Endothelial IGF‐1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity

Abstract Changes in composition of the intestinal microbiota are linked to the development of obesity and can lead to endothelial cell (EC) dysfunction. It is unknown whether EC can directly influence the microbiota. Insulin‐like growth factor‐1 (IGF‐1) and its receptor (IGF‐1R) are critical for coupling nutritional status and cellular growth; IGF‐1R is expressed in multiple cell types including EC. The role of ECIGF‐1R in the response to nutritional obesity is unexplored. To examine this, we use gene‐modified mice with EC‐specific overexpression of human IGF‐1R (hIGFREO) and their wild‐type littermates. After high‐fat feeding, hIGFREO weigh less, have reduced adiposity and have improved glucose tolerance. hIGFREO show an altered gene expression and altered microbial diversity in the gut, including a relative increase in the beneficial genus Akkermansia. The depletion of gut microbiota with broad‐spectrum antibiotics induces a loss of the favourable metabolic differences seen in hIGFREO mice. We show that IGF‐1R facilitates crosstalk between the EC and the gut wall; this crosstalk protects against diet‐induced obesity, as a result of an altered gut microbiota.


Introduction
In the intestine are trillions of microorganisms which are collectively described as the gut microbiota. The traditional dogma that the gut microbiota is pathogenic has evolved with an appreciation of its important role in the maintenance of human health (Lynch & Pedersen, 2016). Recent studies indicate that the gut microbiota is important in the metabolic response to changes in dietary composition (Backhed et al, 2004;Turnbaugh et al, 2006;Vrieze et al, 2012). Obesity secondary to excess calorie intake is a major risk factor for the development of a range of common disorders of human health including the following: type 2 diabetes (Guariguata et al, 2013), fatty liver (Yki-J€ arvinen, 2014) and a number of cancers (Gallagher & Leroith, 2015). While our understanding of the mechanisms underlying the development and complications of obesity remains incomplete, a role for adverse remodelling of the gut microbiota has recently emerged as an important factor in the unfavourable effects of the disorder in a range of tissues and organs (Backhed et al, 2004;Turnbaugh et al, 2006;Khan et al, 2016;Patterson et al, 2016;Castaner et al, 2018) including the vascular endothelium (Koren et al, 2011;Karlsson et al, 2012;Catry et al, 2018;Leslie & Annex, 2018;Amedei & Morbidelli, 2019). The endothelium, previously thought to be an inert monolayer, has emerged as a complex paracrine/autocrine organ, important in the regulation of a range of homeostatic processes (Lee et al, 2007;Ding et al, 2010;Kivel€ a et al, 2019;Tang et al, 2020). It is currently unknown whether the endothelium can influence the composition of the intestinal microbiota.
The insulin-like growth factors (IGF-I and IGF-II) are evolutionally conserved peptide hormones that couple nutrient intake to cellular growth (Jones & Clemmons, 1995). The effects of IGF-I are predominantly mediated by the activation of its plasma membrane receptor-IGF-1R (Adams et al, 2000). During calorie excess, the expression of IGF-1R changes in a range of tissues, including the endothelium, where we have shown it to decline (Mughal et al, 2019). The IGF-1R has also been shown to modulate the intestinal barrier (Dong et al, 2014), and conversely, the microbiome has been shown to modulate IGF-1R signalling in muscle (Schieber et al, 2015) and bone formation (Yan et al, 2016). Therefore, to explore the effects of endothelial IGF-1R on metabolic responses to obesity and the microbiome, we fed mice with endothelial cell overexpression of human IGF-1R (hIGFREO) (Imrie et al, 2012) an obesogenic high-fat high-calorie diet. Feeding hIGFREO an obesogenic diet revealed a hitherto unrecognised mode of communication between the endothelium and the gut wall leading to favourable remodelling of the gut microbiota which protects against the development of diet-induced obesity and its adverse metabolic sequelae.

