Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes

Abstract

Both insulin and the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDEA and increased CIDEC but not CIDEB mRNA expression. Starvation-induced apoptosis in adipocytes was significantly inhibited when insulin decreased the CIDEA mRNA level. Small interfering RNA-mediated depletion of CIDEA inhibited starvation-induced apoptosis similarly to insulin and restored insulin deprivation-reduced adipocyte number, whereas CIDEC depletion did not. Lipid droplet size of adipocytes was increased when insulin increased the CIDEC mRNA level. In contrast, insulin-induced enlargement of lipid droplets was markedly abrogated by depletion of CIDEC but not CIDEA. Furthermore, depletion of CIDEC, but not CIDEA, significantly increased glycerol release from adipocytes. These results suggest that CIDEA and CIDEC are novel genes regulated by insulin in human adipocytes and may play key roles in the effects of insulin, such as anti-apoptosis and lipid droplet formation.

Fig. 1.

Differential expression of CIDE family genes regulated by insulin in human adipocytes. A: Time course of insulin-regulated CIDE family gene expression. Differentiated adipocytes were starved in serum/Dex/insulin-free maintenance medium for 16 h. Cells were then incubated in serum/Dex-free maintenance medium in the presence or absence of 100 nM insulin for the indicated times. The mRNA expression levels of each gene were measured by quantitative real-time PCR, normalized relative to 18S rRNA expression, and shown as mRNA levels relative to zero-time control without insulin. Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01. B: Concentration response effect of insulin on CIDE family gene expression. Differentiated adipocytes were starved in serum/Dex/insulin-free maintenance medium for 16 h and then incubated in serum/Dex-free maintenance medium in the presence or absence of insulin at the indicated concentrations for 24 h. The mRNA expression levels of each gene were measured by quantitative real-time PCR, normalized relative to 18S rRNA expression, and shown as relative mRNA levels. Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01. C: Western blot analysis of CIDEA and CIDEC expression. Differentiated adipocytes were starved in serum/Dex/insulin-free maintenance medium for 16 h. Cells were then incubated in serum/Dex-free maintenance medium in the presence or absence of 100 nM insulin for the indicated times. β-Actin served as a loading control. These experiments were performed three times and the results of one representative experiment are shown.

Fig. 2.

Insulin inhibits starvation-induced apoptosis in human adipocytes. A: Fluorescence microscopy of adipocytes stained with TUNEL, DAPI, and Nile Red. Differentiated adipocytes were incubated in serum/Dex-free maintenance medium in the presence or absence of 100 nM insulin for 48 h. Cells were triple-stained with TUNEL (green), DAPI (blue), and Nile Red (red). TUNEL-positive adipocytes are indicated by the arrowheads. Scale bar, 100 μm. B: Quantification of TUNEL-positive adipocytes. Differentiated adipocytes were starved in serum/Dex-free maintenance medium in the presence or absence of 100 nM insulin for the indicated times. Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01.

Fig. 3.

Suppression of CIDEA expression inhibits starvation-induced apoptosis in human adipocytes. A: Quantification of TUNEL-positive adipocytes. Differentiated adipocytes were treated with control siRNA (siControl), CIDEA siRNA (siCIDEA), or CIDEC siRNA (siCIDEC) in maintenance medium for 7 days. Cells were then incubated in serum/Dex-free maintenance medium in the presence or absence of 100 nM insulin for 48 h and analyzed for apoptosis. Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01. B: Expression analysis of CIDEA and CIDEC mRNA by real-time PCR. The mRNA expression levels of each gene were normalized relative to 18S rRNA expression and shown relative to control siRNA (siControl) without insulin. Data are presented as means ± SEM of three independent experiments. **, P < 0.01 versus siControl without insulin; ^##, P < 0.01 versus siControl with insulin.

Fig. 4.

Suppression of CIDEA expression restores insulin deprivation-reduced adipocyte number. A: Phase contrast microscopy of adipocytes. Differentiated adipocytes were treated with control siRNA (siControl), CIDEA siRNA (siCIDEA), or CIDEC siRNA (siCIDEC) in the maintenance medium in the presence or absence of 100 nM insulin for 15 days. Scale bar, 100 μm. B: Quantification of adipocyte number. Data are presented as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01. C: Expression analysis of CIDEA and CIDEC mRNA by real-time PCR. The mRNA expression levels of each gene were normalized relative to 18S rRNA expression and shown relative to siControl without insulin. Data are presented as means ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01 versus siControl without insulin; ^##, P < 0.01 versus siControl with insulin.

Fig. 5.

Suppression of CIDEC expression inhibits insulin-induced enlargement of lipid droplets and increases glycerol release in human adipocytes. A: Fluorescence microscopy of adipocytes stained with Nile Red. Differentiated adipocytes were treated with control siRNA (siControl), CIDEA siRNA (siCIDEA), or CIDEC siRNA (siCIDEC) in maintenance medium in the presence or absence of 100 nM insulin for 10 days and then stained with Nile Red (red). Scale bar, 30 μm. B: Quantification of lipid droplet size. Data are presented as means ± SEM of three independent experiments. **, P < 0.01 versus siControl without insulin; ^##, P < 0.01 versus siControl with insulin. C: Lipolysis assay. Glycerol released into the medium over 24 h was measured after siRNA-mediated depletion of CIDEA and CIDEC. Data are presented as means ± SEM of three independent experiments. ^##, P < 0.01 versus siControl with insulin. D: Expression analysis of CIDEA and CIDEC mRNA by real-time PCR. The mRNA expression levels of each gene were normalized relative to 18S rRNA expression and shown relative to siControl without insulin. Data are presented as means ± SEM of three independent experiments. **, P < 0.01 versus siControl without insulin; ^##, P < 0.01 versus siControl with insulin.

Fig. 6.

Schematic diagram of insulin action in human adipocytes. Insulin suppresses apoptosis, at least in part, through downregulation of CIDEA mRNA expression but increases lipid droplet formation through upregulation of CIDEC mRNA expression. Chronic insulin action would increase adipocyte number and size through gene regulation of CIDEA and CIDEC and increase the mass of WAT in humans.