SUPPLEMENTARY DATA Data Supplement To

SUPPLEMENTARY DATA Data supplement to

Perivascular adipose tissue controls insulin-stimulated perfusion, mitochondrial protein expression and glucose uptake in muscle through adipomuscular microvascular anastomoses

Surname first author

Turaihi

Authors & Affiliations Turaihi, Alexander H MD1; Serné, Erik H, MD PhD2; Molthoff, Carla FM PhD3; Koning, Jasper J PhD4; Knol, Jaco PhD6; Niessen, Hans W MD PhD5; Goumans, Marie Jose TH PhD7; van Poelgeest, Erik M MD1; Yudkin, John S MD PhD8; Smulders, Yvo M MD PhD2; Connie R Jimenez, PhD6; van Hinsbergh, Victor WM PhD1; Eringa, Etto C PhD1

Skeletal muscle samples were lysed up in 1D‐sample buffer (10% glycerol, 62.5 mmol/L Tris (pH 6.8), 2% w/v LDS, 2% w/v DTT) and protein concentration was determined using Pierce 660‐nm protein assay (Thermo scientific, Waltham, MA USA 02 451; 22 660) according to the manufacturer's instructions. Heat shock protein 90 immunoblotting was performed by application of samples (5 µg protein) on 4‐15% Criterion TGX gels (Biorad, Veenendaal, the Netherlands, 5 671 084) and semi‐dry blotting onto PVDF membranes (GE Healthcare‐Fisher, RPN1416F), incubated overnight with rat monoclonal HSP90 antibody (1:1000 dilution) after blocking with 5% milk in TBS‐T (137 mM NaCl, 20 mmol/L Tris pH 7.0 and 0.1% (v/v) Tween [Sigma‐Aldrich, P7949]). After 2 hours incubation with anti-rat, horse radish peroxidase-coupled secondary antibody (Thermo Fisher 62-9520), the blot was stained using ECL‐prime (Fisher scientific, 10 308 449) and analysed on an AI‐600 imaging system (GE Healthcare, Life Sciences). Four samples from each group were loaded on gel.

Supplementary Table 1. Body weight and glucose infusion rate (GIR) in all groups.

Body weight (g) Glucose infusion rate (µmol/Kg/min) Intact (Saline infusion) 20.8 ± 1.5 NA Intact (Insulin infusion) 22 ± 0.4 180.2 ± 19.8 PVAT Removed 21.2 ± 0.4 203.8 ± 21.4 Sham 22 ± 0.8 201.7 ± 21.4 PVAT Disconnected 21.8 ± 1.7 215.9 ± 50.8

Control Control No PVAT No PVAT Fold UniProt Symbol Protein Name P-value Mean SD Mean SD increase P27612 Plaa Phospholipase A-2-activating protein 0.0 0.0 1.9 1.2 >10 0.001

Q8BVZ5 Il33 Interleukin-33 0.0 0.0 1.2 1.1 >10 0.013

Q5XKN4 Jagn1 Protein jagunal homolog 1 0.0 0.0 0.8 0.8 >10 0.038

Q8BY89-2 Slc44a2 Choline transporter-like protein 2 0.3 0.5 1.7 1.4 6.7 0.035

Q9D154 Serpinb1a Leukocyte elastase inhibitor A 2.3 3.9 5.2 2.2 2.2 0.042

Q64727 Vcl Vinculin 7.6 2.5 15.2 6.1 2.0 0.020

P02469 Lamb1 Laminin subunit beta-1 5.5 2.6 9.7 2.8 1.8 0.030

P01966 Hba-x Hemoglobin subunit zeta 5.6 1.2 13.0 5.8 2.3 0.014

P15327 Bpgm Bisphosphoglycerate mutase 2.6 1.3 5.6 0.8 2.1 0.035

P01942 Hba-a1 Hemoglobin subunit alpha 86.6 20.2 136.1 38.0 1.6 0.015

Q61316 Hspa4 Heat shock protein 70 kDa 4 1.5 1.8 4.5 2.3 2.9 0.029

P11499 Hsp90ab1 Heat shock protein HSP 90-beta 56.5 6.2 77.3 13.6 1.4 0.008

Q80XI4 Pip4k2b Phosphatidylinositol 5-phosphate 4-kinase type-2 beta 0.0 0.0 0.8 0.8 >10 0.038

P35283 Rab12 Ras-related protein Rab-12 0.3 0.6 1.7 0.4 6.2 0.026

P16332 Mut Methylmalonyl-CoA mutase, mitochondrial 0.3 0.5 2.4 1.7 9.5 0.010

Q9D8S9 Bola1 BolA-like protein 1 0.8 1.0 2.7 1.5 3.5 0.035

Q6PB66 Lrpprc Leucine-rich PPR motif-containing protein, mitochondrial 3.4 3.3 7.2 3.1 2.1 0.047

