Keywords: endothelium, hypoxia-inducible factor, insulin resistance, insulin signaling
Abstract
Endothelial cell insulin resistance contributes to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIFs) modulate insulin sensitivity, and we have previously shown that a negative regulator of HIF activity, CREB-binding protein/p300 (CBP/p300) interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined whether CITED2 regulates endothelial insulin sensitivity. In endothelial cells isolated from mice with a “floxed” mutation in the Cited2 gene, loss of CITED2 markedly enhanced insulin-stimulated Akt phosphorylation without altering extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation. Similarly, insulin-stimulated Akt phosphorylation was increased in aortas of mice with endothelial-specific deletion of CITED2. Consistent with these observations, loss of CITED2 in endothelial cells increased insulin-stimulated endothelial nitric oxide synthase phosphorylation, Vegfa expression, and cell proliferation. Endothelial cells lacking CITED2 exhibited an increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, whereas IRS-1 was unchanged. Conversely, overexpression of CITED2 in endothelial cells decreased IRS-2 protein by 55% without altering IRS-1, resulting in impaired insulin-stimulated Akt phosphorylation and Vegfa expression. Overexpression of HIF-2α significantly increased activity of the Irs2 promoter, and coexpression of CITED2 abolished this increase. Moreover, chromatin immunoprecipitation (ChIP) showed that loss of CITED2 increased occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results show that CITED2 selectively inhibits endothelial insulin signaling and action through the phosphoinositide 3-kinase (PI3K)/Akt pathway via repression of HIF-dependent IRS-2 expression. CITED2 is thus a promising target to improve endothelial insulin sensitivity and prevent the vascular complications of diabetes.
NEW & NOTEWORTHY Endothelial cell insulin resistance is a major contributor to the development of diabetic complications. In this study, we have shown that CITED2, a transcriptional coregulator, inhibits endothelial insulin signaling through the PI3K/Akt pathway via repression of HIF-dependent IRS-2 expression, and that deletion of CITED2 enhances insulin signaling. Thus, CITED2 represents a novel and promising target to improve insulin sensitivity in endothelial cells and prevent vascular complications in diabetes.
INTRODUCTION
Insulin resistance is a central feature of type 2 diabetes, and insulin action is impaired in several tissues, including the vasculature (1). Insulin action on vascular endothelial cells regulates blood vessel dilation and capillary recruitment, leukocyte adhesion, and angiogenesis (1). Insulin resistance negatively impacts these endothelial functions, and in animal models, it contributes to the development of diabetes complications such as atherosclerosis (2), tumor formation (3), and poor recovery from tissue ischemia (4). Conversely, enhanced endothelial insulin signaling has been shown to reduce the severity of atherosclerosis (5) and improve wound healing (6) in type 2 diabetes. Therefore, increasing insulin action on endothelial cells is a promising strategy for prevention of vascular complications of diabetes, and there is a need to identify novel targets to enhance endothelial insulin sensitivity.
In hypoxia, cells adapt to reduced oxygen availability by increasing glucose uptake and glycolytic flux (7). There is evidence that this metabolic reprogramming is achieved, in part, via regulation of insulin sensitivity. Hypoxia has been shown to potentiate insulin signaling in liver (8) and skeletal muscle (9) and to increase whole body insulin sensitivity in people with diabetes (10). Hypoxia-inducible factors (HIFs), the master coordinators of the cellular response to hypoxia, may mediate these changes; for example, HIF-2α enhances insulin signaling in hepatocytes by increasing expression of insulin receptor substrate (IRS)-2 (8, 11). This indicates that factors that regulate HIF will in turn regulate insulin sensitivity.
We have recently shown that a negative regulator of HIF activity, CREB-binding protein/p300 (CBP/p300) interacting transactivator-2 (CITED2), is increased in the vasculature of mouse models and of patients with type 2 diabetes (12). We therefore decided to investigate whether CITED2 regulates insulin signaling in endothelial cells and to evaluate its potential as a target for enhancing insulin sensitivity in the vasculature.
METHODS
Animals
Mice with endothelial-specific deletion of CITED2 (Cdh5-cre Cited2fl/fl) were generated as described previously (12). All protocols for animal use and euthanasia were carried out in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institutional Animal Care & Use Committee of the Joslin Diabetes Center.
Cell Culture
Primary mouse lung endothelial cells and MS1 endothelial cells were cultured as described previously (12).
