Apolipoprotein A5 regulates intracellular triglyceride metabolism in adipocytes

Abstract

It has previously been demonstrated that apolipoprotein A5 (apoA5) can be internalized by human adipocytes and significantly decreases intracellular triglyceride content. In the present study, endocytosis of apoA5 by adipocytes under different conditions, and the underlying mechanism by which apoA5 regulates cellular triglyceride storage, was investigated. The results revealed that the apoA5 protein was detected in human subcutaneous abdominal adipose tissues. In addition, the uptake of apoA5 was attenuated in human obese adipose tissues and in cultured adipocytes with hypertrophy or insulin resistance. Low‑density lipoprotein receptor protein 1 (LRP1) knockdown in adipocytes resulted in a decrease in internalized apoA5 content, suggesting that LRP1 serves a role in apoA5 uptake. Treatment of adipocytes with apoA5 decreased the expression of the lipid droplet‑associated proteins such as cidec and perilipin. ApoA5‑treated adipocytes demonstrated an increase in lipolysis activity and expression of uncoupling protein 1, which is the molecular effector of thermogenesis in brown adipocytes. These results suggested that decreased triglyceride accumulation in adipocytes induced by apoA5 may be associated with enhanced lipolysis and energy expenditure, which may result from reduced expression of cidec and perilipin. In conclusion, the present study demonstrated a novel role of apoA5 in regulating the intracellular triglyceride metabolism of adipocytes. The results of the present study suggested that apoA5 may serve as a potential therapeutic target for the treatment of obesity and its related disorders.

Figure 1.

Morphological alterations in 3T3-L1 adipocytes during adipocyte hypertrophy. Oil red O staining was performed at day 8 (non-hypertrophied cells) and day 21 (hypertrophied cells). Original magnification, ×200.

Figure 2.

Endocytosis of apoA5 in hypertrophied adipocytes (21 days of differentiation) is reduced compared with the non-hypertrophied control (8 days of differentiation). (A) Western blot analysis of apoA5 expression in hypertrophied adipocytes. Differentiated 3T3-L1 adipocytes at day 8 and day 21 were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence of 600 ng/ml recombinant human apoA5 for 4 h. β-actin served as a loading control. ^#P<0.05 vs. control. (B) Differentiated 3T3-L1 adipocytes at day 8 and 21 were pulsed with ¹²⁵I-apoA5 for 2 h in serum-free medium and chased for various time-points up to 24 h. Radioactivity (cpm) was normalized to cell protein, and data are expressed as total cpm/mg protein at each time-point. Data are expressed as the mean ± standard error of 3 independent experiments. ApoA5, apolipoprotein A5.

Figure 3.

Expression of apoA5 by adipocytes in an insulin-resistant state. Fully differentiated 3T3-L1 adipocytes for 8 days were treated for 3 days with PBS (control adipocytes) or with 3 ng/ml recombinant murine TNF-α to render them insulin resistant. (A) Western blot analysis of the uptake of apoA5 in control and insulin-resistant adipocytes following incubation with 600 ng/ml recombinant human apoA5 for 4 hrs. β-actin served as a loading control. ^#P<0.05 vs. control. (B) Normal and insulin-resistant adipocytes were pulsed with ¹²⁵I-apoA5 for 2 h in serum-free medium and chased for various times after various time-points up to 24 h. Radioactivity (cpm) was normalized to cell protein, and data are expressed as total cpm/mg protein at each time-point. Data are expressed as the mean ± standard error of 3 independent experiments. ApoA5, apolipoprotein A5.

Figure 4.

ApoA5 protein levels in obese and non-obese human adipose tissue. Western blot analysis of apoA5 in subcutaneous abdominal adipose tissue from obese (BMI ≥30) (n=3) and non-obese (BMI <25) humans (n=3). The experiment was performed three times. *P<0.05 vs. control. ApoA5, apolipoprotein A5.

Figure 5.

