Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults

Abstract

Background: Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells.

Methods and results: Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation.

Conclusions: Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease.

Keywords: endoplasmic reticulum stress; endothelium; free fatty acids; leukocyte.

Figure 1

Adaptive and proapoptotic ER stress pathways. The early adaptive response involves activation of ATF6 and IRE1. ATF6 is transferred to the Golgi apparatus, where it is cleaved to form the transcription factor, ATF6α. IRE1 is dimerized and phosphorylated and then functions as an endoribonuclease leading to the spicing and production of the transcription factor, sXBP‐1. These pathways increase the production of chaperones improving the ER's protein folding capacity and activate ERAD, which limits ER stress. More‐prolonged ER stress activates PERK, which phosphorylates eIF2α and downregulates overall protein synthesis and also induces the transcription factor, ATF4. During prolonged or severe ER stress, the PERK/eIF2α/ATF4 branch becomes more important and leads to an apoptotic response, mainly through activation of CHOP/GADD34. Additionally, sXBP1 can also induce apoptosis under the same conditions through activation of JNK and caspase 12. ATF6 indicates activating transcription factor 6; eIF2α, eukaryotic initiation factor alpha; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum–associated protein degradation; IRE1, inositol requiring kinase 1; JNK, c‐Jun N‐terminal kinase; PERK, protein kinase‐like ER kinase; sXBP1, spliced X‐box‐binding protein 1.

Figure 2

Effects of intralipid on microvascular function. Reactive hyperemia was assessed in the lower extremity using venous occlusion plethysmography at baseline and after intralipid infusion. Intralipid infusion impaired the hyperemic response (P=0.001 by repeated‐measures ANOVA; N=12).

Figure 3

Changes in ER stress‐related genes after 5 hours of intralipid/heparin infusion in healthy subjects. A, Gene expression was assessed by real‐time quantitative PCR in peripheral blood mononuclear cells, as described in Methods. ATF6 (P=0.027) and spliced XBP1 (P=0.039) increased after intralipid/heparin infusion. B, CHOP (P=0.005) and GADD34 (P=0.005) decreased after infusion. Data are mean±SEM (N=12 for each gene; *P<0.05). ATF6 indicates activating transcription factor 6; ER, endoplasmic reticulum; PCR, polymerase chain reaction; XBP1, X‐box‐binding protein 1.

Figure 4

Effects of intralipid/heparin infusion on ER stress‐related proteins ATF6 and phospho‐IRE1 in PBMCs after 5 hours of intralipid/heparin infusion in healthy subjects. PBMCs were isolated and immunofluorescence microscopy was performed as described in Methods. Nuclei were labeled with DAPI (blue) and ATF6 (A and B) or phospho‐IRE1 (C and D) were labeled with red. As shown, intralipid/heparin infusion was associated with an increase in ATF6 expression and nuclear localization and an increase in phospho‐IRE1 expression (E) without changes in ATF4, Grp78, or GADD34 (F). Data are mean±SEM (n=10 for each protein; *P<0.05). ATF6 indicates activating transcription factor 6; DAPI, 4′,6‐diamidino‐2‐phenylindole; ER, endoplasmic reticulum; IRE1, inositol requiring kinase 1; PBMCs, peripheral blood mononuclear cells.

Figure 5

Changes in ER stress‐related proteins in endothelial cells after 5 hours of intralipid/heparin infusion in healthy subjects. Endothelial cells were fixed on microscope slides and protein expression measured by quantitative immunofluorescence as described in Methods. Representative images show higher ATF6 and pIRE1 after intralipid/heparin infusion (green=von Willebrand's factor; blue=DAPI; red=ATF 6 or pIRE1). Pooled data show higher ATF6 (P=0.048) and pIRE1 (P<0.005) protein expression after infusion. Data are mean±SEM (n=9 for each protein; *P<0.05). ATF6 indicates activating transcription factor 6; DAPI, 4′,6‐diamidino‐2‐phenylindole; ER, endoplasmic reticulum; pIRE1, phospho‐inositol requiring kinase 1.