1 Title Perivascular Adipose Tissue Controls Insulin-Stimulated

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Title

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

Surname first author

Turaihi

Authors & Affiliations Alexander H Turaihi, MD1; Erik H Serné, MD PhD2; Carla FM Molthoff, PhD3; Jasper J Koning, PhD4; Jaco Knol, PhD6; Hans W Niessen, MD PhD5; Marie Jose TH Goumans, PhD7; Erik M van Poelgeest, MD1; John S Yudkin, MD FCRP8; Yvo M Smulders, MD PhD2; Connie R Jimenez, PhD6; Victor WM van Hinsbergh, PhD1; Etto C Eringa, PhD1

Departments of 1Physiology, 2Internal Medicine, 3Radiology & Nuclear Medicine, 4Molecular Cell Biology and Immunology and 5Pathology, Amsterdam Cardiovascular Sciences (ACS), 6Medical Oncology, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, the Netherlands. 7Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. 8University College London, London, UK.

Corresponding author Etto C Eringa, PhD Laboratory for Physiology, VU University Medical Center. Address: O/2 building, 11 W 53, de Boelelaan 1117, 1081 HV Amsterdam, the Netherlands [email protected]

Diabetes Publish Ahead of Print, published online January 31, 2020 Diabetes Page 2 of 41

Manuscript information

Word count abstract: 189

Word count text: 3,996

Reference count: 46

Figure count: 7

Supplemental tables: 3

Supplemental Figures: 5

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Abstract (189 words)

Insulin-mediated microvascular recruitment (IMVR) regulates delivery of insulin and glucose to insulin-sensitive tissues. We have previously proposed that perivascular adipose tissue (PVAT) controls vascular function through outside-to-inside communication and through vessel-to-vessel, or “vasocrine” signaling. However, direct experimental evidence supporting a role of local PVAT in regulating IMVR and insulin sensitivity in vivo is lacking. Here, we studied muscles with and without PVAT in mice using combined contrast-enhanced ultrasonography and intravital microscopy to measure IMVR and gracilis artery (GA) diameter at baseline and during the hyperinsulinemic-euglycemic clamp. We show, using microsurgical removal of PVAT from the muscle microcirculation, that local PVAT depots regulate insulin-stimulated muscle perfusion and glucose uptake in vivo. We discovered direct microvascular connections between PVAT and the distal muscle microcirculation, or adipomuscular arterioles, removal of which abolished IMVR. Local removal of intramuscular PVAT altered protein clusters in the connected muscle, including upregulation of a cluster featuring heat shock proteins 90ab1 and 70 and downregulation of a cluster of mitochondrial protein components of complexes III, IV and V. These data highlight the importance of PVAT in vascular and metabolic physiology, and are likely relevant for obesity and diabetes.

Keywords: Insulin sensitivity; Insulin; Adipose Tissue; Muscle; Microcirculation; Endothelium; Microscopy.

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Introduction The diameter of skeletal muscle resistance arteries and muscle perfusion is regulated by a complex interplay of hemodynamic variables, circulating hormones, the autonomic nervous system and local factors. Perivascular adipose tissue (PVAT), adipose tissue surrounding most peripheral arteries with an internal diameter >100 μm(43), are a source of adipokines within organs that has been proposed to locally regulate vascular function(11, 25) and muscle insulin sensitivity(45). After a meal, a rise in insulin induces dilation of resistance arteries within 10 minutes, a mechanism that regulates microvascular muscle perfusion(8, 41). As a result, insulin increases microvascular blood volume (MBV), i.e. Insulin-induced MicroVascular Recruitment (IMVR) in skeletal muscle(2), the primary target site for insulin-stimulated postprandial glucose uptake(7). IMVR expands the endothelial surface area in direct contact with blood, facilitating extraction of glucose and insulin into the muscle interstitium(2). IMVR relies on endothelium-dependent vasodilation(42), primarily relaxation of pre-capillary arterioles(2, 36). These endothelial effects of insulin have been shown to control ~50 percent of muscle insulin sensitivity(19). Perivascular adipose tissue secretes vasodilator hormones such as adiponectin(4, 23) and vasoconstrictor adipokines such as fatty acid-binding protein (A-FABP)(10, 34). PVAT of healthy, lean humans and mice antagonizes sympathetic tone(11) and stimulates insulin-induced vasodilatation in isolated resistance arteries(23, 25). Loss of these vasodilator effects is a hallmark of PVAT dysfunction in human insulin resistance and type diabetes(11, 25, 34). Vasodilator effects of PVAT are dependent on adiponectin(11, 23), and decreased adiponectin secretion is characteristic of PVAT in type 2 diabetes(24). Adiponectin regulates insulin sensitivity(44) and muscle perfusion(6), and we have proposed that impaired cross-talk between PVAT and microvascular endothelium predisposes to type 2 diabetes and cardiovascular disease(45). As PVAT is not present around the precapillary arterioles regulating IMVR, this proposed relationship includes proximal-to-distal transfer of adipokines, or vasocrine signaling, within microvascular beds. In the current study we investigated in vivo whether PVAT around larger resistance arteries directly communicates with proximal and distal muscle microvessels to control local vasomotor function, perfusion and glucose uptake in muscle, independently from whole-body metabolism. Using a microsurgical approach in mice, we tested this hypothesis by evaluating the effect of physical separation of local PVAT from muscle blood vessels on muscle perfusion. Hereafter, we repeated the measurements after surgically severing the connections between PVAT and the adjacent muscle, leaving PVAT attached to the resistance artery in situ. to assess whether

