Decision letter: Endothelial-specific FoxO1 depletion prevents obesity-related disorders by increasing vascular metabolism and growth

Field: Medicine (Health Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Impaired angiogenesis is a hallmark of metabolically dysfunctional adipose tissue in obesity. However, the underlying mechanisms restricting angiogenesis within this context remain ill-defined. Here, we demonstrate that induced endothelial-specific depletion of the transcription factor Forkhead Box O1 (FoxO1) in male mice led to increased vascular density in adipose tissue. Upon high-fat diet feeding, endothelial cell FoxO1-deficient mice exhibited even greater vascular remodeling in the visceral adipose depot, which was paralleled with a healthier adipose tissue expansion, higher glucose tolerance and lower fasting glycemia concomitant with enhanced lactate levels. Mechanistically, FoxO1 depletion increased endothelial proliferative and glycolytic capacities by upregulating the expression of glycolytic markers, which may account for the improvements at the tissue level ultimately impacting whole-body glucose metabolism. Altogether, these findings reveal the pivotal role of FoxO1 in controlling endothelial metabolic and angiogenic adaptations in response to high-fat diet and a contribution of the endothelium to whole-body energy homeostasis. https://doi.org/10.7554/eLife.39780.001 eLife digest In the body, thread-like blood vessels called capillaries weave their way through our tissues to deliver oxygen and nutrients to every cell. When a tissue becomes bigger, existing vessels remodel to create new capillaries that can reach far away cells. However, in obesity, this process does not happen the way it should: when fat tissues expand, new blood vessels do not always grow to match. The starved fat cells can start to dysfunction, which causes a range of issues, from inflammation and scarring of the tissues to problems with how the body processes sugar and even diabetes. Yet, it is still unclear why exactly new capillaries fail to form in obesity. What we know is that a protein called FoxO (short for Forkhead box O) is present in the cells that line the inside of blood vessels, and that it can stop the development of new capillaries. FoxO controls how cells spend their energy, and it can force them to go into a resting state. During obesity, the levels of FoxO actually increase in capillary cells. Therefore, it may be possible that FoxO prevents new blood vessels from growing in the fat tissues of obese individuals. To find out, Rudnicki et al. created mice that lack the FoxO protein in the cells lining the capillaries, and then fed the animals a high-fat diet. These mutant mice had more blood vessels in their fat tissue, and their fat cells looked healthier. They also stored less fat than normal mice on the same diet, and their blood sugar levels were normal. This was because the FoxO-deprived cells inside capillaries were burning more energy, which they may have obtained by pulling sugar from the blood. These results show that targeting the cells that line capillaries helps new blood vessels to grow, and that this could mitigate the health problems that arise with obesity, such as high levels of sugar (diabetes) and fat in the blood. However, more work is needed to confirm that the same cellular processes can be targeted to obtain positive health outcomes in humans. https://doi.org/10.7554/eLife.39780.002 Introduction Obesity is a growing problem worldwide (Moller and Kaufman, 2005; Tchernof and Després, 2013) and thus an urgent need exists to identify molecular processes and signaling pathways that may serve as novel therapeutic targets to hinder obesity-induced pathologies. Although the underlying causes of obesity-related complications are multifactorial, the dysfunction of adipose tissue plays a central role in the development of peripheral tissue metabolic disturbances, ultimately reflecting systemically in dyslipidemia, insulin resistance and hyperglycemia (Moller and Kaufman, 2005; Fuster et al., 2016). Capillary endothelial cells (EC) are well-known regulators of tissue adaptation to pathologic challenges through their prominent role in blood vessel formation and remodeling. During expansion of visceral adipose tissue, impaired vascular remodeling promotes hypoxia, inflammation, and fibrosis (Corvera and Gealekman, 