Real-Time PCR and Western Blotting
Real-time PCR was performed as previously described (2) with the following primers: Vegfa forward: 5′- GCCAAGTGGTCCCAGGCTGC-3′ and reverse: 5′- TCGGACGGCAGTAGCTTCGCT-3′; Irs1 forward: 5′- ACTGGACATCACAGCAGAATGAA-3′ and reverse: 5′- AAGACGTGAGGTCCTGGTTG-3′; Irs2 forward: 5′- TGCAAGCATCGACTTCCTGT-3′ and reverse: 5′- GCTGGTAGCGCTTCACTCTTT-3′; and Rplp0 forward: 5′- GCTCCAAGCAGATGCAGCA-3′ and reverse: 5′- CCGGATGTGAGGCAGCAG-3′. Western blotting was performed as previously described (2) with the following primary antibodies: CITED2 (R&D MAB5005), β-actin (Cell Signaling 8457), insulin receptor β (Cell Signaling 3025), p-insulin receptor β (Cell Signaling 3026), Akt (Cell Signaling 9272), p-Akt (Cell signaling 4060), ERK1/2 (Cell Signaling 4695), p-ERK1/2 (Cell Signaling 9101), eNOS (BD Biosciences 610297), p-eNOS (BD Biosciences 612392), IRS-1 (Millipore 06–248 and Cell Signaling 2382), and IRS-2 (Millipore MABS15 and Cell Signaling 3089). Expression of the target gene was normalized to expression of the “housekeeping gene” Rplp0 using the ΔΔCT method (13).
Flow Cytometry
Flow cytometry of cultured primary endothelial cells was performed as previously described (12).
In Vivo Insulin Treatment
Mice with endothelial-specific deletion of CITED2 (Cdh5-cre Cited2fl/fl) were given insulin as a bolus injection in the inferior vena cava, and aortas were removed as described previously (2).
Plasmid Construction
The full-length cDNA of Cited2 and 1,268 bp of the Irs2 promoter were amplified using PCR with the primers Cited2 (forward: 5'- AGCTACTAGTGCAGACCATATGATGGCC-3′; reverse: 5′- AGCTACGGTTCAACAGCTGACTCTG-3′) and Irs2 (forward: 5′- ATCGGGTACCCGGCACTATGGAAAC-3′; reverse: 5′- ATATACGCGTCGCCGCCGCTTCAG-3′) and inserted into the pLEX and pGL3 basic vectors, respectively to create Cited2-pLEX and Irs2-pGL3.
Lentiviral Production and Transduction
Lentiviral particles expressing flag-tagged CITED2 (FLAG-CITED2) were produced by cotransfection of HEK 293 T cells with Cited2-pLEX, lentiviral packaging plasmid psPAX2, and VSV-G envelope packaging plasmid pMD2.G. After 5 h, the transfection medium was changed, and recombinant lentiviral particles were harvested after 24 h for subsequent transduction.
MS1 endothelial cells were twice cultured with lentivirus for 12 h. Lentiviral medium was replaced with complete medium for 48 h. Stably transduced MS1 cells were selected with 0.1% puromycin (Sigma) for 72 h.
Luciferase Assay
Normoxia-stable pcDNA3 mHIF-2α MYC (P405A/P530V/N851A), a gift from Dr. Celeste Simon (Plasmid No. 44027, Addgene) (14), Irs2 promoter reporter Irs2-pGL3, and Renilla luciferase plasmids were cotransfected into lentivirus-infected MS1 endothelial cells with Lipofectamine 3000 (Invitrogen). After 24 h, cells were lysed, and the Dual Luciferase Reporter Gene Assay Kit (Yeasen Cat. No. 11402) was used according to the manufacturer’s instructions.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100-616, Novus Biologicals). Relative enrichment of chromatin was determined by real-time PCR using primers for hypoxia-response elements in the Irs2 promoter (forward: 5′- CCGCCGCACAGTGAGTAAC-3′; reverse: 5′- GCAGAGTCACGTGTTGTTTTGC-3′).
Statistical Analysis
Comparisons were made using Student’s t test and one- or two-way ANOVA followed by Tukey’s post hoc test as specified in the figure legends, with P < 0.05 considered statistically significant. All statistical analyses were performed using GraphPad Prism 9.0.2. In text and graphs, data are presented as means ± SE.