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proximal PVAT is directly connected to the distal muscle microcirculation, we studied the microvascular anatomy at the interface between PVAT and the muscle using light and fluorescence microscopy. Our study provides evidence for a role of PVAT in vivo in insulin- induced vasodilatation, local regulation of IMVR and skeletal muscle glucose uptake in vivo. We describe previously unrecognized adipomuscular microvessels that directly transfer PVAT- derived signals to the distal muscle microcirculation, regulating microvascular blood content. Finally, removal of PVAT from healthy muscle induces changes in muscle protein expression that have been shown to contribute to diet-induced insulin resistance.

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Methods Animals

Animal experiments were performed in accordance with the European Community Council Directive 2010/63/EU for laboratory animal care and the Dutch Law on animal experimentation. The experimental protocol was approved by the local committee on animal experimentation of the VU University. In functional assays, we used C57Bl/6 mice (male, age 8 weeks, Charles River International Inc, Sulzfeld, Germany). VeCadherin-CreERT2;mTmG mice were generated by crossing VeCadherin-Cre ERT2 (Tg(Cdh5-cre/ERT2)CIVE23Mlia)(26) and mTmG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo)(27). GFP expression was induced by a single injection of tamoxifen, 24 hours before sacrifice.

Surgical procedures

For manipulation of PVAT in vivo, a 2 cm skin incision was made under sevoflurane anesthesia parallel to the inguinal ligament. Four groups were studied: a group where PVAT was removed (PVAT-removed, figure 3B, n=6), skin and released deep fascia were incised without removing PVAT (Sham, n=5), a group where PVAT was separated from the underlying muscle while leaving it attached to the resistance artery (PVAT disconnect, figure 3C) and a group without surgery (PVAT intact, figure 3A, n=13). The experimental design is shown in figure 1. All mice tolerated the surgical procedures well, with no loss of mice during surgery or in the post-operative period. No PVAT was observed in the operated area two weeks after PVAT removal (Figure 3B). The weight of PVAT was comparable in both hindlimbs (supplementary figure S1A) and body weights were similar between mice in all experimental groups (supplementary table 1).

Hyperinsulinemic euglycemic clamp

Insulin sensitivity was evaluated after an overnight fast using the hyperinsulinemic-euglycemic clamp as described(41), using an insulin infusion rate of 7.5 mU/kg/min) for 60 minutes.

Contrast-enhanced ultrasonography of the muscle microcirculation

Muscle perfusion in the thigh muscles was determined using Contrast-Enhanced Ultrasonography (CEUS) as described(41).

Determination of skeletal muscle glucose uptake

Local muscle glucose uptake was determined by PET-CT scanning of uptake of 18F-2-fluoro-2- deoxy-D-glucose (18FDG) (Cyclotron VU, Amsterdam, The Netherlands). In six mice, we removed

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PVAT in one hindlimb (N=3 in the right hindlimb, N=3 in the left hindlimb) and used Sham surgery in the contralateral hindlimb as an internal control.

During PET-CT (Mediso nanoPET-CT, Budapest, Hungary) a computed tomography (CT) scan was performed for 6 min. After 15 minutes of hyperinsulinemia, 18FDG (7 MBq) was administered i.v. and a dynamic emission scan of 1 hour was performed. PET data were normalized, and corrected for scatter, randoms, attenuation, decay and dead time as described(3). After PET scanning, hindlimb muscle and blood were obtained for determination of radioactivity in blood and muscle(3).

PET data were analyzed using AMIDE software (A Medical Image Data Examiner, version 0.9.2)(22) and fixed size ellipsoidal shaped regions of interest (ROI) (dimensions: 4x4x4mm3) were manually drawn over predetermined areas of the left and right medial upper hindlimb in the last frame of the image (three regions of the upper hindlimb, medial to the femur: proximal, middle, distal corresponding to three different blood supplies to these regions). ROIs were projected onto the dynamic image sequence, and time-activity curve (TAC) data were extracted. TACs were expressed as standardized uptake values (SUV): mean ROI radioactivity concentration normalized for injected dose and body weight.

Assessment of PVAT vasodilator function ex vivo

The effect of PVAT from intact (n=14) and sham-operated muscles (n=5) on insulin-induced vasodilation was analyzed ex vivo as described(24) in gracilis resistance arteries of PVAT- removed mice .

Measurement of PVAT adiponectin secretion

Adiponectin secretion by intramuscular PVAT was measured as described(23) and corrected for PVAT weight.

Fluorescence microscopy of the PVAT microvasculature

Hindlimb muscle of VeCadherin-CreERT2;mTmG mice was collected and fixed for imaging. Scanning laser microscopy was performed on a Nikon A1R+ (Nikon Instruments). Green Fluorescent Protein (GFP) and mTomatoRed were irradiated at 488 and 561 nm respectively. A 20x air objective with NA0.5 aperture was used to image the sample. Detection of the fluorescent signal was performed with GaAsP PMTs.

