Deamidated lipocalin-2 induces endothelial dysfunction in dietary obese mice

Abstract

INTRODUCTION: Lipocalin-2 is a pro-inflammatory adipokine implicated in the pathogenesis of obesity-associated cardiovascular diseases. The expression and production of lipocalin-2 is augmented in obese humans and animals. Mice deficient of lipocalin-2 (Lcn2-KO) are protected from high fat diet-induced endothelial dysfunction. Administration of recombinant lipocalin-2 inhibits endothelium-dependent relaxations but enhanced endothelium-dependent contractions in mice on a high fat diet. However, the same treatment fails to evoke endothelial dysfunction in mice fed with standard chow. The present study investigated the underlying mechanisms. METHODS AND RESULTS: Mass spectrometry analysis revealed that human and murine lipocalin-2 was modified by polyamination. Deamidation of lipocalin-2 occurred during co-incubation with adipose tissue of obese mice. Polyaminated and deamidated lipocalin-2 could both be detected in adipose tissues of obese animal and human subject. While the deamidated species could not be detected in serum of lean mice, the circulating levels of polyaminated and deamidated lipocalin-2 were both elevated in obese mice. After intraperitoneal administration of recombinant lipocalin-2, both species (polyaminated and deamidated) were significantly increased only in serum of wild type mice under high fat diet, but not in those under standard chow. Administration of lipocalin-2 in combination with non-esterified free fatty acids (NEFAs), especially linoleic acid (18:2n-6), resulted in a significant elevation of serum lipocalin-2, in both polyaminated and deamidated forms. The deamidated lipocalin-2 was more stable in circulation than the polyaminated species, due in part to its reduced interaction with matrix metalloproteinase 9 (MMP9). Isothermal titration calorimetry analysis revealed that lipocalin-2 bound to linoleic acid with high affinity. By blocking the interactions with MMP9, linoleic acid promoted the stabilization of lipocalin-2 and facilitated the arterial accumulation of the deamidated species, which subsequently induced endothelial dysfunction and vascular inflammation. A human lipocalin-2 mutant with the cysteine 87 replaced by alanine (C87A) contained much less amount of polyamines and exhibited reduced capacity to form heterodimeric complexes with MMP9. C87A evoked endothelial dysfunction in the absence of combined treatment with linoleic acid. CONCLUSION: These results demonstrate that activation of lipocalin-2 by deamidation plays a causal role in causing endothelial dysfunction under conditions of high NEFAs intake or dietary obesity.