RESULTS
CITED2 Selectively Inhibits Insulin Signaling in Endothelial Cells
Endothelial cells were isolated from lungs of Cited2fl/fl mice by immunomagnetic isolation and infected with an adenovirus-expressing cre recombinase or control adenovirus. Although present in control cultures, CITED2 was not detectable by Western blot following cre-mediated recombination (Fig. 1A). In cells lacking CITED2, the insulin-stimulated increase in Akt phosphorylation at Ser473 (120.3 ± 30-fold, 67.3-fold greater than CITED2 KO baseline) was significantly greater than the increase observed in control cells (2,918% ± 446%, 29.2-fold greater than control cell baseline) (Fig. 1, B and D). However, insulin-stimulated insulin receptor β and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation were similar in control and CITED2 knockout cells (Fig. 1, B, C, and E). No differences in baseline phosphorylation of insulin receptor β, Akt, or ERK1/2 were observed. Thus, CITED2 selectively inhibits insulin signaling via the phosphoinositide 3-kinase (PI3K)-Akt pathway in endothelial cells.
Figure 1.
CITED2 selectively inhibits insulin signaling in endothelial cells in vitro and in vivo. A–E: lung endothelial cells were isolated from Cited2fl/fl mice and treated with adenovirus expressing cre recombinase (Ad-Cre) or GFP only (Ad-GFP), thereby creating CITED2 knockout (CITED2 KO) and control cultures from the same cell isolation. A: CITED2 expression was measured in cell lysate by Western blotting. B: cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein expression was measured in cell lysate by Western blotting. C–E: quantitative analysis of insulin-stimulated phosphorylation of insulin receptor-β (INSRβ), Akt, and ERK based on densitometry of 3–4 independent experiments and normalization to the baseline phosphorylation in control cells. F–G: Cdh5-cre Cited2fl/fl and Cited2fl/fl control mice were injected intravenously with 5 U insulin or vehicle. F: after 5 min, the aorta was isolated and flash-frozen. Levels of Akt, phosphorylated Akt, and β-actin were measured by Western blotting of tissue lysate. G: quantitative analysis of insulin-stimulated Akt phosphorylation based on densitometry of 5 pairs of animals. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2; ERK, extracellular signal-related kinase; GFP, green fluorescent protein.
To determine whether CITED2 regulates insulin sensitivity in vivo, Cdh5-cre Cited2fl/fl mice, which lack CITED2 specifically in endothelial cells, and cre-negative controls were given insulin intravenously, and insulin-induced signaling was assessed in the aorta. Total Akt (Supplemental Fig. S1, see https://doi.org/10.6084/m9.figshare.14516781.v1) and baseline Akt phosphorylation levels (Fig. 1, F and G) were unchanged, but insulin-stimulated Akt Ser473 phosphorylation in the aorta was significantly greater in mice lacking CITED2 in endothelial cells (728% ± 128%) compared with the control mice (529% ± 87%, P = 0.0025; Fig. 1, F and G). This difference may be underestimated because endothelial cells constitute only a small fraction of cells in the aorta. These data demonstrate that CITED2 inhibits endothelial insulin sensitivity in vivo as it does in endothelial cells in culture.
CITED2 Impairs Insulin Action on Endothelial Cells
To assess the functional relevance of the observed increase in insulin signaling through Akt, we measured endothelial nitric oxide synthase (eNOS) phosphorylation (1), expression of vascular endothelial growth factor (VEGF) (16), and endothelial cell proliferation (12), which are well-characterized endothelial responses to insulin treatment. In CITED2 knockout cells, the insulin-stimulated phosphorylation of eNOS at Ser1176 (368% ± 53%, 4.7-fold greater than CITED2 KO baseline) was significantly greater than in control cells (235% ± 25%, 2.3-fold greater than baseline) (Fig. 2, A and B). Similarly, Vegfa mRNA level was increased by 2.1-fold in cells lacking CITED2 4 h after insulin treatment compared with control cells, in which Vegfa expression increased by only 1.5-fold (Fig. 2C). To measure cell proliferation, control and CITED2 knockout cultures were treated with insulin for 16 h; labeled with 5-ethynyl-2′-deoxyuridine (EdU), which is incorporated into newly synthesized DNA; and analyzed by flow cytometry (Fig. 2D). CITED2 knockout cells exhibited a 2.9-fold increase, relative to baseline in CITED2 KO cells, in EdU incorporation with insulin treatment, compared with 1.7-fold in control cells (compared with control cell baseline; Fig. 2E). Therefore, inhibition of PI3K-Akt signaling by CITED2 impairs the proangiogenic actions of insulin on endothelial cells.