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For light sheet fluorescence microscopy, muscle tissue was prepared according to the uDisco protocol(30). An automated surface was applied using the ‘surface’ tool to segment the resistance artery.

Mass spectrometry-based proteomics

Protein expression in hindlimb muscle tissue of four animals of the Sham group, and five animals of the Removed group was analysed using mass spectrometry as described(33). Gel lanes were divided into 5 slices and peptides were extracted for analysis by nanoLC-MS/MS on a Q Exactive mass spectrometer (ThermoFisher, Bremen, Germany). LC-MS/MS, raw data processing and database searching were performed as described(5, 21, 39). MS/MS spectra were searched against a Swissprot Mus musculus reference proteome FASTA file with canonical proteins and isoforms (release August 2017, 25052 entries).

Statistics and bioinformatics

Data are presented as mean±standard deviation (SD). Differences between PVAT Removed, Sham and Intact were determined by one-way ANOVA with Tukey post-hoc correction. For comparison between Removed and Sham legs within the same mouse, a paired t-test was performed. Analyses were done using Graphpad Prism 6.04 (GraphPad Software Inc., San Diego, CA, USA). PET images were analyzed using AMIDE software (Amide's a Medical Image Data Examiner, version 1.0.1, (22)). Differences were considered significant at p<0.05.

For proteomics, differential analysis of normalized spectral counts for identified proteins was performed using the beta-binominal test, which takes into account within- and between-sample variation(32). Protein-protein association data were retrieved from STRING version 10(40) and visualized as networks in Cytoscape version 3.5.1(37). Heatmaps with hierarchical clustering (Euclidean distance, complete linkage) were generated in R version 3.4.3, using the complex Heatmap package version 1.17.1(12).

Data availability

The mass spectrometry data for this publication have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository (www.ebi.ac.uk/pride/archive) and assigned the identifier PXD011179.

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Results

Local removal of perivascular adipose tissue impairs insulin-stimulated vasodilatation and microvascular perfusion in muscle To assess the role of perivascular adipose tissue in regulation of insulin-stimulated muscle perfusion in vivo, we unilaterally removed PVAT from the gracilis muscle resistance artery and vein and the proximal part of the femoral artery (figures 3A and 3B). First, we verified the vasodilator properties of PVAT ex vivo by pressure myography. PVAT uncovered insulin- stimulated vasodilatation in gracilis resistance arteries ex vivo, and microsurgery did not affect this vasodilator effect of PVAT (figure 2). Insulin [1 nM] increased the GA diameter in the presence of PVAT (mean±SD 26±25 %; N=14; p<0.0001) but not in absence of PVAT (mean±SD 0±12 %, N=9). PVAT from the PVAT Removed and Sham groups induced a similar vasodilator effect of insulin (20.2±7%, N=14, p=0.64 vs. control PVAT; figure 2). We examined adiponectin secretion by intramuscular PVAT by measuring adiponectin in PVAT-conditioned medium. In line with previous data showing PVAT is an intramuscular source of vasodilator adipokines, we found abundant adiponectin in PVAT-conditioned medium but no differences between control and insulin-stimulated conditions (mean±SEM 2750±347 pg/ml/mg vs. 2608±306 pg/ml/mg, p=0.7). After confirming adiponectin secretion, vasodilator properties of PVAT and lack of lasting effects of microsurgery thereon, we evaluated effects of PVAT manipulation on the muscle microcirculation in vivo using intravital microscopy (IVM) and contrast ultrasonography (CEUS). We determined resistance artery diameters (figure 3A) before and during hyperinsulinemia in the PVAT-removed and sham-operated hindlimbs (Sham) and compared it to those of mice without any intervention (PVAT intact; figure 3). At baseline, resistance artery diameters were comparable between groups (Figure 3D). In contrast, removal of PVAT fully inhibited insulin-induced vasodilatation of gracilis resistance arteries (figure 3F). After 30 minutes of hyperinsulinemia, insulin increased GA diameter of Intact (14±2% vs. -6±4% in saline-infused mice) and Sham (12±3%) hindlimbs, but not in the PVAT-removed group (1±2%). These differences persisted through 60 minutes of hyperinsulinemia, as insulin increased GA diameter of the Intact (15±2%) and Sham (19±4%) when compared to the PVAT-Removed (2±2 %) and control mice that received saline (-6±2%, p=0.001; Figure 3F). To test whether proximally located PVAT controls distal microvascular recruitment in muscle in response to insulin, we used contrast ultrasonography (CEUS). At baseline, microvascular blood volume (MBV) in muscle was similar between groups (Figure 3E). Similar to

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its effects on proximal muscle resistance arteries, the presence of PVAT determined insulin- mediated microvascular recruitment in muscle. In the Sham group insulin increased microvascular blood volume by 44±10%, comparable to the PVAT intact group (36±12%) and different from mice receiving saline infusion (-11±11%). In the PVAT removed group, IMVR was abrogated (-5±7%) (PVAT-Removed vs. Sham and Intact p=0.007; Figure 3G). PVAT removal from muscle was not accompanied by a change in structural capillary density, which was similar between muscles of sham-operated and PVAT-removed hindlimbs (mean+SEM in sham vs. PVAT removed 539+22 vs. 556+38 capillaries/mm2, n=5 per group, P=0.7). In summary, removal of local PVAT from hindlimb muscle specifically abrogated insulin- induced vasodilatation in proximal resistance arteries and insulin-mediated microvascular recruitment in the distal muscle microcirculation. Baseline resistance artery diameter, capillary density and perfusion were not dependent on the presence of PVAT.