2014; Fuster et al., 2016). Conversely, forced stimulation of vascular growth in adipose tissue of obese rodents improves adipose tissue function (Sun et al., 2012; Robciuc et al., 2016; Seki et al., 2018), counteracting obesity-related metabolic disorders (Sung et al., 2013; Seki et al., 2018). These findings indicate that the remodeling capacity of microvascular ECs during obesity is vital not only for the adipose tissue function but also for the development of systemic metabolic disturbances. However, surprisingly little is understood about the signaling pathways that limit the angiogenic response of EC in obesity. Forkhead Box O1 (FoxO1) signaling is essential to the homeostasis of EC and restricts vascular growth (Wilhelm et al., 2016). In addition to the control of angiogenesis-related genes (Potente et al., 2005; Paik et al., 2007; Milkiewicz et al., 2011; Roudier et al., 2013; Wilhelm et al., 2016), FoxO1 is a gatekeeper of EC metabolism; its overexpression reduces the metabolic rate of EC and enforces a state of endothelial quiescence (Wilhelm et al., 2016). Thus, this transcription factor is one of the major regulators of angiogenic capacity, since the switch from a quiescent to an angiogenic phenotype requires a coordinated increase in EC metabolic activity to meet the higher demand for energy and biomass production associated with proliferation and migration (De Bock et al., 2013; Schoors et al., 2015; Kim et al., 2017). Compelling observational evidence indicates that endothelial FoxO1 dysregulation coincides with obesity-associated metabolic disturbances. For instance, FoxO1 protein levels were elevated in capillaries from skeletal muscle of mice fed a high-fat diet (Nwadozi et al., 2016) and the activity of endothelial FoxO1 correlated with adipose insulin resistance of obese subjects (Karki et al., 2015). Additionally, in vitro conditions that mimic hyperglycemia and insulin resistance increase FoxO1 protein and activity in EC (Tanaka et al., 2009; Nwadozi et al., 2016). Nevertheless, to our knowledge, the contribution of FoxO1 signaling to vascular remodeling during obesity has not been addressed experimentally. To date, only a few reports have assessed the relevance of endothelial FoxO proteins in diet-induced disorders, but none have examined the influence on adipose tissue. Moreover, those studies employed simultaneous EC-specific depletion of multiple FoxOs (FoxO1, FoxO3, and FoxO4) and transgenic lines in which gene targeting was not exclusive to EC (Tanaka et al., 2009; Tsuchiya et al., 2012; Nwadozi et al., 2016), preventing discrimination of the specific functions of endothelial FoxO1. Notably, it has been shown that in vitro conditions associated with FoxO1 dysregulation can also compromise EC metabolism (Zhang et al., 2000; Du et al., 2003; Jais et al., 2016). Although this suggests that the interplay between endothelial FoxO1 levels and EC metabolic activity may be critically implicated in limiting vascular remodeling in obesity, this concept demands validation. The converging roles of FoxO1 in the angiogenic phenotype and the metabolism of quiescent EC led us to hypothesize that FoxO1 is a critical nodal point in determining the response of capillary EC to obesity. Consequently, we postulated that targeted endothelial-specific depletion of FoxO1 would provoke capillary growth, preventing obesity-driven adipose tissue dysfunction, and provide a valuable tool to unmask the role of the microvascular endothelium metabolism in the pathophysiology of obesity. Results Mice with EC-FoxO1 depletion exhibit greater vascular density in visceral adipose tissue To assess the involvement of EC-FoxO1 in the control of vascular growth in the adipose tissue of adult mice, we utilized a mouse model of EC-selective depletion of FoxO1 expression (referred to 'EC-FoxO1 KD' mice hereafter) through inactivation of the Foxo1 gene specifically in EC. Foxo1 floxed (Foxo1f/f) mice were crossbred with Pdgfb-iCreERT2 mice that express tamoxifen-activated Cre recombinase in EC and Cre-mediated recombination of Foxo1 was induced in adult mice. Littermate mice homozygous for the floxed Foxo1 allele but not expressing Cre recombinase were used as controls. After tamoxifen injection, Foxo1 recombination was observed within adipose and skeletal muscle but not within the liver, as endothelial Pdgfb expression is undetectable in this