Figure 2.
CITED2 impairs insulin action on endothelial cells. A: control and CITED2 KO lung endothelial cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Insulin-stimulated eNOS phosphorylation was measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated eNOS phosphorylation based on densitometry of 4 independent experiments and normalization to the baseline phosphorylation in control cells. C: cell cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR and normalized to expression of the housekeeping gene Rplp0 in 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Tukey’s multiple-comparisons test. D: cell cultures were serum-starved for 16 h, treated with 10 nM insulin for 16 h and then labeled with EdU for an additional 4 h. EdU-labeled cells were stained with Alexa Fluor 647 (AF647) using Molecular Probes Click-iT procedure and analyzed by flow cytometry. E: insulin-stimulated EdU incorporation relative to baseline EdU incorporation for each cell type was determined in 3 independent experiments. **P < 0.01, Student’s t test. CITED2, CBP/p300 interacting transactivator-2; CITED2 KO, CBP/p300 interacting transactivator-2 knockout; eNOS, endothelial nitric oxide synthase; GFP, green fluorescent protein.
CITED2 Represses IRS-2 Expression in Endothelial Cells
Differential expression of IRS proteins alters insulin signaling through the PI3K-Akt pathway in endothelial cells (17). Therefore, we measured levels of IRS proteins in CITED2 knockout cells. Notably, CITED2 knockout lung endothelial cells expressed 1.7-fold more Irs2 mRNA and 3.6-fold more IRS-2 protein than control cells (Fig. 3, B, C, and E), whereas no differences in Irs1 mRNA or IRS-1 protein were observed (Fig. 3, A, C, and D). Consistent with these results, overexpression of FLAG-tagged CITED2 (FLAG-CITED2) in MS1 endothelial cells reduced IRS-2 protein by 56.7% (Fig. 3, F and H) but had no effect on IRS-1 (Fig. 3, F and G). These data suggest that CITED2 inhibits insulin signaling via downregulation of IRS-2.
Figure 3.
CITED2 represses IRS-2 expression in endothelial cells. A–E: lung endothelial cells were isolated from Cited2fl/fl mice and treated with adenovirus expressing Cre recombinase (Ad-Cre) or control adenovirus expressing GFP only (Ad-GFP), creating CITED2 KO and control cultures from the same cell isolation. A–B: expression of Irs1 and Irs2 was measured by real-time PCR and normalized to the housekeeping gene Rplp0 and expression in control cells. C: expression of IRS-1 and IRS-2 was measured by Western blotting. D–E: quantitative analysis of IRS-1 and IRS-2 expression based on densitometry of 3 independent experiments and normalized to expression in control cells. F–H: MS1 endothelial cells were cultured with lentivirus expressing FLAG-CITED2 for 12 h, and lentiviral medium was then replaced with complete medium for 48 h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72 h. F: expression of IRS-1 and IRS-2 was measured by Western blotting. G–H: quantitative analysis of IRS-1 and IRS-2 expression based on densitometry of 3 independent experiments and normalized to expression in control cells. *P < 0.05, ** P < 0.01, Student’s t test. CITED2, CBP/p300 interacting transactivator-2; CITED2 KO, CBP/p300 interacting transactivator-2 knockout; eNOS, endothelial nitric oxide synthase; IRS, insulin receptor substrate.
CITED2 Overexpression Recapitulates Endothelial Insulin Resistance
We have previously shown that CITED2 expression is increased in endothelial cells in diabetes (12). To model this situation, we overexpressed FLAG-CITED2 in MS1 endothelial cells and examined the effects on insulin signaling and VEGF expression, a known target of insulin regulation (1, 16). In cells with CITED2 overexpression, phosphorylation of Akt in response to insulin treatment was completely abolished (Fig. 4, A and B). Similarly, insulin-stimulated Vegfa expression was lost with CITED2 overexpression; Vegfa levels increased significantly with insulin treatment (by 1.8-fold) in control cells, but there was no significant change in Vegfa levels in cells with CITED2 overexpression (Fig. 4C). Thus, CITED2 overexpression inhibits endothelial insulin signaling through PI3K-Akt and recapitulates features of endothelial insulin resistance observed in obesity and type 2 diabetes.