Results and Discussion
Endothelial IGF-1R overexpression prevents high-fat diet-associated weight gain To explore the role of IGF-1R in the endothelium under circumstances recapitulating diet-induced obesity, we fed hIGFREO and wild-type littermates (WT) a 60% high-fat diet (HFD) for 8 weeks ( Fig 1A). Endothelial overexpression of hIGF-1R was confirmed using qPCR ( Fig 1B); endothelial insulin receptor expression was similar in hIGFREO and WT ( Fig 1C); this expression pattern was recapitulated at the protein level ( Fig 1D and E). Protein markers of vascular function (eNOS and AKT) in the aorta were unchanged between the genotypes (Fig EV1A and B). On chow diet, hIGFREO had similar weight to WT, as we have previously reported (Imrie et al, 2012); however, on HFD, hIGFREO did not gain as much weight as WT mice ( Fig 1F). MRI was used to assess whole-body adiposity; hIGFREO had significantly less subcutaneous and visceral adipose tissue compared with WT on HFD (Fig 1G and H). Wet organ weight confirmed that hIGFREO had smaller white epididymal adipose depots than WT on HFD, with no difference in heart, spleen or liver weight ( Fig 1I). The IGF-1R is known to be an important regulator of foetal and postnatal growth (Woods et al, 1996;Garcia et al, 2014;Fujimoto et al, 2015;Juanes et al, 2015), and hIGFREO and WT mice had similar body and femur length (Fig 1J), demonstrating that endothelial IGF-1R overexpression did not cause growth retardation.
Overexpression of endothelial IGF-1R prevents obesity-associated glucose intolerance Chow-fed hIGFREO had similar glucose tolerance as WT (Fig EV1C-E). However, when challenged by a HFD, hIGFREO had significantly lower fasting blood glucose compared with WT (Fig 2A) and were also protected from the glucose intolerance seen in WT (Fig 2B and C). hIGFREO on HFD were also more insulin sensitive as shown using the homeostatic model assessment of insulin resistance (HOMA-IR) analysis (Fig 2D), which was associated with an increase in the expression of AKT and phosphorylation of AKT at serine 437 in skeletal muscle of hIGFREO (Fig EV1F and G). hIGFREO and WT had similar fasting plasma concentrations of IGF-I and insulin (Fig 2E and F). HFD-fed hIGFREO handled olive oil gavage more effectively over a 3-hr period postgavage with a significantly smaller increment in plasma triglycerides than WT (Fig 2G and H).
Endothelial IGF-1R overexpression does not lead to changes in activity, food intake or energy expenditure To further probe the mechanisms underpinning the anti-obesity and anti-diabetic effect of endothelial IGF-1R, metabolic cages were used to perform measurement of multiple metabolic parameters.
A Schematic representation of feeding time course. B, C In primary endothelial cells isolated from human IGF-1 receptor endothelial overexpressing mice (hIGFREO) and wild-type littermates (WT), quantitative polymerase chain reaction (qPCR) shows that hIGFREO have increased expression of human IGF-1R but similar levels of murine insulin receptor (IR) gene expression as WT (n = 3-5 mice per group). D, E In primary endothelial cells isolated from WT and hIGFREO, immunoblotting shows that hIGFREO have increased expression of IGF-1R but similar levels of IR protein expression (n = 3-4 mice per group). F Chow-fed hIGFREO had similar body mass to WT; however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n = 6-10 mice per group). G Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1 cm. H Subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) volumes were reduced in hIGFREO (n = 4 per genotype). I hIGFREO had reduced white epididymal adipose depot weight compared with WT; there was no difference in heart, spleen or liver weight (n = 7-11 mice per group). J hIGFREO had similar whole-body and femur length as WT (n = 7-9 mice per group).
Data information: Data shown as mean AE SEM, individual mice are shown as data points, P < 0.05 taken as being statistically significant using Student's t-test and denoted as * (** denotes P ≤ 0.01, ns denotes not significant). Source data are available online for this figure.
A Human IGF-1R endothelial overexpressing mice (hIGFREO) had significantly lower fasting blood glucose compared with wild-type littermates (WT) after HFD (n = 5-7 mice per group). B, C hIGFREO had reduced glucose intolerance compared with WT (as measured by glucose tolerance test and area under the curve (AUC)) (n = 5-7 mice per group). D hIGFREO had improved insulin sensitivity compared with WT as shown by lower HOMA-IR score (n = 9-10 mice per group). E, F hIGFREO and WT had similar fasting plasma IGF-1 and insulin concentrations (n = 6-12 mice per group). G, H Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3-h period postgavage in hIGFREO compared with WT and shown as area under the curve (n = 10-12 mice per group).
