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, 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 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 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 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 blotting. 10g of total protein was stained with antibodies against PGC1 (1:500; sc-13067, Santa Cruz Biotechnology, Santa Cruz, CA), mitochondrial transcription factor A (Tfam or mtTFA) (1:2000; sc-23588, Santa Cruz), ATP synthase (ATPsyn) (1:2000, 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. 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 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.
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 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 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 factor (Cidea), TP53 apoptosis effector (Perp) and Bcl-2 binding component 3 (Bbc), were involved in the among up-regulated genes (SupplementalTable 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 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 (SupplementalTable1C). 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 amongthe 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 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 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).
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.
SupplementalTable 1 Microarray analysis of the skeletal muscle of AngII-infused mice.
A: Thirtygenes 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 / hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 4 / 0.50
NM_009648 / Akap1 / A kinase (PRKA) anchor protein 1 / 0.52
AK028953 / Apool / apolipoprotein O-like / 0.53
NM_031877 / Wasf1 / WASP family 1 / 0.55
AK050856 / Hadha / hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit / 0.58
U63712 / U63712 * / Mus musculus testis-specific HMG-box protein m-tsHMG precursor mRNA, partial cds, and mitochondrial transcription factor m-mtTFA precursor mRNA / 0.59
NM_019880 / Mtch1 / mitochondrial carrier homolog 1 (C. elegans) / 0.60
NM_027570 / Ldhd / lactate dehydrogenase D / 0.60
NM_026542 / Slc25a39 / solute carrier family 25, member 39 / 0.61
NM_008906 / Ctsa / cathepsin A / 0.62
AK054185 / Mrpl3 / mitochondrial ribosomal protein L3 / 0.63
NM_010438 / Hk1 / hexokinase 1 / 0.64
NR_001460 / Rmrp / RNA component of mitochondrial RNAase P / 0.65
NM_145494 / Me2 / malic enzyme 2, NAD(+)-dependent, mitochondrial / 0.65
AK085055 / Clic4 / Chloride intracellular channel 4 (mitochondrial) / 0.68
NM_030225 / Dlst / dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) / 0.69
NM_173011 / Idh2 / isocitrate dehydrogenase 2 (NADP+), mitochondrial / 0.69
AK081256 / 9030617O03Rik / RIKEN cDNA 9030617O03 gene / 0.70
NM_025500 / Mrpl37 / mitochondrial ribosomal protein L37 / 0.70
NM_013543 / H2-Ke6 / H2-K region expressed gene 6 / 0.71
NM_011591 / Timm17b / translocase of inner mitochondrial membrane 17b / 0.71
AK004470 / 1190003J15Rik / RIKEN cDNA 1190003J15 gene / 0.71
NM_138600 / Aldh7a1 / aldehyde dehydrogenase family 7, member A1 / 0.72
NM_019826 / Ivd / isovaleryl coenzyme A dehydrogenase / 0.73
NM_024188 / Oxct1 / 3-oxoacid CoA transferase 1 / 0.73
NM_181407 / Me3 / malic enzyme 3, NADP(+)-dependent, mitochondrial / 0.73
XM_132976 / Caprin2 / caprin family member 2 / 0.74
NM_016774 / Atp5b * / ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit / 0.74
BC030946 / Ndufv2 * / NADH dehydrogenase (ubiquinone) flavoprotein 2 / 0.74
NM_138601 / D10Jhu81e / DNA segment, Chr 10, Johns Hopkins University 81 expressed / 0.74
* genes involved in mitochondrial biogenesis or electron transport.
B: Thirtygenes 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 / solute carrier family 25 (mitochondrial carrier, citrate transporter), member 1 / 6.68
NM_007702 / Cidea * / cell death-inducing DNA fragmentation factor, alpha subunit-like effector A / 6.50
NM_013770 / Slc25a10 / solute carrier family 25 (mitochondrial carrier, dicarboxylate transporter), member 10 / 4.91
NM_146118 / Slc25a25 / solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 25 / 4.89
NM_181315 / Car5b / carbonic anhydrase 5b, mitochondrial / 4.49
NM_146214 / Tat / tyrosine aminotransferase / 4.38
NM_019946 / Mgst1 / microsomal glutathione S-transferase 1 / 3.90
NM_027324 / Sfxn1 / sideroflexin 1 / 3.53
NM_134154 / Slc25a45 / solute carrier family 25, member 45 / 3.04
NM_026405 / Rab32 / RAB32, member RAS oncogene family / 3.02
NM_009653 / Alas2 / aminolevulinic acid synthase 2, erythroid / 2.88
NM_008220 / Hbb-b1 / hemoglobin, beta adult major chain / 2.76
NM_008513 / Lrp5 / Low density lipoprotein receptor-related protein 5 / 2.48
NM_013546 / Hebp1 / heme binding protein 1 / 2.30
NM_008218 / Hba-a1 / hemoglobin alpha, adult chain 1 / 2.23
NM_008797 / Pcx / pyruvate carboxylase / 2.09
