Endothelial Notch signaling controls insulin transport in muscle
2020; Springer Nature; Volume: 12; Issue: 4 Linguagem: Inglês
10.15252/emmm.201809271
ISSN1757-4684
AutoresSana S. Hasan, Markus Jabs, Jacqueline Taylor, Lena Wiedmann, Thomas Leibing, Viola Nordström, Giuseppina Federico, Letícia Prates Roma, Christopher Carlein, Gretchen Wolff, Bilgen Ekim Üstünel, Maik Brune, Iris Moll, Fabian Tetzlaff, Hermann‐Josef Gröne, Thomas Fleming, Cyrill Géraud, Stephan Herzig, Peter P. Nawroth, Andreas Fischer,
Tópico(s)Kruppel-like factors research
ResumoArticle18 March 2020Open Access Endothelial Notch signaling controls insulin transport in muscle Sana S Hasan Sana S Hasan Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Markus Jabs Markus Jabs Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Jacqueline Taylor Jacqueline Taylor Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Lena Wiedmann Lena Wiedmann Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Thomas Leibing Thomas Leibing Department of Dermatology, Venereology, and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Section of Clinical and Molecular Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Viola Nordström Viola Nordström Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Giuseppina Federico Giuseppina Federico Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Leticia P Roma Leticia P Roma Biophysics Department, Center for Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Search for more papers by this author Christopher Carlein Christopher Carlein Biophysics Department, Center for Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Search for more papers by this author Gretchen Wolff Gretchen Wolff Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Search for more papers by this author Bilgen Ekim-Üstünel Bilgen Ekim-Üstünel Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Search for more papers by this author Maik Brune Maik Brune Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Iris Moll Iris Moll Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Fabian Tetzlaff Fabian Tetzlaff Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Hermann-Josef Gröne Hermann-Josef Gröne Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Pharmacology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Thomas Fleming Thomas Fleming Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Cyrill Géraud Cyrill Géraud Department of Dermatology, Venereology, and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Section of Clinical and Molecular Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Peter P Nawroth Peter P Nawroth orcid.org/0000-0002-6134-7804 Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Fischer Corresponding Author Andreas Fischer [email protected] orcid.org/0000-0002-4889-0909 Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Sana S Hasan Sana S Hasan Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Markus Jabs Markus Jabs Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Jacqueline Taylor Jacqueline Taylor Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Lena Wiedmann Lena Wiedmann Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Thomas Leibing Thomas Leibing Department of Dermatology, Venereology, and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Section of Clinical and Molecular Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Viola Nordström Viola Nordström Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Giuseppina Federico Giuseppina Federico Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Leticia P Roma Leticia P Roma Biophysics Department, Center for Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Search for more papers by this author Christopher Carlein Christopher Carlein Biophysics Department, Center for Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Search for more papers by this author Gretchen Wolff Gretchen Wolff Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Search for more papers by this author Bilgen Ekim-Üstünel Bilgen Ekim-Üstünel Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Search for more papers by this author Maik Brune Maik Brune Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Iris Moll Iris Moll Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Fabian Tetzlaff Fabian Tetzlaff Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Hermann-Josef Gröne Hermann-Josef Gröne Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Pharmacology, Philipps University of Marburg, Marburg, Germany Search for more papers by this