organ (Hellström et al., 1999). The endothelial cell specificity of the recombinase activity was confirmed in microvascular EC isolated from adipose tissue (Figure 1A). Consequently, Foxo1 transcript level, as measured by qPCR, was decreased by 50% in microvascular EC of EC-FoxO1 KD mice relative to control littermates 8 weeks after the administration of tamoxifen, confirming effective and stable Foxo1 depletion in these cells (Figure 1B). Of note, Foxo3 mRNA expression was unaltered in microvascular EC (Figure 1B), demonstrating a lack of compensation by this FoxO family member in response to the depletion of Foxo1. Moreover, consistent with the previously described absence of Pdgfb-Cre activity within macrophages (Claxton et al., 2008), no significant changes in Foxo1 mRNA levels were detected in CD16/CD32+ immune cells from white adipose tissue (Figure 1C), indicating that Cre-mediated recombination did not occur in these stromal cells. The depletion of EC-Foxo1 in microvascular beds of EC-FoxO1 KD mice also was validated via assessment of FoxO1 protein levels by Western blotting. In agreement with the lower mRNA levels observed in microvascular ECs from adipose tissue, protein levels of FoxO1 were diminished by 70% in capillary fragments from skeletal muscle of EC-FoxO1 KD mice compared to control littermates 6 weeks after the administration of tamoxifen (Figure 1D). Together, these results not only imply that successful Foxo1 depletion was constrained to the endothelial cell compartment, particularly microvascular beds, but also support the use of EC-FoxO1 KD mice as an appropriate model to assess the relevance of endothelial FoxO1 for vascular remodeling during adipose tissue expansion. Figure 1 Download asset Open asset Endothelial-specific depletion of Foxo1 induced in adult male mice effectively reduces FoxO1 levels in skeletal muscle and adipose microvascular beds. (A) PCR of genomic DNA from multiple organs of control (Cre-; Foxo1f/f) and EC-FoxO1 KD mice using primers for the floxed and deleted (E1∆E3) alleles. (B–C) Gene expression analysis of microvascular EC and CD16/CD32+ cells isolated from white adipose tissue of Control (n = 6) and EC-FoxO1 KD (n = 3–5) mice. (D) Representative Western blot images and quantitative analysis of FoxO1 and β-actin levels in capillary fragments isolated from skeletal muscle (n = 3–4). Results are expressed relative to β-actin levels. Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test. https://doi.org/10.7554/eLife.39780.003 EC-FoxO1 KD mice maintained on a normal chow (NC) diet for 16 weeks exhibited no gross abnormalities and similar body weight gain compared to control counterparts (8.32 ± 1.3 vs. 7.75 ± 1.17 g, n = 6/group), but significantly increased mRNA levels of the EC marker Pecam1 in eWAT (Figure 2A). When blood vessels were visualized by whole-mount staining with G. simplicifolia lectin, it was evident that the vascular density of visceral adipose tissue from EC-FoxO1 KD mice (Figure 2B–C) was significantly higher. EC-FoxO1 depletion did not alter the number of vessel branch points (Figure 2D). On the other hand, vessels in the adipose of EC-FoxO1 KD mice were significantly enlarged, showing increased vessel diameter, compared to control littermates (Figure 2E), which was consistent with the reported influence of EC-Foxo1 depletion in vascular development in retinas (Wilhelm et al., 2016). No difference in the expression of Pecam1 was detected in other assessed tissues, such as skeletal muscle and liver (Figure 2A). Figure 2 Download asset Open asset Greater vascular density in visceral adipose tissue of normal chow-fed EC-FoxO1 KD mice. (A) Pecam1 mRNA levels in various tissues of Control and EC-FoxO1 KD mice after 16 weeks of normal chow (NC) diet (Control n = 6, EC-FoxO1 KD n = 5). (B) Representative confocal images of adipose tissue whole-mount staining with BODIPY 493/503 (green) and G. simplicifolia lectin (red) (×20 magnification; scale bar = 100 μm). (C–E) Lectin area (C), capillary branch density (D) and microvessel diameters (E) were quantified from confocal images (Control n = 5, EC-FoxO1 KD n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test. https://doi.org/10.7554/eLife.39780.004 EC-FoxO1 depletion provokes greater microvascular remodeling under the stimulus of a high-fat diet To determine whether EC-FoxO1 depletion evokes vascular growth during