Endothelial IGF‐1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202050767
ISSN1469-3178
AutoresNatalie J Haywood, Cheukyau Luk, Katherine Bridge, Michael Drozd, Natallia Makava, Anna Skromna, Amanda D. V. MacCannell, Claire H Ozber, Nele Warmke, Chloe Wilkinson, Nicole T. Watt, Joanna Koch‐Paszkowski, Irvin Teh, Jordan H. Boyle, Sean Smart, Jürgen E. Schneider, Nadira Yuldasheva, Lee D. Roberts, David J. Beech, Piruthivi Sukumar, Stephen B. Wheatcroft, Richard M. Cubbon, Mark T. Kearney,
Tópico(s)Dietary Effects on Health
ResumoReport2 May 2021Open Access Source DataTransparent process Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity Natalie J Haywood orcid.org/0000-0002-8762-7257 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Cheukyau Luk Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Katherine I Bridge Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Michael Drozd Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Natallia Makava Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Anna Skromna Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Amanda Maccannell Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Claire H Ozber Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nele Warmke Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Chloe G Wilkinson Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nicole T Watt orcid.org/0000-0002-7429-7367 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Joanna Koch-Paszkowski Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Irvin Teh Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Jordan H Boyle Faculty of Engineering, School of Mechanical Engineering, University of Leeds, Leeds, UK Search for more papers by this author Sean Smart Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Jurgen E Schneider Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nadira Y Yuldasheva Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Lee D Roberts Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author David J Beech Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Piruthivi Sukumar Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Stephen B Wheatcroft Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Richard M Cubbon Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Mark T Kearney Corresponding Author [email protected] orcid.org/0000-0002-9376-8814 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Natalie J Haywood orcid.org/0000-0002-8762-7257 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Cheukyau Luk Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Katherine I Bridge Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Michael Drozd Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Natallia Makava Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Anna Skromna Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Amanda Maccannell Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Claire H Ozber Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nele Warmke Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Chloe G Wilkinson Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nicole T Watt orcid.org/0000-0002-7429-7367 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Joanna Koch-Paszkowski Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Irvin Teh Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Jordan H Boyle Faculty of Engineering, School of Mechanical Engineering, University of Leeds, Leeds, UK Search for more papers by this author Sean Smart Department of Oncology, University of Oxford, Oxford, UK Search for more papers by this author Jurgen E Schneider Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Nadira Y Yuldasheva Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Lee D Roberts Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author David J Beech Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Piruthivi Sukumar Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Stephen B Wheatcroft Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Richard M Cubbon Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Mark T Kearney Corresponding Author [email protected] orcid.org/0000-0002-9376-8814 Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Search for more papers by this author Author Information Natalie J Haywood1, Cheukyau Luk1, Katherine I Bridge1, Michael Drozd1, Natallia Makava1, Anna Skromna1, Amanda Maccannell1, Claire H Ozber1, Nele Warmke1, Chloe G Wilkinson1, Nicole T Watt1, Joanna Koch-Paszkowski1, Irvin Teh1, Jordan H Boyle2, Sean Smart3, Jurgen E Schneider1, Nadira Y Yuldasheva1, Lee D Roberts1, David J Beech1, Piruthivi