Direct arteriolar connections between PVAT and adjacent muscle

Using a second microsurgical approach, we unexpectedly discovered that anatomical connections between proximal PVAT and the adjacent muscle are critical to the effect of PVAT on IMVR in the distal capillaries. In 6 mice, we severed the connections between PVAT and the underlying muscle (PVAT disconnect group, figure 3C). In these mice the local vasodilator response to insulin in the GA was preserved (20±3%; figure 3F), yet IMVR was fully inhibited (- 2±7%), similar to the PVAT-Removed group (Figure 3G). As we observed microbleeds at the interface between PVAT and muscle during the disconnection of PVAT from adjacent muscle, we hypothesized that the effect of PVAT on distal IMVR is mediated by direct microvascular connections between PVAT and muscle (adipomuscular arterioles). In line with this, we observed small branches of the resistance artery, extending through the PVAT into the underlying muscle (Figure 4A). To examine the microvascular connections between PVAT and muscle in detail, we generated reporter mice expressing GFP in vascular endothelium (see Methods). Using this technique, we found GFP- positive microvessels surrounding the perivascular adipocytes and capillaries running in parallel to muscle fibers were readily visible (Supplementary figure S2). When visualized in 3D, vascular structures extending from the adipocytes within PVAT to muscle fibers were seen (Figure 4B-D, supplementary figure S2 and supplementary movies 1 and 2). These adipomuscular arterioles were 20-40 microns in diameter and appeared to come together proximal to the PVAT-muscle interface and muscle fibers.

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PVAT removal from muscle blood vessels reduces muscle glucose uptake After observing that PVAT controls insulin-induced vasodilatation and muscle IMVR in vivo, we investigated whether these regulatory effects of PVAT contribute to local insulin-stimulated glucose uptake in muscle. To this end, we compared the rate of glucose uptake in skeletal muscle between the PVAT Removed and Sham hindlimbs using positron emission tomography with 18F- deoxyglucose (18FDG). Whole-body insulin sensitivity, measured as the glucose infusion rate (GIRs) during the hyperinsulinemic-euglycemic clamp, was comparable between the Intact (180±20 µmol/Kg/min), PVAT Removed (204±10 µmol/kg/min), PVAT Disconnect groups (216±51 µmol/Kg/min) and Sham (216±23 µmol/Kg/min).

In contrast to whole-body insulin sensitivity, local uptake of 18FDG in PVAT Removed hindlimbs was markedly decreased compared to Sham hindlimbs (figure 5). In muscle proximal to the area where PVAT was removed, there was no significant difference in 18FDG uptake between the sham-operated hindlimb and PVAT-removed hindlimb (mean difference at 45 minutes after 18FDG injection +SD -20+36%, P=0.54; fig 5A). In the central area around the surgery site corresponding to the region perfused by the GA, 18FDG was decreased by 38+28% and 23+18% at 45 and 60 minutes after 18FDG injection, respectively (mean+SD; p=0.009 and p=0.04, N=5; fig 5B). In the distal ROIs corresponding to the area perfused by small arteries branching from the saphenous artery, 18FDG uptake was reduced in the PVAT-removed hindlimb after 60 minutes (-20+12%; P=0.04; fig 5C). After the hyperinsulinemic clamp, total 18FDG content in proximal muscle biopsies taken from PVAT-Removed and Sham hindlimbs was determined. In these biopsies, 18FDG content was similar between muscle from PVAT-Removed and Sham hindlimbs (2.0±0.2 vs. 2.1±0.2%ID/g, p=0.58).

PVAT removal from muscle downregulates a cluster of protein components of mitochondrial complexes III, IV and V

To gain insight into the mechanisms underlying the effects of PVAT removal on muscle physiology, we examined protein expression by mass spectrometry-based proteomics. In hindlimb muscle tissue a total of 1720 proteins were identified with at least one MS/MS spectrum (raw MaxQuant protein groups export in Supplementary Tables 2 and 3). Forty-five proteins were significantly upregulated (P<0.05) in the PVAT Removed group (figure 6A) and 63 proteins were significantly downregulated compared to the Sham group (figure 6B). Abundance of proteins was

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also visualized with heat maps (supplementary figures S3 and S4). As expected, the adipocyte proteins fatty acid binding proteins 4 and 9 were reduced in PVAT-removed muscles.