Expression of apoA5 by adipocytes following LRP1 siRNA transfection. (A) Efficiency of LRP1 siRNA knockdown as determined by western blot analysis. *P<0.05 vs. control. (B) Western blot analysis of apoA5 protein expression in differentiated 3T3-L1 adipocytes transfected with LRP1 siRNA or nonsilencing siRNA following incubation with 600 ng/ml recombinant human apoA5 for 4 h. *P<0.05 vs. control. (C) Transfected cells were pulsed with ¹²⁵I-apoA5 for 2 h in serum-free medium and collected at the end of pulse period. *P<0.05 vs. control. Data are expressed as the mean ± standard error of 3 independent experiments. ApoA5, apolipoprotein A5; LRP1, low-density lipoprotein receptor protein 1; siRNA, small interfering RNA.

Figure 6.

ApoA5 treatment reduces the TG concentration but did not affect lipid droplet formation in human adipocytes. Differentiated adipocytes were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence or absence of 200 ng/ml apoA5 for 48 h. (A) TG concentration in adipocytes was determined. ***P<0.001 vs. control. (B) Cells were stained with Oil Red O and 4′,6-diamidino-2-phenylindole. Representative images from confocal microscopy analysis are presented. Scale bar, 30 µm. (C) Quantification of droplet number per cell (left graph) and mean droplet size (right graph) after treatment with or without apoA5. Data are expressed as the mean ± standard error of at 5 independent experiments. ApoA5, apolipoprotein A5; TG, triglyceride.

Figure 7.

ApoA5 reduces the expression levels of the genes encoding cidec and perilipin in human adipocytes. Differentiated adipocytes were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence or absence of 200 ng/ml apoA5 for various time-points up to 48 h. mRNA expression levels of (A) CIDEC and (B) PLIN1. The mRNA expression levels of each gene normalized relative to 18S rRNA expression, and presented as mRNA levels relative to the 0 h control (without apoA5). ***P<0.001 vs. control. (C) Western blot and densitometric analysis of cidec, perilipin and β-actin (control) expression levels in adipocytes 24 h after 200 ng/ml apoA5 treatment. Data are expressed as the mean ± standard error of 3 independent experiments. *P<0.05 and **P<0.01 vs. control. ApoA5, apolipoprotein A5; CIDEC, cidec gene; PLIN1, perilipin gene.

Figure 8.

Effects of apoA5 treatment on glycerol concentration in human adipocytes. Differentiated adipocytes were incubated in serum-free medium in the presence or absence of 200 ng/ml apoA5 for 24 h. Cells were then incubated for 3 h in the absence (basal) or presence of 10 µM isoproterenol, after which concentrations of glycerol in the culture medium were measured. Data are expressed relative to the value for control cells, and are expressed as the mean ± standard error of 3 independent experiments. *P<0.05 vs. control. ApoA5, apolipoprotein A5.

Figure 9.

Effects of apoA5 treatment on the expression of lipid synthesis-associated genes in human adipocytes. mRNA expression levels of (A) lipolysis-associated genes, (B) TG hydrolysis-associated genes, (C) mitochondrial biogenesis or oxidative phosphorylation-associated genes and (D) BAT-specific genes in human adipocytes, 48 h after 200 ng/ml apoA5 treatment. Data were normalized to 18S rRNA mRNA expression levels, and are expressed as the mean ± standard error of 5 independent experiments. *P<0.05 vs. control. ApoA5, apolipoprotein A5; DGAT, diacylglycerol acyltransferase; SCD1, stearoyl-CoA desaturase 1; TG, triglyceride; HSL, hormone-sensitive lipase; ATGL, adipose triglyceride lipase; NRF1, nuclear respiratory factor 1; COXIV, subunit IV of cytochrome c oxidase; PGC1a, PPARγ coactivator 1α; UCP1, uncoupling protein 1; FOXC2, forkhead box C2; BAT, brown adipose tissue.