Page 1 of 41 Diabetes 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] 1 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 2 Page 3 of 41 Diabetes 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. 3 Diabetes Page 4 of 41 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 4 Page 5 of 41 Diabetes 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. 5 Diabetes Page 6 of 41 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 6 Page 7 of 41 Diabetes 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