A major protein in the up-regulated network (figure 6A) was Hsp90ab1, which showed a moderate fold change of 1.4. Proteins associated with HSP90ab1 included proteins linked to vesicular trafficking (Rab12, Nsf, Plaa, Pip4kb2, Hspa4, Vcl) albeit that several of these were detected at very low level. In addition, the cytoskeletal proteins Vcl (vinculin) and Arhgdia, a regulator of Rho GTPases such as Rac1 and Cdc42, and Capn2 (calpain 2), were also upregulated. Some of the upregulated proteins can also be involved in cellular stress and immune responses (Hsp90ab1, Hsp70 family member Hspa4, Ywhaq/14-3-3 protein theta, Il33, Serpinb1a). The effect of PVAT removal on expression of the most abundant upregulated muscle protein, HSP90, was confirmed by Western blotting (supplementary figure S5).

Several upregulated proteins have a mitochondrial localization (respiratory complex I components Ndufb4, Ndufs5, Ndufa6, Ndufa7; Cbr2, Qdpr, Gfm1, Mtx2, Mut, Bola1, Lrpprc). Surprisingly, three proteins involved in erythrocyte development (Hba-x, Hba-a1, Bpgm) showed ~twofold upregulation in muscle deprived of PVAT (figure 4A), although total Hb was not affected by PVAT removal. The up-regulated Gyg/glycogenin protein is a primer for glycogen synthesis.

The major cluster of downregulated proteins following PVAT removal involved a different set of mitochondrial proteins, including respiratory complex III, complex IV and complex V (ATP synthase) components (figure 6B). Furthermore, PVAT removal reduced abundance of three proteins involved in regulation of glucose metabolism: the regulator complex protein LAMTOR3, the vesicle-associated membrane protein Vamp5 that regulates trafficking of Glut4 in insulin- treated cells(35) (4-fold down-regulation, P=0.02), and Cisd1/CDGSH iron-sulfur domain- containing protein 1 (1.4-fold down-regulation, P=0.01), which binds Pioglitazone(29).

In summary, removal of local PVAT from the proximal end of the muscle microvascular bed induced changed the protein composition of the muscles connected, highlighted by upregulation of protein components of mitochondrial complex I, downregulation of a cluster of protein components of mitochondrial complexes III, IV and V and downregulation of Vamp5.

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Discussion

Here, we demonstrate a role of PVAT in regulation of muscle perfusion, protein expression and glucose uptake in vivo. We present three main findings (figure 7): First, PVAT in muscle is required for insulin-stimulated vasodilatation and muscle IMVR. Second, the vasoregulatory function of PVAT is mediated by vascular connections extending from the larger arterioles to the underlying muscle through PVAT. Third, removal of normal PVAT reduces insulin-stimulated skeletal muscle glucose uptake in vivo and changes muscle protein expression, downregulating a cluster of mitochondrial proteins. Insulin controls NO production through phosphatidylinositol-3 kinase (PI3K) and Akt(1) and endothelin release through extracellular signal-regulated kinase 1/2(6). We have shown here that muscle resistance arteries do not dilate in response to insulin in the absence of PVAT. These data extend earlier in vitro evidence for vasodilator effects of PVAT(23) and figure 2). While this vasodilator effect critically depends on adiponectin in vitro(23), the presence of blood-borne adiponectin in the vascular lumen within muscles without PVAT in vivo is insufficient to permit insulin-induced vasodilatation (fig 3F), As such, locally rather than systemically generated adiponectin determines insulin-stimulated vasodilatation in muscle. We did not observe differences in basal muscle MBV nor basal resistance artery diameter between the PVAT Removed, PVAT disconnected, Sham, PVAT intact groups (figure 3D). This suggests that PVAT does not control basal tone of resistance arteries in vivo but specifically controls arteriolar responses to insulin. In support, isolated muscle resistance arteries also showed no differences in basal tone or general endothelium-dependent vasodilatation whether the artery was incubated with PVAT or not.

Role of adipomuscular arterioles in vasocrine signaling A mechanism we have previously proposed to mediate a regulatory action of PVAT is vasocrine, or vessel-to-vessel, signaling(45). As originally envisioned, PVAT-derived adipokines were secreted into the arteriolar lumen to dilate distal arterioles in response to insulin. Our present data support vasocrine signaling by PVAT, yet suggest a more elegant mechanism. Instead of adipokine secretion into the arteriolar lumen and subsequent dilution in plasma before reaching terminal arterioles, adipomuscular arterioles enable PVAT-derived adipokines to reach the muscle tissue directly (fig 4). These microvessels may converge with the precapillary muscle arterioles, functioning primarily as arteriolar-capillary connections that deliver adipokines.

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To test the function of the adipomuscular arterioles, we severed these microvessels by separating the outer surface of PVAT from the attached muscle tissue, while leaving the PVAT attached to the gracilis artery (PVAT-disconnect, figure 3C). This intervention abrogated IMVR in the distal muscle microcirculation (figure 3G), but preserved proximal insulin-induced vasodilation (figure 3E), arguing that the paracrine effect of PVAT is still intact. The discrepancy between the effects of insulin on the proximal and distal parts of the muscle microcirculation after PVAT disconnect surgery supports a functional role for adipomuscular arterioles in vasocrine signaling by PVAT.

