Endothelial dysfunction in adipose triglyceride lipase deficiency

Abstract

Systemic knockout of adipose triglyceride lipase (ATGL), the pivotal enzyme of triglyceride lipolysis, results in a murine phenotype that is characterized by progredient cardiac steatosis and severe heart failure. Since cardiac and vascular dysfunction have been closely related in numerous studies we investigated endothelium-dependent and -independent vessel function of ATGL knockout mice. Aortic relaxation studies and Langendorff perfusion experiments of isolated hearts showed that ATGL knockout mice suffer from pronounced micro- and macrovascular endothelial dysfunction. Experiments with agonists directly targeting vascular smooth muscle cells revealed the functional integrity of the smooth muscle cell layer. Loss of vascular reactivity was restored ~50% upon treatment of ATGL knockout mice with the PPARα agonist Wy14,643, indicating that this phenomenon is partly a consequence of impaired cardiac contractility. Biochemical analysis revealed that aortic endothelial NO synthase expression and activity were significantly reduced in ATGL deficiency. Enzyme activity was fully restored in ATGL mice treated with the PPARα agonist. Biochemical analysis of perivascular adipose tissue demonstrated that ATGL knockout mice suffer from perivascular inflammatory oxidative stress which occurs independent of cardiac dysfunction and might contribute to vascular defects. Our results reveal a hitherto unrecognized link between disturbed lipid metabolism, obesity and cardiovascular disease.

Keywords: Adipose triglyceride lipase; Endothelial NO synthase; Endothelial dysfunction; Perivascular inflammation; Vascular proteasome.

Graphical abstract

Fig. 1

ATGL deficiency causes severe endothelial dysfunction. (A) Aortic relaxation to ACh was impaired in AKO mice. The relaxant response was partially restored by treatment with the PPARα agonist Wy14,643. Vasorelaxation to DEA/NO was not affected. (C) Treatment with the NADPH oxidase inhibitor gp91ds-dat (50 μM) or a combination of PEG-SOD (100 U/ml) and PEG-cat (500 U/ml) did not restore the relaxant response to ACh. (D) Neither gp91ds-dat nor PEG-SOD/PEG-cat affected relaxation to DEA/NO. (E) Relaxation of coronary arteries to BK was severely impaired in ATGL-deficient hearts. (F) The dilatory response of coronary vessels to DEA/NO was not affected. Results represent mean values ± SEM of 5 individual experiments. *P < 0.05 vs WT; §P < 0.05 vs AKO.

Fig. 2

NO/cGMP signaling is blunted in AKO aortas. (A) ATGL is functionally expressed in WT aortas. Triglyceride hydrolase activity of aortas was compared to that of WAT; n = 4–5; *P < 0.05; aorta vs WAT. (B) Protein expression and Ser¹¹⁷⁷ phosphorylation of eNOS were significantly decreased in AKO aortas; *P < 0.05 vs WT; n = 12–14. (C) Vascular NOS activity (measured as formation of l-citrulline) was comparably reduced in ATGL deficiency. The effect on enzyme activity was completely reversed in aortas of AKO mice treated with the PPARα agonist Wy14,643. *P < 0.05 vs WT; §P < 0.05 AKO (untreated) vs AKO (Wy 14,643-treated); n = 4–9. (D) Aortic eNOS mRNA levels; n = 5–8. (E) Protein expression and phosphorylation of VASP at Ser²³⁹ were decreased in AKO aortas; *P < 0.05 vs WT; n = 12–14. (F) Aortic expression of ubiquitinated proteins was markedly decreased in ATGL deficiency. (G) Proteasomal 20S core protein expression was significantly increased in aortas of AKO mice; *P < 0.05 vs WT; n = 9. (I) Expression of TNFα mRNA was upregulated in ATGL deficiency; *P < 0.05 vs WT; n = 4–5. Except in Fig. A, results are expressed relative to WT controls (= 1). Data illustrated in Fig. C were analyzed for statistical significance by ANOVA using Student–Newman–Keuls as post hoc test. Data represent mean values ± SEM of n experiments.