Figure 4.
CITED2 overexpression recapitulates endothelial insulin resistance. A–C: MS1 endothelial cells were cultured with lentivirus expressing FLAG-CITED2 for 12 h, and then, lentiviral medium was replaced with complete medium for 48 h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72 h. A: control and FLAG-CITED2-overexpressing cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein levels were measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated phosphorylation of Akt based on densitometry of 4 independent experiments. C: control and FLAG-CITED2-overexpressing cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR of 3 independent experiments, and relative Vegfa mRNA expression was determined by normalization to the housekeeping gene Rplp0. *P < 0.05, **P < 0.01, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2.
CITED2 Limits IRS-2 Expression in Endothelial Cells through Inhibition of HIF-2α
Hypoxia inducible factor (HIF)-2α has been shown to regulate levels of IRS-2, but not IRS-1, in hepatocytes and livers during hypoxia (8, 11). To determine whether HIF-2α regulates IRS-2 in endothelial cells, we used a luciferase reporter of Irs2 promoter activity. Expression of this reporter in MS1 endothelial cells showed that overexpression of constitutively active HIF-2α significantly increased Irs2 promoter activity (Fig. 5A). Coexpression of CITED2 in these cells abolished Irs2 promoter activity (Fig. 5A), consistent with previous evidence that CITED2 inhibits HIF transactivation in endothelial cells (12).
Figure 5.
CITED2 limits HIF-2α-mediated transcription of Irs2. A: MS1 endothelial cells transduced with lentivirus expressing FLAG-CITED2 were cotransfected with Renilla luciferase and normoxia-stable pcDNA3.1 mHIF-2α MYC P405A/P530V/N851A (HIF2α-DNA3.1), Irs2 promoter reporter (Irs2-pGL3), or empty vector control (pGL3, pcDNA3.1) plasmids. After 24 h, cells were lysed and luminescent signal measured with the Dual Luciferase Reporter Gene Assay kit. B: CITED2 KO and control endothelial cells were cross-linked, lysed, and sonicated to produce 200–1,000-bp chromatin fragments as analyzed by agarose gel electrophoresis. Immunoprecipitations were performed with 10 μg of p300 antibody (NB100-616, Novus Biologicals) or mouse IgG. Relative enrichment of chromatin was determined by real-time PCR. C: CITED2 inhibition of the p300/CBP-HIF-2α transcriptional complex decreases Irs2 transcription and IRS-2 expression, leading to impaired insulin signaling in endothelial cells. **P < 0.01, Student’s t test. ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple-comparisons test. CBP, CREB-binding protein; CITED2, CBP/p300 interacting transactivator-2; ChIP, chromatin immunoprecipitation; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; INSR, insulin receptor.
Chromatin immunoprecipitation (ChIP) followed by real-time PCR showed that occupancy of p300 on the Irs2 promoter is increased in CITED2 knockout endothelial cells. p300 binding was observed in a region of the Irs2 promoter containing hypoxia response elements; this binding increased by 4.5-fold in CITED2 knockout cells (Fig. 5B). This demonstrates that CITED2 interferes with p300 binding to the Irs2 promoter but does not directly show the involvement of HIF-2α. However, when coupled with the results of the luciferase assay, these data suggest that CITED2 downregulates IRS-2 in endothelial cells through inhibition of the HIF-2α transcriptional complex, which leads to inhibition of insulin signaling and impairment of insulin-regulated endothelial functions (Fig. 5C).
DISCUSSION
The current study identifies CITED2 as a potent regulator of insulin signaling in endothelial cells, thereby establishing CITED2 as a novel target for improving vascular insulin sensitivity in obesity and diabetes. This has significant health implications, as reversal of endothelial cell insulin resistance has the potential to prevent a range of vascular pathology in diabetes and obesity, including atherosclerosis (5), recovery from cardiac ischemia (4), and wound healing (6). It may also have implications beyond vascular biology, as CITED2 is ubiquitously expressed and may be a target for improving insulin resistance in other insulin-sensitive tissues.