PVAT regulation of insulin sensitivity and muscle protein expression Impairment of insulin-induced vasodilation and IMVR are commonly observed in insulin resistance and type 2 diabetes(9,20), reducing postprandial insulin delivery to insulin-sensitive tissues. We have shown that removing PVAT from muscle microvessels blunts both IMVR and muscle glucose uptake in vivo. The decrease in insulin-induced glucose uptake was evident in the central and distal ROIs drawn in the hindlimb corresponding to the vascular regions of the GA and distal small arteries from the saphenous artery(16). Total hindlimb muscle glucose uptake was not significantly different between muscles with and without PVAT, which may be explained by the fact that biopsies were taken from a muscle region supplied by the proximal deep profunda artery(16) that was not affected by the surgery. Indeed, several collateral routes exist in the innate muscle vasculature (13, 46). Taken together, local PVAT depots surrounding muscle resistance arteries have a crucial role in IMVR and when absent, insulin-induced muscle glucose uptake is decreased. The differences in skeletal muscle glucose reported in this study cannot be explained by systemic changes in insulin secretion or liver insulin sensitivity. Removal of PVAT induced changes in myocellular protein expression typical of insulin resistance, particularly decreased expression of mitochondrial electron transport chain components (figure 6B). Mitochondrial dysfunction is a hallmark of muscle insulin resistance in type 2 diabetes(14) and decreased expression of cytochrome C and ATPase has been observed in muscles of insulin-resistant human subjects(31) and mice(38). Furthermore, mitochondrial function has been shown to be regulated by adiponectin and AdipoR1 receptors(17,28). Heat shock protein 90ab1 (HSP90β), which was upregulated in muscles after PVAT removal (fig 6A), is also upregulated in diet-induced insulin resistance(15) reduces muscle mitochondrial function and glucose uptake(15). These data are consistent with a model wherein intramuscular PVAT regulates muscle glucose metabolism by secreting adiponectin, reducing HSP expression and increasing expression of mitochondrial proteins. In health, this regulation is independent from

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oxidative stress, as expression of oxidative stress markers such as superoxide dismutase and catalase was normal in muscle without PVAT. These metabolic effects of PVAT add to the growing body of evidence on endocrine effects of adipose tissue in the pathogenesis of type 2 diabetes. While intra-abdominal adipose tissue has established roles in liver insulin resistance and impaired insulin secretion(18, 34), our data suggest that paracrine signaling by intramuscular PVAT contributes to regulation of muscle insulin sensitivity, in addition to endocrine regulation by circulating adipokines.

Study limitations A few limitations of our study should be considered. It is difficult to standardize the amount of PVAT removed by different investigators, and the surgery itself could have influenced local perfusion and metabolism through inflammation. To control for effects of mechanical trauma, we applied sham surgery to the contralateral hindlimb (figure 3). In addition, we used a 2-week recovery period between surgery and measurements. Finally we tested GA from the PVAT Removed group ex vivo in the pressure myograph to validate preserved PVAT function and found normal vasodilation in response to insulin (figure 2) and acetylcholine. Absence of a chronic inflammatory response after microsurgery is supported by protein expression of the inflammatory proteins Macrophage migration inhibitory factor 1 (MIF-1), the mast cell markers chymase and mast cell carboxypeptidase A and the stress protein MAPK14, which were all similar between muscles of sham and PVAT-removed hindlimbs. Classic inflammatory mediators such as TNFα, IL-6, IL-1β and MCP-1 were undetectable in the muscles studied. The critical products of PVAT mediating local actions in vivo as well as their targets remain to be determined, as nonsurgical interventions to alter PVAT function still have to be developed. As a final limitation, it should be noted that insulin was the only vasoactive agent used and effects of PVAT on responses to other vasoactive agents in vivo are uncertain.

In conclusion, our data demonstrate a local regulatory role for PVAT in muscle perfusion, mitochondrial function and glucose metabolism, mediated by dedicated microvascular connections. These data open new avenues for treatment of type 2 diabetes and the associated organ failure.

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Acknowledgments E.C.E. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. We are grateful to Elisa Meinster, Nanne Paauw, Jeroen Kole, Mariska Verlaan, Ricardo Vos and Zeineb Gam for expert technical assistance. This work was supported by the Netherlands Organization for Scientific Research (VIDI grant 917.133.72). We thank prof A.K. Groen (Dept. of Internal Medicine, Amsterdam UMC, Amsterdam, the Netherlands) for valuable discussion of our manuscript. None of the authors has a conflict of interest to declare. Part of the data were presented as an oral presentation at the Scientific Sessions of the American Diabetes Association in June 2016 and as a poster at the Scientific Sessions of the American Diabetes Association in June 2018.

Author contributions

A.H.T wrote the manuscript, researched and analyzed data. J.J.K. analyzed data, and edited the manuscript. Y.M.S., V.W.M.v.H., and E.H.S. supervised, contributed to discussions, and edited the manuscript. C.F.M.M., M.J.G., J.S.Y., E.v.P., H.W.N. contributed to discussions and edited the manuscript. E.C.E. supervised, contributed to discussions, and edited the manuscript. E.C.E. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Legends

Figure 1. Study protocol. Microsurgical manipulation of muscle PVAT was performed at day 0, followed by a 2-week recovery period before in vivo and ex vivo phenotyping of muscles with and without PVAT. IVM, intravital microscopy; CEUS, contrast-enhanced ultrasound; HEC, hyperinsulinemic-euglycemic clamp; 18FDG PET, 18Fluoro-deoxyglucose positron emission tomography.