adipose expansion in response to excess caloric consumption, we challenged mice with a prolonged high-fat diet (HF) and assessed tissue angiogenesis. Gene expression analysis indicated that EC-FoxO1 depletion resulted in higher Pecam1 mRNA levels in multiple adipose tissue depots: eWAT, subcutaneous and brown adipose tissue (BAT, Figure 3A). In line therewith, transcript levels of other EC markers, von Willebrand factor (Vwf) and endothelial nitric oxide synthase (Nos3) were elevated in the eWAT of HF-fed EC-FoxO1 KD mice (Figure 3B). Whole-mount staining of adipose tissue revealed remarkable increases in vascular area and number of vessel branch points in the eWAT of HF-fed EC-FoxO1 KD (Figure 3C–E). Consistently, quantitative histological analysis showed that capillary number per adipocyte (capillary to adipocyte ratio) was significantly higher in eWAT of HF-fed EC-FoxO1 KD mice, further validating the greater microvascular content in eWAT of these mice compared to HF-fed control counterparts (Figure 3F–G). Furthermore, EC-FoxO1 depletion led to significant capillary enlargement in eWAT (Figure 3H–I). Of note, the increase of vessel diameter in HF-fed EC-FoxO1 KD mice was greater than observed in NC-fed EC-FoxO1 KD mice (1.8 vs. 1.3-fold increase), suggesting that the enlargement of capillaries promoted by EC-FoxO1 depletion is exacerbated by HF feeding. Figure 3 Download asset Open asset EC-Foxo1 depletion strongly induces vascular growth within adipose tissue in response to HF diet. (A) Pecam1 mRNA levels in different adipose tissue depots of Control and EC-FoxO1 KD mice after 16 weeks of high-fat (HF) diet (Control n = 5–7, EC-FoxO1 KD n = 7). (B) Gene expression analysis of eWAT of HF-fed Control and EC-FoxO1 KD mice (Control n = 7, EC-FoxO1 KD n = 6–7). (C) Representative confocal images of adipose tissue whole-mount staining with BODIPY 493/503 (green) and G. simplicifolia lectin (red) (C - scale bar = 100 μm). (D,E and I). Lectin area (D) and capillary branch density were quantified from these images (Control, n = 6; EC-FoxO1 KD, n = 5 or 6). (F–G) G. simplicifolia lectin (green) and Wheat germ aggluttinin (red) staining of paraffin-sectioned adipose tissue (F - scale bar = 100 μm) was used to assess capillary to adipocyte ratio (G). (H) Representative confocal images of adipose tissue whole-mount staining with Isolectin alone (greyscale; scale bar = 20 μm). (I) Microvessel diameters were quantified from confocal images (Control, n = 6; EC-FoxO1 KD, n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, ***p < 0.001, calculated with two-tailed unpaired t-test. https://doi.org/10.7554/eLife.39780.005 Subsequent gene expression analysis showed that EC-FoxO1 depletion did not change transcript levels of Pecam1 in the liver (corresponding with the lack of Cre recombination in this organ) but did upregulate its expression in skeletal muscle, suggesting that under the stimulus of HF diet, EC-FoxO1 depletion also induces microvascular remodeling in this tissue (Figure 4A). Skeletal muscle of HF-fed EC-FoxO1 KD mice displayed a trend towards higher capillary:fiber ratio (p = 0.06) compared to control mice (Figure 4B–C). Transmission electron microscopy revealed increased capillary endothelial cross-sectional area and capillary lumen diameters in skeletal muscle of EC-FoxO1 KD mice, demonstrating a modest expansion of the size of individual capillaries (Figure 4D–F). Taken together, these data indicate that EC-FoxO1 depletion results in remarkable vascular growth in response to HF diet, which is particularly pronounced within visceral adipose tissue. Figure 4 Download asset Open asset EC-Foxo1 depletion also favors microvascular expansion in skeletal muscle under HF diet feeding. (A) Pecam1 mRNA levels in liver and skeletal of HF-fed Control (n = 5–6) and EC-FoxO1 KD (n = 6–7) mice. (B) Images of EDL muscle stained with Isolectin-FITC to identify capillaries (scale bar = 50 μm). (C) Capillary to fiber (C:F) ratios were calculated from 3 to 4 independent fields of view per mouse (Control n = 6, EC-FoxO1 KD n = 6). (D) Representative EM images of capillaries within skeletal muscle from HF-fed Control and EC-FoxO1 KD mice (×6.5k magnification; scale bar = 2 μm). (E–F) EC cross-sectional area (E) and capillary luminal diameter were quantified from EM images from n = 4 mice per group, with individual capillary measurements shown (F). Data in all panels are expressed as mean ± SEM; *p < 0.05, calculated with two-tailed unpaired t-test. https://doi.org/10.7554/eLife.39780.006 EC-FoxO1 KD mice exhibit a healthier adipose tissue expansion in response to HF diet The vasculature is critical for maintenance of adipose tissue homeostasis during obesity-driven adipocyte enlargement. Thus, we inferred that the increased vascular density observed with EC-FoxO1 depletion may hinder adipose tissue expansion and dysfunction induced by high-fat diet. Although HF-fed EC-FoxO1 KD mice showed only a trend towards reduced body weight gain (p = 0.06), these mice displayed less fat accumulation, showing lower trunk fat content, smaller retroperitoneal (rWAT) and subcutaneous fat pads compared to control mice (Figure 5A–C and Table 1). The phenotype was not explained by changes in food consumption (Figure 5—figure supplement 1A). HF-fed EC-FoxO1 KD mice also displayed lower fed levels of serum triglycerides and glycerol, and less hepatic lipid accumulation (Figure 5—figure supplement 1B–D), suggesting an improvement in the capacity to handle dietary nutrient excess in these mice. Moreover, histological analysis revealed that increased vascular growth in adipose tissue of HF-fed EC-FoxO1 KD mice was associated with smaller-sized and generally spherical adipocytes, whereas adipocytes from HF-fed control mice were large with irregular polygonal shapes (Figure 5D–E), which was previously related to cellular stress (Giordano et al., 2013). Of note, adipocytes from EC-FoxO1 KD mice retained a unilocular structure (Figure 5D) rather than the hallmark multilocular morphology of brown fat. Furthermore, no change in the mRNA levels of browning markers Ucp1 and Prdm16 (Figure 5F) was detected with EC-FoxO1 depletion. Correspondingly, we did not observe any difference in mitochondrial protein content of eWAT nor in ADP-stimulated respiration through either Complex I (pyruvate/malate, glutamate) or Complex II (succinate) (Figure 5—figure supplement 2A–C). Isoproterenol-stimulated phosphorylation of the hormone-sensitive lipase (HSL) was unaffected (Figure 5—figure supplement 2D–E), indicating that EC-FoxO1 depletion did not impact the adipose tissue sensitivity to lipolytic stimuli. In contrast, and consistent with an improved function, eWAT from HF-fed EC-FoxO1 KD mice displayed enhanced Akt phosphorylation in response to insulin (Figure 5G–H), which was accompanied by higher Adiponectin mRNA levels and concomitant lower Leptin expression (Figure 5I). Collectively, these findings demonstrate that depletion of EC-FoxO1 signaling exerts a protective effect against obesity-induced metabolic remodeling of adipose tissue without promoting a browning phenotype. Notably, the transcripts levels of Vegfa and Apelin were also higher in eWAT from HF-fed EC-FoxO1 KD mice (Figure 5J), providing evidence that the improvements in adipose phenotype include a more pro-angiogenic adipose tissue microenvironment. Figure 5 with 2 supplements see all Download asset Open asset EC-FoxO1 KD mice exhibit a healthier adipose tissue expansion in response to HF diet. (A) Body weights during 16 weeks of HF feeding. (B) Summarized weight gain over the course of 0–14 weeks (Control n = 7, EC-FoxO1 KD n = 7). (C) Abdominal transverse micro-CT images of HF-fed Control (n = 5) and EC-FoxO1 KD (n = 6) mice (upper panel). Fat content (shown in white) was calculated as % of total trunk volume. (D) Representative and images of adipose tissue from the fat (scale bar = 100 μm). (E) adipocyte cross-sectional area (Control n = 5 EC-FoxO1 KD n = 5). mRNA for browning markers Ucp1 and Prdm16 relative to (Control n = 7, EC-FoxO1 KD n = 7). Representative Western blot images and quantitative analysis (H) of and total Akt levels in eWAT after in the absence or of Results are expressed relative to total Akt levels (Control n = EC-FoxO1 KD n = 7). mRNA for and and angiogenic markers Vegfa and in eWAT relative to (Control n = EC-FoxO1 KD n = Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired t-test. Table 1 weights of Control and EC-FoxO1 KD mice after 16 weeks of HF diet weight ± ± ± ± ± ± adipose ± ± ± ± ± ± ± ± ± ± ± ± ± ± adipose retroperitoneal adipose brown adipose tissue Data are expressed as mean ± n = per was using unpaired < Control EC-FoxO1 depletion provokes a metabolic glucose in HF-fed mice To the metabolic of EC-FoxO1 