author Thomas Fleming Thomas Fleming Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Cyrill Géraud Cyrill Géraud Department of Dermatology, Venereology, and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Section of Clinical and Molecular Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Peter P Nawroth Peter P Nawroth orcid.org/0000-0002-6134-7804 Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Fischer Corresponding Author Andreas Fischer [email protected] orcid.org/0000-0002-4889-0909 Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Author Information Sana S Hasan1,‡, Markus Jabs1,‡, Jacqueline Taylor1,2, Lena Wiedmann1,2, Thomas Leibing3,4, Viola Nordström5, Giuseppina Federico5, Leticia P Roma6, Christopher Carlein6, Gretchen Wolff7, Bilgen Ekim-Üstünel7, Maik Brune8, Iris Moll1, Fabian Tetzlaff1, Hermann-Josef Gröne5,9, Thomas Fleming8, Cyrill Géraud3,4,10, Stephan Herzig7,8, Peter P Nawroth7,8 and Andreas Fischer *,1,8,10 1Division Vascular Signaling and Cancer (A270), German Cancer Research Center (DKFZ), Heidelberg, Germany 2Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany 3Department of Dermatology, Venereology, and Allergology, University Medical Center and Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 4Section of Clinical and Molecular Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 5Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany 6Biophysics Department, Center for Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany 7Institute for Diabetes and Cancer (IDC) and Joint Heidelberg-IDC Translational Diabetes Program, Helmholtz Center Munich, Neuherberg, Germany 8Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany 9Institute of Pharmacology, Philipps University of Marburg, Marburg, Germany 10European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 1622 1424150; E-mail: [email protected] EMBO Mol Med (2020)12:e09271https://doi.org/10.15252/emmm.201809271 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 The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes. Synopsis Insulin flux from blood plasma to muscle cells across the endothelium is a critical step in insulin-mediated lowering of blood glucose levels. This study highlights the role of Notch signaling in regulating systemic glucose homeostasis. Notch signaling activity was higher in endothelial cells (ECs) isolated from obese mice compared to non-obese mice. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels in mice. EC-specific inhibition of canonical Notch signaling increased insulin sensitivity and improved glucose tolerance in a high-fat diet-induced insulin resistance model. Glucose tolerance improvement was associated with increased transcription of genes involved in caveolae formation (Cav1 and Cavin1), higher number of caveolae in ECs and insulin uptake rates. The paper explained Problem Metabolic diseases such as diabetes mellitus frequently lead to subsequent blood vessel damage, impairing endothelial cell function. These damages pose a major risk for further metabolic complications and cardiovascular events. Despite their contribution to disease prevalence, the role of endothelial cells in actively regulating systemic metabolism is poorly understood. Results Here, we investigated how endothelial cells are involved in the control of systemic glucose metabolism. Our experiments revealed that Notch signaling in endothelial cells controls gene expression of proteins required for caveolae formation, which are in turn essential for insulin transport to muscle cells. Chronic over-activation of Notch signaling impaired insulin sensitivity and increased blood glucose levels. On the contrary, inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance. Impact Our data imply that the endothelium actively contributes to the control of insulin sensitivity and glucose uptake in muscle. Therefore, blood vessels not only transport insulin and glucose but also regulate their transport across the vessel wall. Introduction The inner lining of blood vessels is composed of endothelial cells (ECs) which provide an anti-thrombotic surface for transportation of blood to distal organs. In addition, ECs also regulate tissue regeneration, stem cell renewal and differentiation, and tumor progression through paracrine (angiocrine) interactions in an organ-specific manner (Rafii et al, 2016; Augustin & Koh, 2017). As ECs form a barrier between blood and all other cell types in the human body, they possess a unique spatial advantage to act as gatekeepers and maintain