Figure 2. Ex vivo characterization of muscle PVAT. Muscle PVAT triggered insulin-stimulated vasodilation, which was similar between PVAT from control muscles and muscles exposed to sham surgery. *P<0.05, **P<0.01.

Figure 3. Removal of healthy perivascular adipose tissue specifically impairs insulin- induced vasodilatation and microvascular recruitment in muscle. A-C: gross appearance of the proximal gracilis muscle vasculature after sham (A), PVAT removal (B) and PVAT disconnect surgery (C). In panel A, “P” indicates PVAT, “V” the muscle venule and “A” the muscle resistance artery. Intravital microscopy of the muscle resistance artery was performed on the artery segment indicated by the blue rectangle. In panel C, the blue arrows indicate the locations of the surgical cuts. D and E: Basal resistance artery diameter (D) and microvascular blood volume (E) in muscle were not affected by surgical removal of PVAT, nor by microsurgical disconnection of PVAT from adjacent muscle. F and G: effects of PVAT manipulation on effects of insulin’s microvascular actions in vivo (infusion rate 7.5 mU/kg/min) in the proximal and distal muscle microcirculation. F: PVAT removal, but not separation from adjacent muscle (disconnect) abolishes insulin-induced vasodilatation of the proximal gracilis artery in vivo. G: Both PVAT removal and disconnection from adjacent muscle abolish insulin-stimulated distal microvascular recruitment in muscle. All data are represented as mean±SD and were tested with one-way ANOVA with Tukey post-hoc analysis. *P<0.05.

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mTomato Red-positive muscle fibers and PVAT depicted in white. C and D: 3-dimensional visualization of adipomuscular arterioles determined by light sheet fluorescence microscopy. Stacked light sheet fluorescence microscopy image (left) of a cleared biopsy from the medial hindlimb (segment shown in online supplement) showing microvessels connecting PVAT around the gracilis artery to the adjacent muscle. The enlarged segment in panel D is indicated by the blue rectangle. D Segmented stacked image (right) shows the gracilis artery in green, PVAT and muscle in white.

Figure 5. PVAT removal decreases insulin-induced muscle glucose uptake in vivo. Right panel: Representative coronal PET images of 18FDG in mice (60 minutes after injection). A-C: of 18FDG between the sham-operated and PVAT-removed hindlimb (mean+SD; N=5). In off line analysis, three circular regions of interest (ROI’s, 16 mm2 each) were determined a priori and drawn on each hindlimb medial to the femur: Proximal (starting distal to the bladder), middle and distal (proximal to the knee). Panels A-C: differences in Standardized Uptake Values (ΔSUV) of 18FDG between the PVAT-Removed hindlimb and Sham hindlimb, expressed as percentage of uptake in the sham-operated hindlimb. Data were tested using paired t-test; * p=0.04, ** p=0.009.

Figure 6. Protein network visualization of PVAT-regulated proteins in muscles with and without PVAT. A: Proteins with significantly increased abundance after removal of local PVAT from muscle compared to sham-operated muscle. Node colors indicate fold increase in muscle without PVAT, and node sizes are scaled to protein abundance as deduced from the mean spectral count in the Removed group. B: Proteins significantly decreased in muscle after PVAT removal, with node sizes scaled to protein abundance in the Sham group. Protein-protein associations were obtained from the STRING database.

Figure 7. Summary of key findings. Local PVAT regulates muscle perfusion and glucose uptake in vivo through vessel-to-vessel signaling. PVAT communicates locally with arteries via paracrine signaling (blue dashed arrows) and with the distal microcirculation via adipomuscular arterioles (indicated by blue circles).