whole-body metabolic functions were for in a of HF-fed mice. EC-FoxO1 KD mice exhibited reduced and increased during the (Figure with production and activity levels compared to control mice (Figure These data indicated that EC-FoxO1 KD mice increased of relative to as an energy suggesting that EC-FoxO1 depletion whole-body energy homeostasis towards glucose with these HF-fed EC-FoxO1 KD mice displayed more glucose from the blood during glucose tolerance (Figure However, higher glucose tolerance was not associated with whole-body insulin on insulin tolerance (Figure or Akt phosphorylation in the skeletal muscle (Figure supplement 1). the on glucose metabolism observed in HF-fed EC-FoxO1 KD mice, no change in whole-body glucose metabolism was detected in NC-fed EC-FoxO1 KD mice compared to control counterparts (Figure supplements 1 and fasting glycemia was significantly lower (Figure whereas serum lactate levels were elevated in HF-fed EC-FoxO1 KD mice compared to their littermates ± vs. ± = n = These findings imply that whole-body glucose metabolism of EC-FoxO1 KD mice on a HF may be to higher glucose us to that increased glycolytic at the tissue level to the metabolic phenotype of EC-FoxO1 KD mice. To this we assessed the expression of glycolytic the glucose the 2 and and the mRNA levels of glycolytic with the of were in the eWAT from HF-fed EC-FoxO1 KD mice compared to control mice, (Figure Furthermore, mRNA levels of the lactate 5, were also increased in eWAT of HF-fed EC-FoxO1 KD mice (Figure consistent with an increased glycolytic of glucose to lactate in the adipose tissue of these mice. Figure 6 with 2 supplements see all Download asset Open asset EC-Foxo1 depletion improves glucose homeostasis in HF-fed mice. consumption production ratio - (C) and activity (D) were measured during using a Control n = 5, EC-FoxO1 KD n = 6). (E) tolerance of HF-fed Control and EC-FoxO1 KD mice was examined by glucose tolerance after weeks of HF diet and 16 under the Control n = 7, EC-FoxO1 KD n = 7). sensitivity of HF-fed Control and EC-FoxO1 KD mice was assessed by insulin tolerance after weeks of HF diet and 4 (H) over the Control n = EC-FoxO1 KD n = (I) glucose levels of HF-fed Control (n = and EC-FoxO1 KD (n = mice after 4 eWAT gene expression analysis by (Control n = EC-FoxO1 KD n = 6). Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, calculated with two-tailed unpaired or when a was detected by model EC the changes in glucose metabolism EC on to support angiogenesis (De Bock et al., 2013) and a reported that FoxO1 overexpression EC metabolism (Wilhelm et al., 2016). Therefore, we that the changes in glucose were at in to increased metabolic activity of EC from EC-FoxO1 depletion. To this we isolated the EC from white adipose tissue depots of mice fed a HF diet for weeks and assessed gene expression of glycolytic with the findings observed with adipose tissue, increased mRNA levels of glycolytic genes and were detected in the EC from adipose tissue of HF-fed EC-FoxO1 KD mice (Figure also whether the elevated gene expression of glycolytic markers in EC from EC-FoxO1 KD mice would with greater glycolytic capacity, as assessed by cellular glucose and changes in glucose consumption and the accumulation of In agreement with higher transcript levels of EC isolated from adipose tissue of HF-fed EC-FoxO1 KD mice displayed increased glucose than EC from floxed controls (Figure Moreover, of glucose consumption and lactate production were higher in EC with FoxO1 depletion compared to control cells (Figure Additionally, we observed elevated mRNA in the EC from EC-FoxO1 KD mice, indicating that an enhanced proliferative state coincides with the glycolytic activity of these EC (Figure Figure Download asset Open asset FoxO1 is a critical of glucose metabolism in EC. Gene expression analysis of EC from adipose tissue from Control (n = and EC-FoxO1 KD (n = mice fed a HF diet for (C–E) glucose (C) glucose consumption (D) and lactate production (E) in EC from HF-fed EC-FoxO1 KD (n = mice compared to Control (n = Data in all panels are expressed as mean ± SEM; *p < 0.05, **p < 0.01, calculated with two-tailed unpaired or when a was detected by model To that dysregulation of FoxO1 signaling is in of glycolytic we skeletal muscle EC in and high glucose as in vitro studies have shown that hyperglycemia can increase FoxO1 activity (Tanaka et al., and