Normal glucose uptake in the brain and heart requires an endothelial cell-specific HIF-1α-dependent function

Abstract

Although intimately positioned between metabolic substrates in the bloodstream and the tissue parenchymal cells that require these substrates, a major role of the vascular endothelium in the regulation of tissue metabolism has not been widely appreciated. We hypothesized that via control of transendothelial glucose transport and contributing paracrine mechanisms the endothelium plays a major role in regulating organ and tissue glucose metabolism. We further hypothesized that the hypoxia-inducible factor -1α (HIF-1α) plays an important role in coordinating these endothelial functions. To test these hypotheses, we generated mice with endothelial cell-specific deletion of HIF-1α. Loss of HIF in the endothelium resulted in significantly increased fasting blood glucose levels, a blunted insulin response with delayed glucose clearance from the blood after i.v. loading, and significantly decreased glucose uptake into the brain and heart. Endothelial HIF-1α knockout mice also exhibited a reduced cerebrospinal fluid/blood glucose ratio, a finding consistent with reduced transendothelial glucose transport and a diagnostic criterion for the Glut1 deficiency genetic syndrome. Endothelial cells from these mice demonstrated decreased Glut1 levels and reduced glucose uptake that was reversed by forced expression of Glut1. These data strongly support an important role of the vascular endothelium in determining whole-organ glucose metabolism and indicate that HIF-1α is a critical mediator of this function.

Fig. 1.

Endothelial deletion of HIF-1α results in elevated basal glucose levels and abnormal glucose tolerance. Mice with deletion of HIF-1α in the endothelium (EC–HIF^−/−) were generated by crossing HIF-1α^loxP/loxP with Tie-2-Cre mice. (A) Body weights of EC–HIF^−/− and control littermates were not different. (B) EC-HIF^−/− mice demonstrated significantly elevated basal fasting blood glucose (FBG) levels (n ≥ 40 age- and sex-matched mice/group). (C) >40% of EC-HIF^−/− and 8% of control (EC–HIF^+/+) mice had FBG levels ≥126 mg/dL (D) Glucose tolerance testing (i.v. glucose bolus of 2 mg/g) demonstrated a significantly reduced rate of BG clearance in EC–HIF^−/− mice and a delayed increase in circulating insulin levels in response to the glucose load (E), although basal insulin levels were not different between the groups. (F and G) There were no histological abnormalities in pancreatic architecture or islets or in brain or heart histology in EC–HIF^−/− mice (F, Upper panels) and no basal differences in vascular density in multiple tissues by immunohistochemical analysis (heart, skeletal muscle, and brain shown), consistent with our previous findings (24). *P < 0.05.

Fig. 2.

Expression of HIF-1α in endothelial cells is required to maintain normal glucose uptake and oxidation in the heart and brain. [2-³H]DG was administered i.v. to EC–HIF^−/− and littermate control mice. The in vivo tissue-specific glucose uptake index (Rg) was determined, revealing significantly reduced rates of glucose uptake into the brains (A) and hearts (B) of EC–HIF^−/− mice. Data were normalized to plasma [2-³H]DG disappearance curves obtained by measuring the ³H-specific activity of arterial plasma samples at the time points indicated (C). As a complementary approach, glucose uptake was measured in isolated perfused EC–HIF^−/− and control hearts (the same loading, perfusion, and heart rates in both groups), eliminating any contribution of blood-borne cells or noncardiac EC cells (D). In these same hearts, glucose oxidation and lactate production was determined at baseline during experimental ischemia (0.5 mL/min coronary perfusion) and after reperfusion (3 mL/min). Glucose oxidation was significantly decreased in EC–HIF^−/− hearts under all measured conditions (E), similar to the reduction seen in hearts in which HIF-1α was deleted specifically from cardiac myocytes (cmHIF^−/−). (F) Lactate production was decreased at baseline in EC–HIF^−/− hearts, and significantly increased during ischemia and reperfusion to levels similar to controls. cmHIF^−/− hearts did not alter lactate production in response to ischemia or reperfusion. *P < 0.05; P < 0.005; n ≥ 7 per group.