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20. Kusters YHAM, Schalkwijk CG, Houben AJHM, Kooi ME, Lindeboom L, Op ’t Roodt J, Joris PJ, Plat J, Mensink RP, Barrett EJ, Stehouwer CDA. Independent tissue contributors to obesity-associated insulin resistance. JCI Insight 2: 2017. 21. Liu H, Sadygov RG and Yates JR, 3rd. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Analytical chemistry 76: 4193-4201, 2004. 22. Loening AM and Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2: 131-137, 2003. 23. Meijer RI, Bakker W, Alta CL, Sipkema P, Yudkin JS, Viollet B, Richter EA, Smulders YM, van H, V, Serne EH, Eringa EC. Perivascular Adipose Tissue Control of Insulin-Induced Vasoreactivity in Muscle Is Impaired in db/db Mice. Diabetes 62: 590-598, 2013. 24. Meijer RI, Bakker W, Alta CL, Sipkema P, Yudkin JS, Viollet B, Richter EA, Smulders YM, van Hinsbergh VW, Serne EH, Eringa EC. Perivascular adipose tissue control of insulin-induced vasoreactivity in muscle is impaired in db/db mice. Diabetes 62: 590-598, 2013. 25. Meijer RI, Serne EH, Korkmaz HI, van der Peet DL, de Boer MP, Niessen HW, van Hinsbergh VW, Yudkin JS, Smulders YM, Eringa EC. Insulin-induced changes in skeletal muscle microvascular perfusion are dependent upon perivascular adipose tissue in women. Diabetologia 58: 1907-1915, 2015. 26. Monvoisin A, Alva JA, Hofmann JJ, Zovein AC, Lane TF, Iruela-Arispe ML. VE-cadherin- CreERT2 transgenic mouse: a model for inducible recombination in the endothelium. Developmental dynamics : an official publication of the American Association of Anatomists 235: 3413-3422, 2006. 27. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis 45: 593-605, 2007. 28. Okada-Iwabu M, Yamauchi T, Iwabu M, Honma T, Hamagami K, Matsuda K, Yamaguchi M, Tanabe H, Kimura-Someya T, Shirouzu M, Ogata H, Tokuyama K, Ueki K, Nagano T, Tanaka A, Yokoyama S, Kadowaki T. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature 503: 493-499, 2013. 29. Paddock ML, Wiley SE, Axelrod HL, Cohen AE, Roy M, Abresch EC, Capraro D, Murphy AN, Nechushtai R, Dixon JE, Jennings PA. MitoNEET is a uniquely folded 2Fe–2S outer mitochondrial membrane protein stabilized by pioglitazone. Proceedings of the National Academy of Sciences of the United States of America 104: 14342-14347, 2007. 30. Pan C, Cai R, Quacquarelli FP, Gasemigharagoz A, Erturk A. Whole organ and organism tissue clearing by uDISCO. 2016.

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Figure 3. Removal of healthy perivascular adipose tissue specifically impairs insulin-induced vasodilatation and microvascular recruitment in muscle. A-C: gross appearance of the proximal gracilis muscle vasculature after sham (A), PVAT removal (B) and PVAT disconnect surgery (C). The blue arrows in figure 3C indicate the locations of the surgical cuts. D and E: Basal resistance artery diameter (D) and microvascular blood volume (E) in muscle are not affected by surgical removal of PVAT, nor by microsurgical disconnection of PVAT from adjacent muscle. F and G: effects of PVAT manipulation on effects of insulin’s microvascular actions in vivo (infusion rate 7.5 mU/kg/min) in the proximal and distal muscle microcirculation. F: PVAT removal, but not separation from adjacent muscle (disconnect) abolishes insulin-induced vasodilatation of the proximal gracilis artery in vivo. G: Both PVAT removal and disconnection from adjacent muscle abolish insulin-stimulated distal microvascular recruitment in muscle. All data are represented as mean±SD and were tested with one-way ANOVA with Tukey post-hoc analysis. *P<0.05.

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Figure 4. Arteriolar connections between perivascular adipose tissue and adjacent muscle. A: adipomuscular arteriole between PVAT and muscle observed using light microscopy. Arrows indicate the arteriole connecting the gracilis artery (GA) and the adjacent muscle (M) in the medial hindlimb. Large-magnification images are zoomed in in the lower panel. B: 2-Dimensional image of the PVAT-muscle interface of the gracilis muscle with adipomuscular arterioles (blue arrows), identified by VE-Cadherin-Cre-driven endothelial expression of GFP (indicated in green). mTomato Red-positive muscle fibers and PVAT depicted in white. C and D: 3-dimensional visualization of adipomuscular arterioles determined by light sheet fluorescence microscopy. Stacked light sheet fluorescence microscopy image (left) of a cleared biopsy from the medial hindlimb (segment shown in online supplement) showing microvessels connecting PVAT around the gracilis artery to the adjacent muscle. The enlarged segment in panel D is indicated by the blue rectangle. D Segmented stacked image (right) shows the gracilis artery in green, PVAT and muscle in white.

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Figure 6. Protein network visualization of PVAT-regulated proteins in muscles with and without PVAT. A: Proteins with significantly increased abundance after removal of local PVAT from muscle compared to sham- operated muscle. Node colors indicate fold increase in muscle without PVAT, and node sizes are scaled to protein abundance as deduced from the mean spectral count in the Removed group. B: Proteins significantly decreased in muscle after PVAT removal, with node sizes scaled to protein abundance in the Sham group. Protein-protein associations were obtained from the STRING database.

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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

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Western immunoblotting 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

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Supplementary Table 2. Proteins upregulated in muscles without PVAT compared to muscles with PVAT. Control Control No PVAT No PVAT Fold UniProt Symbol Protein Name P-value increase Mean SD Mean SD

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

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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

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Supplementary Table 3. Proteins downregulated in muscles without PVAT compared to muscles with PVAT. Control Control No PVAT No PVAT Fold UniProt Symbol Protein Name P-value Mean Decrease Mean SD SD

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

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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

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Supplementary figure S1

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.

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Supplementary figure S2

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.

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Supplementary figure S3: heat map of upregulated proteins in muscle after removal of PVAT

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Supplementary figure S4: heat map of downregulated proteins in muscle after removal of PVAT

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100 kD HSP90

75 kD

GAPDH

Ponceau 37 kD Sham PVAT-Removed

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