Sukumar1, Stephen B Wheatcroft1, Richard M Cubbon1 and Mark T Kearney *,1 1Faculty of Medicine and Health, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK 2Faculty of Engineering, School of Mechanical Engineering, University of Leeds, Leeds, UK 3Department of Oncology, University of Oxford, Oxford, UK *Corresponding author. Tel: +44 113 343 8834; E-mail: [email protected] EMBO Rep (2021)22:e50767https://doi.org/10.15252/embr.202050767 See also: Z Bouman Chen & N Kaur Malhi (May 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. SYNOPSIS It remained unclear if gut endothelial cells can directly influence the microbiota. Here, endothelial specific over-expression of IGF-1R is shown to promote advantageous remodelling of the gut microbiota upon high fat diet, which protects against the development of obesity. Mice overexpressing insulin like growth factor-1 receptors in the endothelium (hIGFREO) are protected against obesity. Protection against obesity is in part due to remodelling of the intestinal microbiota due to endothelium-enterocyte crosstalk. Altered microbial diversity in the gut leads to a relative increase in the beneficial genus Akkermansia. Microbiota depletion using broad-spectrum antibiotics attenuates advantageous metabolic phenotypes of hIGFREO mice. 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-Järvinen, 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; Kivelä 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. Figure 1. Endothelial IGF-1R overexpression prevents high-fat diet (HFD)-induced weight gain 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 ± 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. Source Data for Figure 1 [embr202050767-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Endothelial IGF-1R overexpression has no effect on glucose tolerance in chow-fed mice A, B. In aorta from human IGF-1R endothelial overexpressing mice (hIGFREO) and wild-type littermates (WT) after 8 weeks HFD, immunoblotting shows that hIGFREO have similar protein expression of phospho-eNOS at Serine 1177 and phospho-AKT at Serine 473 (n = 8–10 mice per group). C. hIGFREO had comparable glucose fasting blood glucose levels compared with WT on chow diet (n = 5–10 mice per group). D, E. hIGFREO had comparable glucose intolerance, compared to WT on chow diet (as measured by glucose tolerance test and area under the curve (AUC)) (5–10 mice per group). F, G. In muscle from WT and hIGFREO after 8 weeks of HFD, immunoblotting shows that hIGFREO have increased protein expression of total and phospho-AKT at Serine 473 (n = 3 per genotype). Data information: Data shown as mean ± SEM and individual mice are shown as data points, P < 0.05 taken as being statistically significant using Student’s t-test. ns denotes not significant. Source data are available online for this figure. Download figure Download PowerPoint Overexpression of endothelial IGF-1R prevents obesity-associated glucose intolerance Chow-fed hIGFREO had similar glucose tolerance as WT (Fig EV1-EV5). 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). Click here to expand this figure. Figure EV2. Protection from high-fat diet (HFD)-induced weight gain is not due to altered energy expenditure in mice overexpressing human IGF-1R in the endothelium (hIGFREO) A–C. hIGFREO exhibit no difference in oxygen consumption, CO2 production or respiratory exchange ratio using indirect calorimeter assessment after HFD when compared to WT after 8 weeks of HFD (n = 4 per genotype). D, E. hIGFREO had comparable concentrations of fasting plasma leptin and adiponectin to WT after 8 weeks of HFD (n = 5–7 mice per group). Data information: Data shown as mean ± 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 * and ns denotes not significant. For indirect calorimetry, ANOVA testing was performed using mass as a co-variant (ANCOVA testing) using calrapp.org. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Adipose remodelling was no different in human IGF-1 receptor endothelial overexpressing mice (hIGFREO) in the setting of high-fat diet (HFD)-induced obesity A–C. (A), Histological examination of brown and white adipose tissue in hIGFREO compared with WT after 8 weeks of HFD. Scale bar = 300 µm. (B), hIGFREO mice have increased abundance of smaller adipocytes than WT. (C), There is no difference in average size of adipocytes in brown adipose tissue of hIGFREO compared with WT using haematoxylin and eosin stain (n = 6 per genotype). D. Representative confocal microscopy images of whole mount white and brown adipose tissue. Adipocytes stained with LipidTOX (green) and endothelial