IRS-2 has been previously proven to be more important for endothelial cell function than IRS-1 (18). Here, we show that CITED2 limits expression of IRS-2 in endothelial cells likely through repression of HIF-2α activity, while having no effect on expression of IRS-1. A previous report demonstrated that IRS-2 is a direct target of HIF-2α, but not HIF-1α, and that the IRS-2 promoter contains several hypoxia-response element motifs required for HIF binding (11), but additional study is required to confirm the necessity of HIF-2α for the observed changes in insulin signaling. The IRS-2 isoform-specific regulation by CITED2 likely contributes to the selective inhibition of insulin signaling observed in this study. Further study will be important to demonstrate whether such isoform-specific regulation of IRS and HIF is similar in tissues where IRS-1 is the dominant isoform, such as liver (19) and muscle (20).
Our findings also reveal pathway-specific effects of CITED2, as it inhibits insulin signaling through the PI3K-Akt pathway, whereas activation of the ERK pathway is unchanged. This pattern is consistent with selective inhibition of IRS-2 expression; endothelial cells lacking IRS-2 exhibit reduced Akt and eNOS phosphorylation in response to insulin stimulation, whereas those lacking IRS-1 have no defects in PI3K-Akt signaling (18). Moreover, IRS-2 is dispensable for insulin-stimulated endothelin-1 expression in endothelial cells, indicating that reduced expression of IRS-2 has little impact on ERK pathway activation (18).
Selective impairment of insulin signaling through PI3K-Akt, but not through ERK, is a feature of insulin resistance in endothelial cells and other cell types in obesity and type 2 diabetes (1). Because CITED2 regulates signaling through PI3K-Akt but not ERK, it is a particularly attractive target for ameliorating endothelial dysfunction due to insulin resistance. Potentiating signaling through PI3K-Akt has been shown to improve atherosclerosis and wound healing in obese mice with endothelial-specific overexpression of IRS-1 (5, 6). As in those studies, targeting CITED2 may enhance the antiatherosclerotic and proangiogenic effects of insulin-PI3K-Akt signaling without the concomitant increases in the proatherosclerotic effects of insulin-ERK signaling (1). If, as in previous studies of endothelial insulin resistance (2, 5), insulin sensitivity in large-vessel endothelial cells is regulated in the same manner as in lung endothelial cells, CITED2 may represent a regulator of the development of coronary artery and aortic atherosclerosis.
Hypoxia-induced changes in glucose metabolism are well documented and may rely, in part, on regulation of insulin signaling. Our work identifies a way to leverage this regulatory pathway, which until now has received relatively little attention (8, 11). Furthermore, inhibition of CITED2 presents an opportunity to improve selective insulin resistance without amplifying ERK signaling, making it a valuable target for improving vascular complications of type 2 diabetes.
DATA AVAILABILITY
The datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.
SUPPLEMENTAL DATA
Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.14516781.v1.
GRANTS
B.K. was supported by a postdoctoral fellowship from the National Institutes of Health (5T32DK007260) and the Mary K. Iacocca Visiting Interdisciplinary Fellowship provided by the Iacocca Family Foundation. S.M.L. was supported by a Non-Clinical Open Funding Programme Fellowship from the Diabetes Research and Wellness Foundation. X.W. received National Natural Science Foundation of China Grant No. 81873645 and Science and Technology Commission of Shanghai Municipality Grant No. 18140902100. C.R.M. was supported by a pilot and feasibility grant from the Boston Area Diabetes Endocrinology Research Center (BADERC), a subcontract from NIH Grant No. 5P30DK057521-18. Flow cytometry and real-time PCR were performed using cores in the Diabetes Research Center, supported by NIH Grant Nos. 5P30DK036836 and S10OD021740 at Joslin Diabetes Center.
DISCLAIMERS
The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
DISCLOSURES
C.R. is currently on a leave of absence from Joslin Diabetes Center and employed by Sanofi US. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.
AUTHOR CONTRIBUTIONS
B.K., X.W., and C.R. conceived and designed the research; B.K., K.C., S.M.L., X.W., and C.R. performed experiments; B.K., K.C., S.M.L., and X.W. analyzed the data; B.K., K.C., S.M.L., and X.W. interpreted results of experiments; B.K., K.C., S.M.L., and X.W. prepared the figures; B.K., S.M.L., and C.R. drafted the manuscript; B.K., S.M.L., and C.R. edited and revised manuscript; B.K., K.C., S.M.L., X.W., and C.R. approved final version of the manuscript.
ACKNOWLEDGMENTS
Dr. Sally L. Dunwoodie of the Victor Chang Cardiac Research Institute, Sydney, Australia, generously provided the Cited2fl/fl mice. The graphical abstract and schematic in Fig. 5C were created with BioRender.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.