Data information: Data shown as mean AE SEM and individual mice are shown as data points, P < 0.05 taken as being statistically significant using Student's t-test and denoted as * (** denotes P ≤ 0.01, ns denotes not significant). hIGFREO on HFD showed no difference in activity levels ( Fig 3A), food consumption (Fig 3B), oxygen consumption ( Fig EV2A), carbon dioxide production (Fig EV2B), energy expenditure ( Fig 3C) or respiratory exchange ratio ( Fig EV2C) compared with WT on HFD. IGF-1R are thought to contribute to temperature homeostasis and may contribute to regulation of energy homeostasis during calorie restriction (Cintron-colon et al, 2017). Going against this possibility adipose tissue expression of browning markers ( Fig 3D) and body temperature ( Fig 3E) were all unchanged in hIGFREO compared to WT. Plasma leptin and adiponectin were also no different (Fig EV2D and E). There was also no difference in adipose tissue remodelling, shown by similar adipocyte size (Fig EV3A-C), adipose tissue vascularity (Fig EV3D and E) and adipose tissue inflammatory markers, in hIGFREO and WT on HFD (Fig EV3F-I).
There was no difference in hepatic steatosis ( Fig EV4A-H  Endothelial IGF-1R overexpression alters the gut microbiota and augments the abundance of the beneficial genus Akkermansia We then asked whether IGF-1R facilitated endothelial communication with the gut wall to influence the microbiota. Faith's phylogenetic diversity (PD), a measure of faecal microbial diversity, was significantly different in hIGFREO compared with WT after HFD ( Fig 4A and B). Chao-1 analysis, a complementary measure of faecal microbial diversity and abundance, was also significantly different (Fig 4C and D). To further investigate these changes to the microbiota and assess the contribution of each genus to the difference between hIGFREO and WT, partial least squares discriminant analysis (PLS-DA) modelling and the variable importance in projection (VIP) score were performed. This demonstrated that hIGFREO mice on HFD have increased abundance of Escherichia Shigella, Coriobacteriaceae UCG-002, Faecalibaculum, Peptococcus, Akkermansia and Dehalobacerium. hIGFREO mice on a HFD are depleted in Enterococcus, Barnesiella, Helicobacter, Streptococcs, Tyzzerella, Lachnospiraceae NK4A136 and Bilophila, as well as several genera from the Ruminococcaceae family ( Fig 4E and F).
Of particular relevance to our findings was the increase in relative abundance of the genus Akkermansia (Derrien, 2004) seen in highfat-fed hIGFREO. Akkermansia is thought to have anti-obesity and anti-diabetic effects in both humans and rodents (Everard et al, 2013;Cani & de Vos, 2017;Plovier et al, 2017;Depommier et al, 2019). Specifically, Akkermansia muciniphila reduces diet-induced weight gain, fat mass development, fasting hyperglycaemia and improves glucose tolerance without affecting food intake in mice (Everard et al, 2013), the same phenotype observed in hIGFREO. Increased levels of Akkermansia muciniphila are also associated with better clinical outcomes, such as insulin sensitivity, after a calorie restricted diet in overweight/obese adults (Dao et al, 2016). More recently, a proof-of-concept clinical trial in obese humans demonstrated that supplementation with Akkermansia muciniphila was a safe, well-tolerated intervention which improved several metabolic parameters (Depommier et al, 2019). However, it is also noteworthy that Bilophila was depleted in high-fat-fed hIGFREO; Bilophila has previously been shown to contribute to HFD-induced metabolic dysfunction (Natividad et al, 2018). Dehalobacerium was enhanced in high-fatfed hIGFREO mice and has previously been shown to be protective against atherosclerosis and reduced cholesterol (Chan et al, 2016). It is difficult to speculate further about the contribution of these other genera as little more is known about their role in obesity and metabolic disease; further studies would be of interest. Interestingly, when hIGFREO mice were unchallenged on a chow diet, there was no difference in microbial diversity compared with WT (Fig EV5D-G).
To dissect potential mechanisms underpinning the altered microbial diversity, we examined the expression of genes known to modulate the microbiota (Chang & Kao, 2019). We saw several changes in gene expression in the gut wall (Fig EV5H-J), raising the possibility that crosstalk between endothelial cells and the gut wall can influence gene expression. It is well established that endothelial cells can act in a paracrine/autocrine fashion (Lee et al, 2007;Ding et al, 2010; Kivel€ a et al, 2019) and equally well established that enterocytes respond to microbial metabolites (Nuenen et al, 2005;Garrett, 2020). To examine a role for secreted factors from endothelial cells in the altered gene expression seen in hIGFREO small intestine, we used primary endothelial cells from hIGFREO to condition culture media to treat Caco-2 cells, as a model of the intestinal epithelial barrier. Caco-2 cells