Oxidized LDL Modify the Human Adipocyte Phenotype to an Insulin Resistant, Proinflamatory and Proapoptotic Profile

Abstract

Little information exists in humans on the regulation that oxidized low-density lipoprotein (oxLDL) exerts on adipocyte metabolism, which is associated with obesity and type 2 diabetes. The aim was to analyze the oxLDL effects on adipocytokine secretion and scavenger receptors (SRs) and cell death markers in human visceral adipocytes. Human differentiated adipocytes from visceral adipose tissue from non-obese and morbidly obese subjects were incubated with increasing oxLDL concentrations. mRNA expression of SRs, markers of apoptosis and autophagy, secretion of adipocytokines, and glucose uptake were analyzed. In non-obese and in morbidly obese subjects, oxLDL produced a decrease in insulin-induced glucose uptake, a significant dose-dependent increase in tumor necrosis factor-α (TNF-α), IL-6, and adiponectin secretion, and a decrease in leptin secretion. OxLDL produced a significant increase of Lox-1 and a decrease in Cxcl16 and Cl-p1 expression. The expression of Bnip3 (marker of apoptosis, necrosis and autophagy) was significantly increased and Bcl2 (antiapoptotic marker) was decreased. OxLDL could sensitize adipocytes to a lower insulin-induced glucose uptake, a more proinflammatory phenotype, and could modify the gene expression involved in apoptosis, autophagy, necrosis, and mitophagy. OxLDL can upregulate Lox-1, and this could lead to a possible amplification of proinflammatory and proapoptotic effects of oxLDL.

Keywords: adipocyte; apoptosis; inflammation; oxidized low-density lipoprotein; scavenger receptors.

Figure 1

(A) Oil red-O staining of human mesenchymal stem cells at day 0 of differentiation; (B) Oil red-O staining and quantification in human visceral in vitro differentiated adipocytes after 15 days of differentiation; (C) immunofluorescence staining of FABP4 and adiponectin in human visceral in vitro differentiated adipocytes after 15 days of differentiation; (D) OxLDL uptake by visceral in vitro differentiated adipocytes. In vitro differentiated adipocytes were incubated without oxLDL (0 μg/mL oxLDL or negative control) and with 50 μg/mL oxLDL for 4 h.

Figure 2

(A) Total cholesterol, esterified cholesterol and free cholesterol levels in human visceral in vitro differentiated adipocytes from non-obese (n = 4) and morbidly obese subjects (n = 3) incubated with different doses of oxLDL (0, 25, and 50 μg/mL oxLDL). ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL. (B) levels of mRNA expression of Lxrα and Abca1 in human visceral in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25, and 50 μg/mL of oxLDL. ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL within non-obese subjects. ^b p < 0.05: significant differences with regard to 0 μg/mL oxLDL within morbidly obese subjects. * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects.

Figure 3

(A) Glucose uptake in human visceral in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 4) and morbidly obese subjects (n = 4) incubated with 0, 25, and 50 μg/mL of oxLDL for 24 h. ^a p < 0.05: significant differences between 50 μg/mL with 0 and 25 μg/mL oxLDL. ¹ p < 0.05: significant differences with regard to its control (non-insulin treated). * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects; (B) levels of mRNA expression of insulin receptor (Insr) in human visceral in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25, and 50 μg/mL of oxLDL for 24h. ^a p < 0.05: significant differences between 50 μg/mL with 0 and 25 μg/mL oxLDL. ^b p < 0.05: significant differences between 0 and 25 μg/mL oxLDL.

Figure 4

Levels of adipocitokines in the culture medium of in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25, and 50 μg/mL of oxLDL for 24h. ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL within non-obese subjects. ^b p < 0.05: significant differences with regard to 0 μg/mL oxLDL within morbidly obese subjects. * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects.

Figure 5

Levels of mRNA expression of scavenger receptors in the in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25 and 50 μg/mL of oxLDL for 24h. ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL within non-obese subjects. ^b p < 0.05: significant differences with regard to 0 μg/mL oxLDL within morbidly obese subjects. * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects.

Figure 6

Levels of mRNA expression of scavenger receptors in visceral mature adipocytes obtained from visceral adipose tissue of non-obese (n = 10) and morbidly obese subjects (n = 10). ^a p < 0.05: significant differences with regard to non-obese subjects.

Figure 7

(A) Levels of mRNA expression of Bcl2, Casp3, Atg3 and Bnip3 in the in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25, and 50 μg/mL of oxLDL for 24h. ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL within non-obese subjects. ^b p < 0.05: significant differences with regard to 0 μg/mL oxLDL within morbidly obese subjects. * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects; (B) levels of mRNA expression Bcl2, Casp3, Atg3 and Bnip3 in visceral mature adipocytes obtained from visceral adipose tissue of non-obese (n = 10) and morbidly obese subjects (n = 10). ^a p < 0.05: significant differences with regard to non-obese subjects.

Figure 8

(A) Levels of mRNA expression of Nf-kB and Nrf2 and (B) NF-κB-target genes (Tnfα, Il6 and Mcp1) in the in vitro differentiated adipocytes obtained from HMSC from non-obese (n = 6) and morbidly obese subjects (n = 6) incubated with 0, 25 and 50 μg/mL of oxLDL for 24h. ^a p < 0.05: significant differences with regard to 0 μg/mL oxLDL within non-obese subjects. ^b p < 0.05: significant differences with regard to 25 μg/mL oxLDL within non-obese subjects. ^c p < 0.05: significant differences with regard to 0 μg/mL oxLDL within morbidly obese subjects. ^d p < 0.05: significant differences with regard to 25 μg/mL oxLDL within morbidly obese subjects. * p < 0.05: significant differences for each dose between non-obese and morbidly obese subjects; (C) levels of mRNA expression of Nf-kB and Nrf2 in visceral mature adipocytes obtained from visceral adipose tissue of non-obese (n = 10) and morbidly obese subjects (n = 10). ¹ p < 0.05: significant differences with regard to non-obese subjects.