metabolic homeostasis by controlling the access of nutrients and hormones from blood to the surrounding tissue. ECs have different characteristics, which contribute to organ-specific vascular beds. For example, fenestrated endothelium containing pores allow for rapid exchange of water and small solutes in kidneys while sinusoidal endothelium form gaps that facilitate passive transport of larger molecules to hepatocytes in liver. On the other hand, continuous endothelium, e.g., in muscle, brain, and skin, provides a restrictive transcellular flux of nutrients and hormones (Aird, 2007; Augustin & Koh, 2017). Circulating hormones like insulin must cross the continuous endothelium to reach its target cells. It has been suggested that insulin transport across the endothelium to muscle tissue (the major site of insulin-mediated glucose uptake) is the rate-limiting step in insulin-mediated lowering of blood glucose (Yang et al, 1989). Although a model of non-saturable fluid-phase insulin transport has recently been described (Williams et al, 2018), there is strong evidence that the rapid insulin transport to muscle cells occurs through ECs and requires the presence of insulin receptors on ECs (Chernick et al, 1987; Bar et al, 1988; Baura et al, 1993; Konishi et al, 2017). The mechanism by which insulin crosses the continuous endothelium is critical to understand insulin action and insulin resistance. Trans-endothelial insulin transport varies among vascular beds and requires either clathrin-coated vesicles or caveolae (Wang et al, 2011; Azizi et al, 2015). Caveolae are specialized lipid rafts whose formation is dependent on proteins of the Caveolin (Cav) and Cavin families. Insulin binds to its receptor on ECs, is internalized by caveolae (Wang et al, 2006; Barrett et al, 2009), and is released at the basolateral EC membrane, where it diffuses in the interstitium and activates insulin-mediated pathways in muscle cells (Barrett et al, 2011). The caveolar protein Cav1 is essential for the formation of caveolae irrespective of the cell type (Sowa, 2012). In vitro, overexpression of Cav1 increases EC insulin transport rates, whereas a reduction in Cav1 expression impairs insulin flux (Wang et al, 2011). However, in vivo the situation is more complex. Inactivation of the Cav1 gene leads to lower numbers of caveolae, but this also opens paracellular routes to compensate for the impaired transcellular flux (Schubert et al, 2002). Likewise, overexpression of Cav1 alone does not increase the abundance of caveolae, implying that additional proteins are involved in the control of caveolae numbers (Razani et al, 2002; Bauer et al, 2005). The muscular endothelium expresses multiple proteins linked to caveolae formation, including Cav2, which interacts with Cav1, and Cavin1, an adaptor protein crucial for caveolae stabilization (Hansen et al, 2013). Even though in recent years extensive research has emerged into mechanisms of organ-specific vascular development, the knowledge about signaling pathways that control endothelial transport of hormones and nutrients is still very preliminary. Very recently, we have shown that endothelial-specific Notch signaling is required for fatty acid transport to muscle cells (Jabs et al, 2018). Notch signaling cascade is a juxtacrine communication system that requires the binding of ligands from the Delta-like (Dll) and Jagged family to Notch receptors on adjacent cells. This in turn induces receptor cleavage and leads to translocation of Notch intracellular domain (NICD) to the nucleus where it interacts with transcriptional co-activators like Mastermind like-1 (MAML1) and Rbp-jκ on gene promoters (Kopan & Ilagan, 2009). The activity of Notch signaling is influenced by the nutritional status and certain plasma metabolites and has been shown to be crucial for controlling glucose metabolism in hepatocytes and adipocytes (Pajvani et al, 2011, 2013; Bi et al, 2014; Briot et al, 2015). Therefore, we speculated that Notch signaling in ECs could also be involved in the control of systemic glucose metabolism. Results Obesity induces endothelial Notch signaling Notch signaling activity is altered by metabolites in several cell types (Pajvani et al, 2011, 2013; Bi et al, 2014; Briot et al, 2015). Therefore, we hypothesized that ECs, which are in intimate contact with plasma, respond to altered plasma metabolite concentrations. To examine this, we utilized diet-induced obesity (DIO) mouse models where C57BL/6J male mice were put on high-fat diet (HFD, 60% fat), high-fat-and-sucrose