treated with conditioned media from hIGFREO showed a significant increase in regenerating islet-derived III-c (REG3G) compared with WT gene expression ( Fig EV5K). REG3G belongs to the family of C-type lectins and is one of several antimicrobial peptides produced by Paneth cells and enterocytes (Chang & Kao, 2019;Shin & Seeley, 2019). REG3G destroys grampositive bacteria by binding to the peptidoglycan layer, exerting bactericidal activity by oligomerising to form hexameric transmembrane pores (Shin & Seeley, 2019), thus providing one explanation ◀ Figure 3. Protection from high-fat diet (HFD)-induced weight gain in human IGF-1R endothelial overexpressing mice (hIGFREO) is not due to changes in activity, food intake, energy expenditure, adipose browning or gut transit time.
A-C hIGFREO exhibit no difference in activity levels, food consumption or energy expenditure using indirect calorimeter assessment after HFD compared with wild-type littermates (WT) after HFD. (n = 4 per genotype). D Adipose expression of browning markers is also no different in white epididymal adipose tissue and brown adipose tissue compared with WT (n = 6 per genotype). E Core body temperature is no different in hIGFREO compared with WT (n = 6-8 mice per group). F, G Gut transit time is also unaltered in hIGFREO compared with WT as shown by no change in small intestine length (F) (n = 7-9 mice per group), or total gut transit time after a carmine red gavage (G) (n = 12-13 mice per group).
Data information: The light/dark cycle for graphs A-C is shown as follows: light in yellow and dark in brown. Data shown as mean AE SEM and individual mice are shown as data points. For indirect calorimetry, ANOVA testing was performed using mass as a co-variant (ANCOVA testing) using calrapp.org. ns denotes not significant.
▸ Figure 4. Endothelial IGF-1R overexpression alters the gut microbiota and augments the abundance of the beneficial genus Akkermansia.
A, B Faith's phylogenetic diversity (PD) was used to measure the faecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wild-type littermates (WT) mice after high-fat diet feeding (n = 4-5 mice per group). C, D Chao-1 analysis was used to measure the faecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n = 4-5 mice per group). E, F Partial least squares discriminant analysis (PLS-DA) model and used the variable importance in projection (VIP) score was used to assess the contribution of each genus, shown as a scores plot in (E), and a loading plot of PLS-DA of genus abundances in (F). VIP score cut-off of 1 (n = 4-5 mice per group).
Data information: Data shown as mean AE SEM and individual mice are shown as data points. Diversity analyses were run on the resulting OTU/feature.biom tables to provide both phylogenetic and non-phylogenetic metrics of alpha and beta diversity. Additional data analysis (PLS-DA) and statistics were performed with R. P < 0.05 taken as being statistically significant using Student's t-test and denoted as *.  as to why hIGFREO display reduced microbiota diversity and possibly providing an explanation as to why relative levels of Akkermansia, a gram-negative bacteria, are enhanced. This raises the intriguing possibility that endothelial cell IGF-1R could be a nutrient sensor responding to nutritional cues to influence the architecture of the intestinal microbiome (Bettedi & Foukas, 2017  Antibiotic administration in the setting of obesity prevents the anti-obesity and anti-diabetic actions of endothelial IGF-1R overexpression To investigate the contribution of the altered microbiota to the antiobesity and anti-diabetic effects of endothelial IGF-1R overexpression, hIGFREO and WT were given broad-spectrum antibiotics in their drinking water (Rodrigues et al, 2017) for the duration of HFD (Fig 5A). The addition of antibiotic treatment alongside HFD abolished the difference in weight gain seen between hIGFREO and WT (Fig 5Bi). However, WT on HFD and antibiotic treatment did not gain as much weight as WT on HFD alone. On chow diet hIGFREO and WT did not tolerate prolonged antibiotic treatment and for welfare reasons had to be culled, thus suggesting that the mice did not completely tolerate antibiotic treatment. Nevertheless, both WT and hIGFREO gained significantly more weight than mice on chow diet (Fig 5Bii). Antibiotic treatment also prevented the difference in glucose intolerance, seen between the genotypes when on HFD alone (Fig 5C-E). Wet organ weights were comparable between hIGFREO and WT ( Fig 5F). Chao-1 analysis was no different between hIGFREO and WT after HFD and antibiotic treatment ( Fig 5G). Alpha diversity in hIGFREO and WT on HFD treated with antibiotics was also similar demonstrating no difference in microbial diversity using a range of approaches (Fig 5H and Table EV1). Taken together, these data confirm a causal role for the microbiota in the favourable changes seen in hIGFREO.

