroxyglutarate dehydrogenase
T-cell lymphoma invasion and metastasis 1
C1q and tumor necrosis factor related protein 2
pleckstrin homology domain-containing, family A
member 4
phosphatidylethanolamine binding protein 1
0.71
0.71
diabetes related ankyrin repeat protein
pleckstrin homology domain containing, family A
(phosphoinositide binding specific) member 1
ankyrin repeat and KH domain containing 1
glucosidase, beta, acid
platelet/endothelial cell adhesion molecule 1
isoform 1
retinal degeneration B2 homolog
phospholipase C, delta 4 isoform 2
adiponectin receptor 1
0.78
0.79
0.71
0.71
0.73
0.75
0.76
0.76
0.77
0.77
0.79
0.80
0.80
0.80
0.81
0.81
* genes involved in fatty acid oxidation
# genes related to positive regulation of fatty acid oxidation
D: Thirty genes included in GO:0006629 (lipid metabolism) that were up-regulated in
the skeletal muscle of AngII-infused mice
Genbank
Gene Symbol
Product
AII/
Vehicle
NM_011044
NM_007469
NM_175640
NM_009605
NM_007639
NM_007702
Pck1 #
Apoc1 #
Plin #
Adipoq
Cd1d1
Cidea
NM_130450 Elovl6
phosphoenolpyruvate carboxykinase 1, cytosolic
apolipoprotein C-I
Perilipin
adiponectin, C1Q and collagen domain containing
CD1d1 antigen
cell death-inducing DNA fragmentation factor,
alpha subunit-like effector A
ELOVL family member 6, elongation of long chain
fatty acids
8.47
7.04
6.95
6.75
6.64
6.50
6.43
NM_007988
AK046712
NM_134037
NM_026212
Fasn *
Npr3
Acly
Agpat2
fatty acid synthase
natriuretic peptide receptor 3
ATP citrate lyase
1-acylglycerol-3-phosphate O-acyltransferase 2
5.78
5.47
5.01
4.57
NM_030210
NM_007408
NM_021890
NM_010333
Aacs
Adfp
Fads3
Edg5
4.28
4.07
3.80
3.50
NM_054088 Pnpla3
acetoacetyl-CoA synthetase
adipose differentiation related protein
fatty acid desaturase 3
endothelial differentiation, sphingolipid
G-protein-coupled receptor, 5
Adiponutrin
NM_009695
NM_009127
NM_008509
NM_024406
NM_009696
NM_010730
NM_009128
NM_008797
NM_007585
NM_011125
apolipoprotein C-II
stearoyl-Coenzyme A desaturase 1
lipoprotein lipase
fatty acid binding protein 4, adipocyte
apolipoprotein E
annexin A1
stearoyl-Coenzyme A desaturase 2
pyruvate carboxylase
annexin A2
phospholipid transfer protein
3.08
2.92
2.84
2.38
2.29
2.25
2.23
2.09
2.02
1.99
monoglyceride lipase
RIKEN cDNA 2300002D11 gene
1.92
1.91
ATP-binding cassette 1, sub-family A, member 1
Fc receptor, IgE, high affinity I, gamma polypeptide
1.76
1.69
Apoc2 #
Scd1 *
Lpl
Fabp4
Apoe #
Anxa1
Scd2 *
Pcx #
Anxa2
Pltp
NM_011844 Mgll
BC049168
2300002D11R
ik
NM_013454 Abca1
NM_010185 Fcer1g
* genes involved in fatty acid synthesis
# other genes related to carbohydrate and lipid anabolism
3.43
Supplemental Figure 1
Confirmation of the changes in gene expressions observed in microarray analysis by
means of quantative PCR. * p<0.05, ** p<0.01 compared to the vehicle group.
Gene expressions
200
Vehicle
AngII
(%Vehicle)
**
**
150
100
**
**
Atp5b
Ndufv2
*
**
50
0
Electron transport
Hadha
Slc27a4
Srebp1c
Fasn
Fatty acid oxidation Fatty acid synthesis