Q99LY9 Ndufs5 NADH dehydrogenase [ubiquinone] iron-sulfur protein 5 6.8 4.0 13.5 5.1 2.0 0.029

Q9Z1P6 Ndufa7 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 18.3 8.1 29.8 5.6 1.6 0.015

Q9CQZ5 Ndufa6 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6 10.5 2.9 15.5 3.1 1.5 0.046

Q9CQC7 Ndufb4 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4 14.2 2.2 20.2 4.3 1.4 0.035

O88441 Mtx2 Metaxin-2 0.5 0.6 2.6 1.4 5.1 0.013

Q8K0D5 Gfm1 Elongation factor G, mitochondrial 0.0 0.0 1.5 1.0 >10 0.004

Q99KK7 Dpp3 Dipeptidyl peptidase 3 0.0 0.0 1.5 0.9 >10 0.003

P46460 Nsf Vesicle-fusing ATPase 0.0 0.0 1.9 1.1 >10 0.002

Q9ESX5 Dkc1 H/ACA ribonucleoprotein complex subunit 4 0.0 0.0 1.0 0.7 >10 0.020

P57759 Erp29 Endoplasmic reticulum resident protein 29 0.0 0.0 0.9 1.0 >10 0.023

Q99JT9 Adi1 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 0.0 0.0 0.8 0.4 >10 0.037

P62908 Rps3 40S ribosomal protein S3 1.8 1.0 4.6 2.2 2.5 0.029

P58252 Eef2 Elongation factor 2 24.8 2.6 36.4 1.9 1.5 0.002

Q8BL97-3 Srsf7 Serine/arginine-rich splicing factor 7 0.0 0.0 0.8 0.4 >10 0.037

D3Z4S3 Ptrhd1 Putative peptidyl-tRNA hydrolase PTRHD1 0.0 0.0 1.1 0.4 >10 0.011

Q9DCS2 Mettl26 Methyltransferase-like 26 0.0 0.0 0.9 1.0 >10 0.023

Q9D024-2 Ccdc47 Coiled-coil domain-containing protein 47 0.0 0.0 1.0 1.0 >10 0.023

P68254 Ywhaq 14-3-3 protein theta 0.3 0.5 1.7 1.0 6.6 0.027

Q60972 Rbbp4 Histone-binding protein RBBP4 0.3 0.5 1.5 0.9 5.9 0.043

Q3UHX2 Pdap1 28 kDa heat- and acid-stable phosphoprotein 1.1 0.0 3.6 1.5 3.4 0.013

P08074 Cbr2 Carbonyl reductase [NADPH] 2 1.0 2.1 3.4 2.7 3.3 0.049

O08529 Capn2 Calpain-2 catalytic subunit 2.1 2.4 5.5 2.5 2.7 0.032

P56812 Pdcd5 Programmed cell death protein 5 2.2 1.6 5.7 1.2 2.7 0.007

P28653 Bgn Biglycan 3.3 4.0 8.5 4.0 2.5 0.031

Q8BVI4 Qdpr Dihydropteridine reductase 5.2 3.7 11.4 3.9 2.2 0.019

Q9DAK9 Phpt1 14 kDa phosphohistidine phosphatase 7.0 3.7 11.7 1.9 1.7 0.030

Q9R062 Gyg Glycogenin-1 9.0 2.4 14.2 1.8 1.6 0.026

Q99PT1 Arhgdia Rho GDP-dissociation inhibitor 1 9.5 1.6 14.7 2.9 1.6 0.029

Control Control No PVAT No PVAT Fold UniProt Symbol Protein Name P-value Mean SD Mean SD Decrease P50136 Bckdha 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial 1.6 1.3 0.0 0.0 >10 0.004

Q9CRA7 Atp5s ATP synthase subunit s, mitochondrial 1.0 0.8 0.0 0.0 >10 0.009

Q8BYM8 Cars2 Probable cysteine--tRNA ligase, mitochondrial 0.8 1.0 0.0 0.0 >10 0.032

Q8VD26-3 Tmem143 Transmembrane protein 143 0.8 1.0 0.0 0.0 >10 0.033

Q91WU5 As3mt Arsenite methyltransferase 0.8 1.0 0.0 0.0 >10 0.033

Q9DC61 Pmpca Mitochondrial-processing peptidase subunit alpha 2.1 0.8 0.6 0.5 3.6 0.043

P56379 Atp5mpl 6.8 kDa mitochondrial proteolipid 11.8 4.2 5.4 1.7 2.2 0.004

Q3ULD5 Mccc2 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial 10.2 3.5 4.8 1.0 2.1 0.005

Q8R404 2410015M20Rik Protein QIL1 7.8 3.7 3.8 0.8 2.0 0.018

P43023 Cox6a2 Cytochrome c oxidase subunit 6A2, mitochondrial 7.3 3.3 3.9 1.4 1.9 0.033