Fig. 3

Generation of microvascular ATGL-deficient endothelial cells. (A, B) Endothelial cells isolated from AKO hearts showed increased capacity of triglyceride storage, impaired lipolytic activity, and (C) increased incorporation of glucose-derived carbon into cellular triglycerides. *P < 0.05; AKO vs WT at 20 h; §P < 0.05; WT 20 h vs 24 h; n = 3. (D) mRNA (n = 5–6) and (E) protein expression of eNOS (n = 6) as well as (F) basal and stimulated enzyme activity (n = 6–11) were not affected in ATGL-deficient cells. (G) Protein expression of NOX 2 (n = 3) and NOX4 (n = 6) as well as (H) NADPH oxidase-dependent lucigenin CL (n = 3–4) was similar in WT and AKO cells; *P < 0.05; untreated vs gp91ds-dat-treated WT; #P < 0.05; untreated vs gp91ds-dat-treated AKO. (I) Expression of ubiquitinated proteins was not affected by endothelial ATGL deficiency; n = 5. Expression of NOX2 and NOX4 protein as well as eNOS mRNA is presented relative to WT controls (= 1). eNOS protein is expressed as μg eNOS per mg total protein using purified eNOS as standard. eNOS activity is expressed as % of [³H]l-citrulline formed from incorporated [³H]l-arginine. Data represent mean values ± SEM of n experiments.

Fig. 4

Characterization of ATGL-deficient PVAT. (A, B) The amount of PVAT surrounding thoracic aortas was substantially increased in AKO mice; *P < 0.05 vs WT; n = 30–32. Adipose mRNA expression of (C) TNFα (n = 15), (D) IL-6 (n = 10–11), and (E) MCP-1 (n = 15) was increased in ATGL-deficient PVAT; *P < 0.05 vs WT. (F) mRNA and protein expression of NOX2 was upregulated in PVAT of AKO mice; *P < 0.05 vs WT; n = 16–18. (G) Protein expression of NOX2-related p47^phox (n = 16–17) and p67^phox (n = 9–10) subunits was increased; *P < 0.05 vs WT. (H) Protein expression of Mac-2 was highly upregulated; *P < 0.05 vs WT; n = 6–9. (I) SOD-1 protein was downregulated in PVAT of AKO mice; *P < 0.05 vs WT; n = 9. (J) Catalase-inhibited H₂O₂ release from PVAT; n = 7. (K) Adipose mRNA levels of HO-1 were upregulated in ATGL deficiency; *P < 0.05 vs WT; n = 7–9. (L) Protein expression and phosphorylation of eNOS were downregulated in PVAT of AKO mice; *P < 0.05 vs WT; n = 6–17. Protein and mRNA expression is presented relative to WT controls (= 1). Data represent mean values ± SEM of n experiments.

Fig. 5

Effect of PVAT on vessel function. (A) Aortas isolated from WT and AKO mice exhibited comparable contractility to K⁺ in the absence and presence of PVAT; n = 4–5. (B) Presence of PVAT reduced the contractile efficacy of U46619 in aortas of AKO mice to a greater extent than in WT mice; *P < 0.05 vs PVAT-denuded WT; §P < 0.05 vs PVAT-containing WT; n = 4–5. Relaxation to (C) ACh and (D) DEA/NO was not affected by PVAT; n = 4–5. Data represent mean values ± SEM of n individual experiments.

Fig. 6

Histological characterization of WT and AKO aortas. Representative images of WT and AKO specimens stained with H&E showed no morphological changes through all sections of the aorta (upper panels). Endothelial cells labeled with CD31 (middle panel) or smooth muscle cells labeled with α-SMA (lower panel) exhibit similar staining intensity and distribution in sections of WT and AKO aortas. Images are representative for 3 individual experiments.

Fig. 7

Scanning electron microscopy of the luminal endothelial surface morphology. Experiments confirm the endothelial integrity of thoracic aorta in WT (A) and AKO (B) mice. Arrows point to round endothelial fenestrae, arrowheads to protruding endothelial cell nuclei. Asterisks mark orifices of abutting intercostal arteries. EC; endothelial cell; horizontal field width: (A) 1.20 mm (left), 110 μm (right); (B) 1.95 mm (left), 60 μm (right). Images are representative for 3 individual experiments.