Conclusion
To our knowledge, this is the first report to demonstrate communication between the endothelium and the gut wall, which in turn can modulate the gut microbiota. We report a novel role for endothelial cell IGF-1R in this crosstalk, which protects against diet-induced obesity and its associated adverse metabolic sequelae, by potentially remodelling the architecture of the microbiota.

Animal husbandry
hIGFREO mice with endothelial cell-specific overexpression of the IGF-1 receptor (previously described Imrie et al, 2012) and their wild-type control littermates (WT) were bred in house. Experiments were carried out under the authority of UK Home Office project licence P144DD0D6. Mice were group housed in cages of up to five, which contained a mix of genotypes. Researchers were blinded to genotype until the data analysis stage. Cages were maintained in humidity and temperature-controlled conditions (humidity 55% at 22°C) with a 12-h light-dark cycle. All interventions were performed within the light cycle. Only male mice were used for experimental procedures to prevent variability associated with the oestrous cycle on adiposity and metabolic readouts (Stubbins et al, 2012;Griffin et al, 2016). Genotyping was carried out by Transnetyx commercial genotyping using ear biopsies.

Metabolic phenotyping
Mice were fasted overnight prior to glucose tolerance or for 2hr prior to insulin tolerance tests. Blood glucose was measured using a handheld Glucose Meter (Accu-Chek Aviva). An intra-peritoneal injection of glucose (1 mg/g) or recombinant human insulin (Actrapid; Novo Nordisk) (0.75 IU/kg) was given and glucose concentration measured at 30-min intervals for 2 h from the point of glucose/insulin administration. Mice were not restrained between measurements (Haywood et al, 2017).
Core body temperature was measured using an Indus rectal temperature probe (Vevo2100 (VisualSonics, FujiFilm).
After 8 weeks of HFD, metabolic parameters were measured by indirect calorimetry using Comprehensive Lab Animal Monitoring ◀ Figure 5. Antibiotic administration in the setting of high-fat diet (HFD) eliminates the anti-obesity and anti-diabetic actions of endothelial IGF-1R overexpression.

A
Schematic representation of antibiotic dosing and feeding time course. B (Bi), Human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wild-type littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. (Bii), Both hIGFREO and WT gained significant weight compared with chow-fed mice (n = 7-9 mice per group). C There was no difference in fasting blood glucose in hIGFREO compared with WT (n = 7-9 mice per group). D, E There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n = 7-9 mice per group). F Wet organ weights were similar in hIGFREO and WT (n = 7-9 mice per group). G Chao-1 analysis was used to measure the faecal microbial diversity and abundance and demonstrates no difference between hIGFREO and WT after HFD + antibiotic treatment (n = 3-5 mice per group). H Alpha diversity P values using Kruskal-Wallis pairwise comparisons show there is no difference in microbial diversity.
Data information: Data shown as mean AE SEM and individual mice are shown as data points, P < 0.05 taken as being statistically significant using Student's t-test and denoted as * or ** for P P < 0.01 and NS denotes not significant. Diversity analyses were run on the resulting OTU/feature.biom tables to provide both phylogenetic and non-phylogenetic metrics of alpha and beta diversity. Additional data analysis (PLS-DA) and statistics were performed with R.
ª 2021 The Authors EMBO reports 22: e50767 | 2021 Systems (CLAMS) (Columbus Instruments). In brief, mice were individually housed for 5 days and measurement of oxygen consumption, carbon dioxide production, food intake and locomotor activity were continuously recorded. For each mouse, a full 24-h period, taking into account sleep and wake cycles, was analysed after an acclimatisation period (Roberts et al, 2014). After 8 weeks of HFD (or at 8 weeks old for chow control mice), all mice were sacrificed using terminal anaesthesia and organ weights measured using a standard laboratory balance.