P99028 Uqcrh Cytochrome b-c1 complex subunit 6, mitochondrial 8.7 1.5 4.6 1.5 1.9 0.018

P56135 Atp5j2 ATP synthase subunit f, mitochondrial 25.0 4.7 15.0 2.5 1.7 0.002

Q7TMF3 Ndufa12 NADH dehydrogenase alpha subcomplex subunit 12 30.1 3.3 19.2 1.3 1.6 0.002

Q8BFR5 Tufm Elongation factor Tu, mitochondrial 25.3 4.3 18.7 3.4 1.4 0.041

Q06185 Atp5k ATP synthase subunit e, mitochondrial 27.9 4.8 20.6 3.6 1.4 0.031

Q9CPQ8 Atp5l ATP synthase subunit g, mitochondrial 21.4 1.5 14.2 2.2 1.5 0.012

P56391 Cox6b1 Cytochrome c oxidase subunit 6B1 29.3 1.8 22.2 1.5 1.3 0.042

Q9D855 Uqcrb Cytochrome b-c1 complex subunit 7 64.8 7.1 50.4 6.3 1.3 0.008

P62897 Cycs Cytochrome c, somatic 94.9 17.0 75.8 7.5 1.3 0.024

Q9DCX2 Atp5h ATP synthase subunit d, mitochondrial 48.7 9.4 39.5 2.5 1.2 0.042

Q9CPQ1 Cox6c Cytochrome c oxidase subunit 6C 76.8 6.2 63.7 5.0 1.2 0.022

Q9R0Y5 Ak1 Adenylate kinase isoenzyme 1 111.2 9.1 89.5 11.5 1.2 0.007

P19536 Cox5b Cytochrome c oxidase subunit 5B, mitochondrial 71.6 11.4 60.5 4.2 1.2 0.048

O88653 Lamtor3 Regulator complex protein LAMTOR3 0.8 0.5 0.0 0.0 >10 0.023

Q9Z2P8 Vamp5 Vesicle-associated membrane protein 5 2.4 1.0 0.6 0.9 4.2 0.022

Q91WS0 Cisd1 CDGSH iron-sulfur domain-containing protein 1 41.8 9.9 29.9 3.0 1.4 0.011 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit P62874 Gnb1 1.0 1.4 0.0 0.0 >10 0.037 beta-1 Q9Z0Y1 Dctn3 Dynactin subunit 3 1.6 1.3 0.2 0.4 8.0 0.027

O08539-2 Bin1 Myc box-dependent-interacting protein 1 42.0 4.0 33.5 2.6 1.3 0.045

P40142 Tkt Transketolase 8.2 2.9 4.0 1.8 2.1 0.017

P14824 Anxa6 Annexin A6 91.5 9.2 73.6 12.7 1.2 0.025

P47199 Cryz Quinone oxidoreductase 1.9 1.0 0.2 0.4 10.2 0.009

P04117 Fabp4 Fatty acid-binding protein, adipocyte 32.6 5.1 19.9 3.4 1.6 0.001

Q8VCT4 Ces1d Carboxylesterase 1D 6.4 2.6 3.3 1.2 1.9 0.038

P82348 Sgcg Gamma-sarcoglycan 1.6 0.6 0.0 0.0 >10 0.001

Q99L88 Sntb1 Beta-1-syntrophin 1.1 0.0 0.0 0.0 >10 0.009

P05787 Krt8 Keratin, type II cytoskeletal 8 1.3 1.0 0.2 0.4 7.3 0.040

Q9JHL1 Slc9a3r2 Na(+)/H(+) exchange regulatory cofactor NHE-RF2 1.3 1.0 0.2 0.4 7.1 0.040

Q3UGC7 Eif3j1 Eukaryotic translation initiation factor 3 1.6 1.8 0.0 0.0 >10 0.009

Q6ZWQ7 Spcs3 Signal peptidase complex subunit 3 0.8 0.5 0.0 0.0 >10 0.023

Q80X50-2 Ubap2l Ubiquitin-associated protein 2-like 0.8 0.5 0.0 0.0 >10 0.023

Q9CQ80 Vps25 Vacuolar protein-sorting-associated protein 25 1.5 1.3 0.2 0.4 8.5 0.026

Q9JJI8 Rpl38 60S ribosomal protein L38 4.2 1.1 0.6 0.9 7.0 0.001

Q9CX56 Psmd8 26S proteasome non-ATPase regulatory subunit 8 1.3 1.0 0.2 0.4 6.6 0.040

Q9CR00 Psmd9 26S proteasome non-ATPase regulatory subunit 9 3.4 2.3 1.0 0.7 3.5 0.020