NM_175177 / Bdh1 / 3-hydroxybutyrate dehydrogenase, type 1 / 2.03
AK003929 / Auh / AU RNA binding protein/enoyl-coenzyme A hydratase / 1.99
NM_138665 / Sardh / sarcosine dehydrogenase / 1.96
NM_022032 / Perp * / PERP, TP53 apoptosis effector / 1.88
AK035739 / Slc25a35 / solute carrier family 25, member 35 / 1.84
NM_010292 / Gck / glucokinase / 1.83
NM_029447 / Nln / neurolysin (metallopeptidase M3 family) / 1.80
NM_010363 / Gstz1 / glutathione transferase zeta 1 (maleylacetoacetate isomerase) / 1.75
NM_019687 / Slc22a4 / solute carrier family 22 (organic cation transporter), member 4 / 1.73
NM_008991 / Abcd3 / ATP-binding cassette, sub-family D (ALD), member 3 / 1.72
NM_133234 / Bbc3 * / Bcl-2 binding component 3 / 1.67
NM_007981 / Acsl1 / acyl-CoA synthetase long-chain family member 1 / 1.67
NM_007830 / Dbi / diazepam binding inhibitor / 1.63
NM_028048 / Slc25a35 / solute carrier family 25, member 35 / 1.60
* 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 / crystallin, lambda 1 / 0.36
NM_013473 / Anxa8 / annexin A8 / 0.41
AK046660 / Recql / RecQ protein-like / 0.45
NM_011110 / Pla2g5 / phospholipase A2, group V / 0.54
AK050856 / Hadha * / hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit / 0.58
NM_027570 / Ldhd / lactate dehydrogenase D / 0.60
NM_011365 / Itsn2 / SH3 domain protein 1B / 0.60
NM_010184 / Fcer1a / Fc receptor, IgE, high affinity I, alpha polypeptide / 0.66
NM_010173 / Faah / fatty acid amide hydrolase / 0.67
AK080908 / Sod1 / superoxide dismutase 1, soluble / 0.68
NM_007460 / Ap3d1 / adaptor-related protein complex 3, delta 1 subunit / 0.69
NM_011989 / Slc27a4 * / solute carrier family 27 (fatty acid transporter), member 4 / 0.70
NM_023538 / Agk / multi-substrate lipid kinase / 0.71
NM_018743 / Agpat6 / lysophosphatidic acid acyltransferase zeta / 0.71
NM_013782 / Ptdss2 / phosphatidylserine synthase 2 / 0.71
NM_016781 / Prkag1 # / AMP-activated protein kinase, noncatalytic gamma-1 subunit / 0.71
NM_026792 / Agpat5 / 1-acylglycerolphosphate acyltransferase-epsilon / 0.73
NM_178882 / D2hgdh / D-2-hydroxyglutarate dehydrogenase / 0.75
NM_009384 / Tiam1 / T-cell lymphoma invasion and metastasis 1 / 0.76
NM_026979 / C1qtnf2 / C1q and tumor necrosis factor related protein 2 / 0.76
NM_148927 / Plekha4 / pleckstrin homology domain-containing, family A member 4 / 0.77
NM_018858 / Pebp1 / phosphatidylethanolamine binding protein 1 / 0.77
NM_153502 / Ankrd23 / diabetes related ankyrin repeat protein / 0.78
AK053879 / Plekha1 / pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 1 / 0.79
AK080941 / Ankhd1 / ankyrin repeat and KH domain containing 1 / 0.79
NM_008094 / Gba / glucosidase, beta, acid / 0.80
NM_008816 / Pecam1 / platelet/endothelial cell adhesion molecule 1 isoform 1 / 0.80
NM_011256 / Pitpnm2 / retinal degeneration B2 homolog / 0.80
NM_148937 / Plcd4 / phospholipase C, delta 4 isoform 2 / 0.81
NM_028320 / Adipor1 # / adiponectin receptor 1 / 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 / Pck1 # / phosphoenolpyruvate carboxykinase 1, cytosolic / 8.47
NM_007469 / Apoc1 # / apolipoprotein C-I / 7.04
NM_175640 / Plin # / Perilipin / 6.95
NM_009605 / Adipoq / adiponectin, C1Q and collagen domain containing / 6.75
NM_007639 / Cd1d1 / CD1d1 antigen / 6.64
NM_007702 / Cidea / cell death-inducing DNA fragmentation factor, alpha subunit-like effector A / 6.50
NM_130450 / Elovl6 / ELOVL family member 6, elongation of long chain fatty acids / 6.43
NM_007988 / Fasn * / fatty acid synthase / 5.78
AK046712 / Npr3 / natriuretic peptide receptor 3 / 5.47
NM_134037 / Acly / ATP citrate lyase / 5.01
NM_026212 / Agpat2 / 1-acylglycerol-3-phosphate O-acyltransferase 2 / 4.57
NM_030210 / Aacs / acetoacetyl-CoA synthetase / 4.28
NM_007408 / Adfp / adipose differentiation related protein / 4.07
NM_021890 / Fads3 / fatty acid desaturase 3 / 3.80
NM_010333 / Edg5 / endothelial differentiation, sphingolipid G-protein-coupled receptor, 5 / 3.50
NM_054088 / Pnpla3 / Adiponutrin / 3.43
NM_009695 / Apoc2 # / apolipoprotein C-II / 3.08
NM_009127 / Scd1 * / stearoyl-Coenzyme A desaturase 1 / 2.92
NM_008509 / Lpl / lipoprotein lipase / 2.84
NM_024406 / Fabp4 / fatty acid binding protein 4, adipocyte / 2.38
NM_009696 / Apoe # / apolipoprotein E / 2.29
NM_010730 / Anxa1 / annexin A1 / 2.25
NM_009128 / Scd2 * / stearoyl-Coenzyme A desaturase 2 / 2.23
NM_008797 / Pcx # / pyruvate carboxylase / 2.09
NM_007585 / Anxa2 / annexin A2 / 2.02
NM_011125 / Pltp / phospholipid transfer protein / 1.99
NM_011844 / Mgll / monoglyceride lipase / 1.92
BC049168 / 2300002D11Rik / RIKEN cDNA 2300002D11 gene / 1.91
NM_013454 / Abca1 / ATP-binding cassette 1, sub-family A, member 1 / 1.76
NM_010185 / Fcer1g / Fc receptor, IgE, high affinity I, gamma polypeptide / 1.69
* genes involved in fatty acid synthesis
# other genes related to carbohydrate and lipid anabolism
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.