diet (HFS, 60% fat and 42 g/l sucrose in drinking water ad libitum), and a matching control diet (CD, 10% fat) for a period of 26 weeks starting at 4 weeks of age. We analyzed primary skeletal muscle ECs freshly isolated from these mice. Expression of Notch target genes was elevated in ECs isolated from obese animals (HFD and HFS) compared to ECs derived from CD fed mice (Fig 1A). In addition, we performed similar analyses with ECs isolated from skeletal muscle of mice on HFD for 3 and 8 weeks. Although these mice had a notable elevation in their blood glucose levels and body weights, the analysis of Notch target genes did not reveal any significant differences (Fig EV1A and B). Furthermore, to distinguish between a chronic and acute response to alterations in plasma metabolites, we examined Notch signaling during physiological feeding and fasting cycles. We analyzed Notch targets in primary ECs freshly isolated from skeletal muscle of mice that were either fed ad libitum or fasted for 24 h or refed for 6 h after a 24 h fast. We did not observe any significant differences in Notch target gene expression in ECs among these groups (Fig 1B). These results support the notion that chronic disturbance of plasma metabolites in obese mice leads to an increase in Notch signaling in ECs. Figure 1. Endothelial Notch signaling regulates systemic glucose metabolism A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates, and 42 g/l sucrose in drinking water). n = 4, data represent mean ± SEM, unpaired t-test. B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 h (Fasted group) and then refed for 4 h (Fasted + Refed group) normalized to mice fed for 24 h (Fed group). n = 5, data represent mean ± SEM, unpaired t-test. C. Blood glucose levels of control (n = 5) and NICDiOE-EC (n = 5) mice 5 weeks after recombination. Data represent mean ± SEM, unpaired t-test. D. Plasma insulin levels of control (n = 5) and NICDiOE-EC (n = 5) mice 5 weeks after recombination. Data represent mean ± SEM, Welch's t-test. E. Blood glucose levels for insulin tolerance test of control (n = 7) and NICDiOE-EC control (n = 7) mice. Data represent mean ± SEM, unpaired t-test. F. Quantification of area under curve for insulin tolerance test in (E). Data represent mean ± SEM, Welch's t-test. G. Blood glucose levels for glucose tolerance test of control (n = 6) or NICDiOE-EC (n = 7) mice. Data represent mean ± SEM, unpaired t-test. H. Quantification of area under the curve for glucose tolerance test in (G). Data represent mean ± SEM, unpaired t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Notch targets in high-fat diet fed mice and characterization of NICDiOE-EC mice A, B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) for 3 weeks (A) and 8 weeks (B). n = 5, data represent mean ± SEM, unpaired t-test. C. Body mass of control (n = 5) or NICDiOE-EC (n = 5) mice 7 weeks after tamoxifen injection. Data represent mean ± SEM, unpaired t-test. D. Expression of Notch target genes in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n = 4, data represent mean ± SEM, unpaired t-test. E. Representative confocal images of isolectin B4-stained blood vessels (red) in skeletal muscle of NICDiOE-EC mice and littermate controls 5 weeks after tamoxifen injection. Scale bar 50 μm. F. Representative confocal images of isolectin B4-stained blood vessels (red) in cardiac muscle of NICDiOE-EC mice and littermate controls 5 weeks after tamoxifen injection. Scale bar 30 μm. Download figure Download PowerPoint Sustained endothelial Notch signaling lowers insulin sensitivity in mice Notch signaling regulates glucose metabolism in liver and adipose tissue, and Notch over-activation in these tissues impairs insulin sensitivity (Pajvani et al, 2011; Bi et al, 2014). To test if sustained over-activation of Notch signaling, specifically in ECs, would also affect systemic glucose metabolism, we employed a mouse model in which constitutively active Notch1 intracellular domain (NICD) is expressed under the EC-specific tamoxifen-inducible Cdh5 (VE-Cadherin) promoter (NICDiOE-EC mice; Ramasamy et al, 2014). Gene recombination was induced in adult mice (Wieland et al, 2017; Jabs et al, 2018). Seven weeks after gene recombination, there were no differences in body weight (Fig EV1C). Canonical Notch targets were significantly increased in ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to controls (Fig EV1D). The range of Hey1, Hey2, and Hes1 gene induction was very similar to the induction seen in diet-induced obese mice (Fig 