Lipid absorption
Mice were fasted overnight and blood samples collected from the lateral saphenous vein (EDTA collection tubes Sarstedt 16.444). Mice underwent oral gavage with 200 µl olive oil, and blood was taken from the saphenous vein every hour for a further 3 h (Zhang et al, 2018). Plasma triglycerides were measured using a commercially available kit (ab65336, Abcam).

Intestinal transit time
Mice were fasted overnight before oral gavage with 300 µl of Carmine solution (6% Carmine red (C1022, Sigma) in 0.5% methyl cellulose (M7140, Sigma-Aldrich)). Mice were then individually caged and monitored until the appearance of the first red faecal pellet (Li et al, 2011).

Magnetic resonance imaging (MRI)
Anaesthesia was induced using 5% isoflurane in 100% oxygen and then maintained using 1.5-3% isoflurane at 2 l/min oxygen flow. Animals were positioned prone on a dedicated mouse cradle. Body temperature was maintained with a custom resistive blanket placed on the back of the animal. Cardiac and respiratory signals were continuously monitored (BIOPAC Systems, Inc., Goleta, USA). Mice were imaged on a 7T preclinical MRI scanner with a 660 mT/m shielded gradient system and a quadrature-driven transmit/receive volume coil with inner diameter of 72 mm (Bruker BioSpin MRI GmbH, Ettlingen, Germany). A 2D cardiac-triggered and respiratorygated 3-point Dixon spoiled gradient-echo sequence was used: TR = 5.65 ms, TE = 2.42/2.75/3.09 ms, Matrix = 256 × 128, fieldof-view = 80 × 30 mm, number of slices = 28 in sagittal orientation, slice thickness = 1 mm, number of signal averages = 8, total scan time~30 min. The data were analysed in MATLAB (Math-Works, Natick, USA) using the hierarchical iterative decomposition of water and fat with echo asymmetry and least squares estimation (IDEAL) method (Tsao & Jiang, 2013). The proton density fat fraction (PDFF, the amount of lipid signal over total signal) was used to segment adipose tissue depots. Subcutaneous and visceral adipose depots were segmented separately using Osirix Lite v11.0.2 (Bernex, Switzerland) 2D threshold region growing algorithm tool with segmentation parameters set to a lower threshold of 80% PDFF.

Gene expression
RNA was isolated from cells and tissue samples using the monarch total RNA mini kit (NEB, T2010S). The concentration of RNA in each sample (ng/µl) was measured using a NanoDrop. cDNA was reverse transcribed (NEB, E3010S). Quantitative PCR (qPCR) was performed using a Roche LightCycler 480 Instrument II, using SYBR Green PCR Master Mix (Bio-Rad, 1725270) and relevant primers (See Table 1). The "cycles to threshold" (cT) was measured for each well, the average of triplicate readings for each sample taken, normalised to GAPDH, and finally, the differential expression of each gene was calculated for each sample.

Quantification of protein expression
Cells were lysed or tissue mechanically homogenised in lysis buffer (Extraction buffer, FNN0011) and protein content quantified using a BCA assay (Sigma-Aldrich, St. Louis, MO). Twenty micrograms of protein was resolved on a 4-12% Bis-Tris gel (Bio-Rad, Hertfordshire, UK) and transferred to nitrocellulose membranes. Membranes were probed with antibodies diluted in 5% BSA as per Table 2, before incubation with appropriate secondary horseradish peroxidase-conjugated antibody. Blots were visualised with Immobilon Western Chemiluminescence HRP Substrate (Merck Millipore, Hertfordshire, UK) and imaged with Syngene chemiluminescence imaging system (SynGene, Cambridge, UK). Densitometry was performed in ImageJ (Haywood et al, 2017).

Primary endothelial cell isolation
Primary endothelial cells (PECs) were isolated from lungs, as previously reported (Abbas et al, 2011;Watt et al, 2017). Briefly, lungs were harvested, washed, finely minced and digested in Hanks' balanced salt solution containing 0.18 units/ml collagenase (10 mg/ ml; Roche) for 45 min at 37°C. The digested tissue was filtered through a 70-lm cell strainer and centrifuged at 400 g for 10 min. The cell pellet was washed with PBS/0.5%BSA, centrifuged, resuspended in 1 ml PBS/0.5% and incubated with 1 × 10 6 CD146 antibody-coated beads (Miltenyi Biotec, at 4°C for 30 min. Bead-bound endothelial cells were separated from nonbead-bound cells using a magnet.