Fig. 3.

Reduced whole-organ glucose uptake in EC–HIF-1α^−/− mice correlates with reduced CSF/blood glucose ratios and Glut1 expression in the endothelium. (A, Upper) Western blot showing decreased Glut1 in EC isolated from EC–HIF^−/− mice (WT = littermate controls). These findings paralleled HIF-1α deletion from EC (but not from non-EC) in EC–HIF^−/− mice (PCR; A, Lower), reduced GLUT-1 and Hif-1α mRNA levels (qRT-PCR) in EC from EC–HIF^−/− vs. control mice (B), and reduced GLUT-1 endothelial immunostaining in EC–HIF^−/− hearts vs. controls (C). (D and E) HUVEC transduced with adenovirus encoding a stabilized HIF-1α (Adv-HIF) showed higher GLUT-1 protein levels and commensurately higher insulin-independent glucose uptake vs. control-transduced HUVECs (Ad-GFP). (F) Lentivirus-mediated shRNA (LVshHIF) in HDMEC resulted in efficient knockdown of HIF-1α mRNA. Concomitant transduction with adenovirus encoding Glut1 (AdGlut1) further reduced HIF-1α (LVshCtl and AdCtl = controls) and markedly increased Glut1 mRNA expression (G). (H) LVshHIF knockdown of HIF-1α in HDMEC decreases glucose uptake, and concomitant adenovirus-mediated expression of Glut1 rescues this LVshHIF-mediated decrease in glucose uptake. (I) The ratio of CSF/BG is significantly reduced in EC–HIF^−/− mice vs. control littermates (P < 0.005; n ≥ 4/group). For in vitro experiments a single asterisk indicates significance of at least P < 0.05; “#” indicates P < 0.05 basal vs. insulin stimulated.

Fig. 4.

Endothelial HIF-1α paracrine effects. (A) Conditioned media (CM) from HUVEC transduced with adenovirus encoding stabilized HIF-1α (Ad-HIF) increased glucose uptake in cardiac muscle-derived H9C2 cells vs. CM from control transduced (Ad-GFP) HUVEC (*P < 0.05 vs. control under identical conditions; #P < 0.05 vs. control without insulin). (B) Transduction of HUVEC with Ad-HIF increased, and lentivirus shRNA knockdown of HIF-1α (LV shHIF1) decreased, apelin and adrenomedullin (ADM) mRNA (*P < 0.05 for increase; #P < 0.05 for decrease). (C) Apelin mRNA is reduced in normoxic EC isolated from EC–HIF-1α^−/− mice, and does not demonstrate induction under hypoxia. (D) Plasma apelin levels (determined by ELISA) in EC–HIF1^−/− mice and control littermates under normoxia (21% O₂) and after 3 d exposure to hypoxia (10% O₂) (#P < 0.05 hypoxia vs. normoxia; *P < 0.05 EC–HIF-1^−/− vs. EC–HIF-1^+/+; n ≥ 5/group). (E and F) CM from HIF-1α–transduced EC induces AMPK phosphorylation (representative Western).

Fig. 5.

Endothelial HIF-1α regulation of glucose transport and parenchymal cell metabolism. Shown is a representation of how endothelial expression of HIF-1α can regulate parenchymal cell glucose metabolism. (A) HIF-1α transcriptionally regulates expression of Glut1, the major glucose transporter in the endothelium. Glut1 is a rate-limiting determinant of glucose uptake into EC and mediates the transport of glucose from blood to tissue across the endothelium (B). HIF regulates the expression of a wide repertoire of metabolism-related genes, including the major glycolytic enzymes, and plays a significant role in defining glucose utilization within the endothelium (C). HIF regulates the expression of several proteins secreted from the endothelium, including apelin, that can affect the metabolic state of neighboring cells via a paracrine mechanism and potentially of remote tissues via endocrine effects (D).