cells stained with Isolectin B4 (IB4-647) (red). Scale bar = 50 µm (n = 4–5 mice per group). E. There is no difference in vascular density in white or brown adipose tissue from hIGFREO compared with WT after 8 weeks of HFD (7 per genotype). F. Macrophage infiltration, shown by crown-like structure (CLS) analysis, was similar in hIGFREO and WT after 8 weeks of HFD (n = 7–8 mice per group). G–I. There is also no difference in resident adipose CD45+ cells, CD11b+ cells or F4/80 in hIGFREO compared with WT after 8 weeks of HFD (n = 7–8 mice per group). Data information: Data shown as mean ± SEM and individual mice are shown as data points, P < 0.05 taken as statistically significant using Student’s t-test and denoted as * and ** for P < 0.01. ns denotes not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. There was no difference in hepatic steatosis in human IGF-1 receptor endothelial overexpressing mice (hIGFREO) in the setting of high-fat diet-induced obesity A. Representative images of haematoxylin- and eosin-stained liver from hIGFREO compared with wild-type littermates (WT). Scale bar = 200 µm. B. No difference in extent of hepatic fibrosis in hIGFREO compared with WT after 8 weeks of HFD (n = 7–9 mice per group). C. This was confirmed using MR images showing that there was no difference in hepatic fat content when comparing hIGFREO to WT after 8 weeks of HFD (n = 4 per genotype). D–F. Hepatic levels of free fatty acids, triglycerides and cholesterol are similar when comparing hIGFREO to WT after 8 weeks of HFD (n = 7–11 mice per group). G, H. Fasted plasma concentration of free fatty acids and triglycerides is also similar in hIGFREO and WT (n = 4–8 mice per group). I, J. (I) Pancreatic lipase levels and lipase activity (J) were similar in hIGFREO and WT after 8 weeks of HFD (n = 4–6 mice per group). K. Hepatic expression of CYP7A and ABCB11 is also similar in hIGFREO and WT after 8 weeks of HFD (n = 5–6 mice per group). Data information: Data shown as mean ± SEM and individual mice are shown as data points, P < 0.05 taken as being statistically significant using Student’s t-test. ns denotes not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Endothelial cells from human IGF-1 receptor endothelial overexpressing mice (hIGFREO) can communicate with the gut wall A–C. (A), Representative images of haematoxylin- and eosin-stained villi from hIGFREO compared with WT after 8 weeks of HFD. No difference in villi lipid content (B) or villi length (C) in hIGFREO compared with WT (n = 5–12 mice per group). D, E. Faith’s phylogenetic diversity (PD) was used to measure the faecal microbial diversity and demonstrates no difference between hIGFREO mice and WT on chow diet (n = 5-8 mice per group). F, G. Chao-1 analysis was used to measure the faecal microbial diversity and abundance and again demonstrates no difference between hIGFREO and WT mice on chow diet (n = 5–8 mice per group). H–J. Targeted gene expression of the small intestine from hIGFREO normalised to WT after 8 weeks of high-fat diet demonstrating a significant increment in Muc2 and decrement in Cd36, Sar1b, Apob and Defb1 (n = 6–8 mice per group). K. Gene expression of Caco-2 cells treated with conditioned media from hIGFREO endothelial cells showed a significant increase in Reg3g expression (n = 3–6 mice per group). Data information: Data shown as mean ± 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 *. 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. Download figure Download PowerPoint Figure 2. Endothelial IGF-1R overexpression prevents high-fat diet (HFD)-induced glucose intolerance 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 ± 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). Download figure Download PowerPoint 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. 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 EV3-EV5), adipose tissue vascularity (Fig EV3D and E) and adipose tissue inflammatory markers, in hIGFREO and WT on HFD (Fig EV3-EV5). There was no difference in hepatic steatosis (Fig EV4, EV5), pancreatic lipase or gene expression of cholesterol 7alpha-hydroxylase (Cyp7a) and ATP Binding Cassette Subfamily B Member 11(Abcb11) in liver when comparing hIGFREO to WT (Fig EV4, EV5K). There was no difference in small intestine length (Fig 3F), villi histology (Fig EV5A–C) or gut transit time (Fig 3G). 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 ± 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. Download figure Download PowerPoint 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 complement
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