1A). Furthermore, there were no differences in skeletal and cardiac muscle microvessel density and morphology in NICDiOE-EC mice when compared to littermate controls (Fig EV1E and F). However, upon EC Notch1 over-activation, the mice had increased plasma glucose and insulin levels (Fig 1C and D), a typical sign of impaired insulin sensitivity. To confirm this observation, we performed an insulin tolerance test (ITT). Compared to controls, insulin lowered blood glucose less efficiently in NICDiOE-EC mice (Fig 1E and F). In addition, intraperitoneal glucose tolerance test (GTT) showed a similar trend (Fig 1G and H). To rule out impaired secretion of insulin or other hormonal regulators of glucose metabolism from pancreas and liver, we checked for vascular alterations in stained tissue sections. We did not observe any significant difference in pancreatic islet area or vessel coverage in pancreas between control and NICDiOE-EC mice (Fig EV2A and B). Moreover, histological analysis of liver vasculature also did not reveal any significant difference between the two groups (Fig EV2C and D), confirming that the observed insulin resistance in NICDiOE-EC mice is not an outcome of defective pancreas or liver function. Taken together, these data indicate that induced Notch signaling in ECs contributes to systemic insulin resistance. Click here to expand this figure. Figure EV2. Pancreas and liver vasculature in NICDiOE-EC mice A. Representative confocal images showing CD31+ blood vessels (red) and insulin-positive (green) islets in pancreas sections from NICDiOE-EC mice and littermate controls 5 weeks after tamoxifen injection. Scale bar 50 μm. B. Quantification of blood vessel and islet area in control (n = 5) and NICDiOE-EC (n = 7) mice. Data represent mean ± SEM, unpaired t-test. C. Representative confocal images showing collagen IV+ blood vessels (red) in liver sections from NICDiOE-EC mice and littermate controls 5 weeks after tamoxifen injection. Scale bar 50 μm. D. Quantification of microvessel density and average vessel size in control (n = 3) and NICDiOE-EC (n = 3) mice. Data represent mean ± SEM, Mann–Whitney, and Welch's t-test. Download figure Download PowerPoint Inactivation of endothelial Notch signaling improves insulin sensitivity To test if inhibition of EC Notch signaling would improve insulin sensitivity, we inactivated the Rbpj gene encoding Rbp-jκ, the essential transducer of signal transduction downstream of all four Notch receptors, specifically in ECs. Tamoxifen-driven genetic deletion of Rbpj in adult mice (RbpjiΔEC) (Ramasamy et al, 2014; Jabs et al, 2018) did not affect body weight compared to littermate controls (Fig EV3A) but led to changes in vascular morphology and microvessel density in skeletal muscle tissue as we have previously described (Jabs et al, 2018). Five weeks after gene inactivation, RbpjiΔEC mice had lower glucose and insulin levels in blood (Fig 2A and B). In addition, insulin-mediated lowering of blood glucose was more pronounced and lasted longer in RbpjiΔEC mice compared to controls (Fig 2C and D). Moreover, RbpjiΔEC mice showed better tolerance to glucose (Fig 2E and F). Importantly, these metabolic alterations occurred before the onset of heart failure, which we had observed in our previous study around 7 weeks after gene inactivation (Jabs et al, 2018). Click here to expand this figure. Figure EV3. Characterization of vasculature and pancreas function in RbpjiΔEC mice A. Body mass of control (n = 8) or RbpjiΔEC (n = 6) mice 7 weeks after tamoxifen injection. Data represent mean ± SEM, unpaired t-test. B. Representative confocal images showing CD31+ blood vessels (red) and insulin-positive (green) islets in pancreas sections from RbpjiΔEC mice and littermate controls 6 weeks after tamoxifen injection. Scale bar 50 μm. C, D. Quantification of islet area (C) and blood vessel area (D) in control (n = 4) and RbpjiΔEC (n = 3) mice. Data represent mean ± SEM, Welch's t-test. E, F. Total insulin content (E) and ex vivo glucose stimulated insulin secretion (F) from pancreatic islets isolated from control (n = 5) and RbpjiΔEC (n = 4) mice. Data represent mean ± SEM, unpaired t-test. G, H. Plasma C-peptide levels (G) and blood glucose levels (H) in control and RbpjiΔEC mice after glucose stimulation. n = 4, data represent mean ± SEM, unpaired t-test. Download figure Download PowerPoint Figure 2. Endothelial-specific Notch deletion improves insulin sensitivity A. Blood glucose levels of control (n = 8) and Rbpji∆
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