Q91WQ3 Yars Tyrosine--tRNA ligase, cytoplasmic 6.6 0.8 2.7 1.5 2.4 0.007

P27773 Pdia3 Protein disulfide-isomerase A3 12.9 1.9 7.0 2.4 1.8 0.007

Q9CQC9 Sar1b GTP-binding protein SAR1b 17.0 2.1 11.3 2.2 1.5 0.029

P09103 P4hb Protein disulfide-isomerase 26.9 5.8 18.7 4.0 1.4 0.019

P97443 Smyd1 Histone-lysine N-methyltransferase Smyd1 47.6 5.4 33.9 5.1 1.4 0.003

P62983 Rps27a Ubiquitin-40S ribosomal protein S27a 58.9 4.7 45.7 7.6 1.3 0.010

P03953-2 Cfd Complement factor D 0.8 1.0 0.0 0.0 >10 0.034

P60603 Romo1 Reactive oxygen species modulator 1 1.3 1.0 0.0 0.0 >10 0.005

Q80X50-2 Ubap2l Ubiquitin-associated protein 2-like 0.8 0.5 0.0 0.0 >10 0.023

Q2TPA8 Hsdl2 Hydroxysteroid dehydrogenase-like protein 2 3.7 1.3 1.2 0.5 3.2 0.014

O35215 Ddt D-dopachrome decarboxylase 7.4 1.6 3.4 1.2 2.1 0.012

P37804 Tagln Transgelin 9.7 3.1 5.4 2.2 1.8 0.022 Calcium/calmodulin-dependent protein kinase type II subunit P11798 Camk2a 12.1 2.4 7.6 3.6 1.6 0.036 alpha Q78IK2 Usmg5 Up-regulated during skeletal muscle growth protein 5 12.1 1.0 7.7 1.7 1.6 0.037

Q08642 Padi2 Protein-arginine deiminase type-2 17.0 6.1 11.2 1.7 1.5 0.030

P26883 Fkbp1a Peptidyl-prolyl cis-trans isomerase FKBP1A 21.6 6.3 15.2 2.4 1.4 0.034

P28654 Dcn Decorin 37.9 6.3 28.0 7.4 1.4 0.036

Q3TJD7-2 Pdlim7 PDZ and LIM domain protein 7 56.8 7.3 42.3 8.5 1.3 0.012

©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1066/-/DC1 SUPPLEMENTARY DATA Supplementary figure S1. Weight of PVAT is similar in the right and left hindlimbs. PVAT was removed in either the right or left gracilis muscle as described in methods and subsequently weighed.

©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1066/-/DC1 SUPPLEMENTARY DATA Supplementary figure S2. Direct microvascular connections between PVAT and adjacent muscle determined using confocal laser microscopy. The biopsy was excised from the medial hindlimb and contains the gracilis artery with surrounding PVAT and the underlying muscle. In subsequent Z-stack layers (Images A-J), images with different depths were obtained. Images shown represent the transition from PVAT (positioned left in the image) to the underlying muscle (positioned right in the image; letter M in figure J). mTomato-red is ubiquitously expressed; expression of GFP was induced specifically in endothelial cells using a tamoxifen- inducible VE-Cadherin promoter). A and E (GFP only): Arrows indicate a vascular meshwork surrounding the adipocytes in PVAT. B-C and F-G: the vascular meshwork (arrow) connects with another vessel (dashed arrow) that extends through the PVAT-muscle interface (solid arrow). D and H: The vessel then continues on muscle fibers (solid arrows) to connect with muscle circulation in (*) which are depicted with ( ) in images E and F. mTomato-red is not shown in images I and J because of saturated signal due to high expression in muscle fibers. K: parts of the gracilis muscle that isolated for confocal fluorescence microscopy, indicated by blue rectangles.

©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1066/-/DC1 SUPPLEMENTARY DATA Supplementary figure S5. PVAT removal from skeletal muscle increases protein expression of heat shock protein 90. Methods of PVAT removal described in the Methods section, Western immunoblotting procedures in the Supplementary Methods.

Supplementary movie 1. stacked image of gracilis muscle biopsy with proximal end of the microcirculation of mTmG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) mice with a section of the gracilis resistance artery, its surrounding PVAT and microvascular anastomoses at the PVAT-muscle interface. Blood vessels were identified by VE-Cadherin-Cre-driven endothelial expression of GFP (indicated in green). mTomato Red-positive muscle fibres and PVAT depicted in white.

Supplementary movie 2. 3D image of gracilis muscle biopsy with proximal end of the microcirculation of mTmG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) mice with a section of the gracilis resistance artery, its surrounding PVAT and microvascular anastomoses at the PVAT-muscle interface, obtained using light sheet fluorescence microscopy as described in supplemental methods. Blood vessels were identified by VE-Cadherin- Cre-driven endothelial expression of GFP (indicated in green). mTomato Red-positive muscle fibres and PVAT depicted in white