Quantification of white and brown adipose tissue vascularity
White adipose tissue (WAT) and brown adipose tissue (BAT) (< 0.5 g) were harvested into cold 1% paraformaldehyde (PFA) and allowed to fix for 2hrs at room temperature. Samples were incubated overnight with Isolectin B4 Alexa Fluor 647 (I32450, Thermo Fisher Scientific) and diluted 1:100 in 5% BSA in phosphatebuffered saline (PBS) at 4°C. After washing with PBS, they were incubated with HCS LipidTOX (H34475, Thermo Fisher Scientific) diluted 1:200 in PBS for 20mins at room temperature. Whole tissue was then mounted onto slides beneath coverslips using a silicone spacer (Grace bio-labs, 664113), with Prolong Gold (P36930, Thermo Fisher Scientific). Slides were then imaged using laser scanning confocal microscopy (LSM880, Zeiss), with 8 areas of each sample imaged. Vascular density (the proportion of each image stained with IB4) was measured using thresholding in ImageJ.
Histological assessment of adipocyte size, non-alcoholic fatty liver disease and villi structure Samples for histology were fixed in 4% PFA for at least 24 h and then processed into paraffin blocks. 5-µm sections were taken and collected onto 3-triethoxysilylpropylamine (TESPA) coated slides. After drying, slides were stained with haematoxylin and eosin to assess gross morphology AE oil red o (ORO) for lipid staining. Slides were imaged using an Olympus BX41 microscope at 10× and 20× magnification. For assessment of adipocyte size, three separate fields of view for each sample were assessed. For each one, the average of 20 randomly selected independent cells measured using ImageJ.
For assessment of non-alcoholic fatty liver disease (NAFLD) in sections of murine liver, a validated rodent NAFLD scoring system was used (Liang et al, 2014), which takes into account micro-and macro-steatosis, inflammation and hypertrophy. Each sample was assessed by at least two blinded independent verifiers (NH, KB or NW) and the average score per sample taken.

Flow cytometry
To isolate the stromal vascular fraction, epididymal fat pads were harvested, washed, finely minced and digested in Hanks' balanced salt solution containing collagenase (1 mg/ml; Roche) for 45 min at 37°C. The digested tissue was agitated using a cannula and centrifuged at 1,000 rpm for 10 min. The upper lipid phase was removed and the aqueous phase with pellet was filtered through a 70-µM cell strainer and centrifuged at 1,000 rpm for 7 min. The pellet was resuspended in PBS containing 0.5% BSA (Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich) and was filter through a 30-µM cell strainer and further centrifuged at 1,000 rpm for 7 min.

Pancreatic lipase activity
Tissue was harvested under terminal anaesthesia. 40 mg of pancreas was homogenised and used in a lipase activity assay (Abcam, ab102524).

Liver and plasma lipid measurements
100mg of tissue was weighed and homogenised in 1 ml of 5% Igepal (I8896, Sigma) and heated to 80°C for 5 min, cooled and reheated again before centrifuging for 2 min. The supernatant was used to measure, triglycerides, free fatty acids and cholesterol (Abcam, ab65336, ab65341 and ab65359, respectively).

Conditioning media
Conditioned media experiments require a large number of EC, and pulmonary EC provides an appropriate yield of cells to perform these experiments. Therefore, when PECs reached confluency, supplemented growth media was removed and replaced with basal endothelial growth medium-MV2 for 24 h. Conditioned media was then removed and used in further experiments as described.

Data analysis
All data are shown as mean AE standard error of mean (SEM) unless stated, with individual mice presented as data points. All image analysis was performed in ImageJ unless stated. Student unpaired ttest was used for statistical analyses and performed with GraphPad Prism software version 8 unless stated. For plasma concentrationtime profile experiments, area under the curve analyse was used and performed with GraphPad Prims. For metabolic parameters measured by indirect calorimetry, ANOVA testing was performed using mass as a co-variant (ANCOVA testing) using calrapp.org. P < 0.05 taken as statistically significant.

Data availability
No primary data sets have been generated and deposited.
Expanded View for this article is available online. services, whose research was supported by NIH grant U24-DK092993 (MMPC-University of California Davis Microbiome and Host Response Core, RRID:SCR_ 015361). MTK 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.