gamma-secretase inhibitor induces adipogenesis of adipose-derived stem cells by regulation of Notch and PPAR-gamma

Abstract

Objective: To determine the inhibitory effect and mechanism of Notch signalling on adipogenesis of mouse adipose-derived stem cells (mASCs).

Materials and methods: Varied concentrations of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT) were added to mASCs 3 days before adipogenic induction with insulin-containing differentiation medium. The process of adipogenesis and ability of lipid droplet accumulation were analysed using oil red-O staining. The Notch signalling pathway (Notch-1, -2, -3, -4, Hes-1 and Hey-1) and adipogenesis-related factors (PPAR-gamma, DLK-1/Pref-1 and Acrp) were tested using real-time PCR, Western blot analysis and immunofluorescence staining assays.

Results: We demonstrated that Notch-2-Hes-1 signalling pathway was inhibited dose-dependently by DAPT in mASCs. In addition, transcription of PPAR-gamma was promoted by DAPT before adipogenic induction, while inhibitor of adipogenesis DLK-1/Pref-1 was further depressed. At early stages of differentiation (2-4 days), adipogenesis in mASCs was advanced and significantly enhanced in 5 and 10 mum DAPT pre-treated cases. On day 4, in differentiated mASCs cases with DAPT pre-treatment, we also found promotion of activation of de-PPAR-gamma and depression of HES-1, DLK-1/Pref-1 mRNA and protein expression.

Conclusions: We conclude that blocking Notch signalling with DAPT enhances adipogenesis of differentiated mASCs at an early stage. It may be due to depression of DLK-1/Pref-1 and promotion of de-PPAR-gamma activation, which work through inhibition of Notch-2-Hes-1 pathway by DAPT.

Figure 1

DAPT (1, 2, 5 and 10 μm) treatment (3 days) inhibits transcription of Notch‐2, Hes‐1, Hey‐1 and DLK‐1/Pref‐1 in mASCs; 0.5% DMSO was used as control. (a) Transcription of Hes‐1 and DLK‐1/Pref‐1 was simultaneously reduced by DAPT in a dose‐dependent manner within 1–5 and 10 μm respectively (^#,*P < 0.05). (b) Notch‐2 mRNA was dose‐dependently depressed by DAPT of 1–5 μm (^#,*P < 0.05), while Notch‐1, ‐3 and ‐4 were not significantly inhibited (^# P > 0.05). ^#,*Significantly different from control level and specified DAPT group respectively (P < 0.05 by ^#Dunnett t‐test and *LSD t‐test, n = 9). Transcription level of genes was analysed using real‐time PCR as described in the Materials and methods section.

Figure 2

DAPT groups significantly promoted adipogenetic ability. (a) At 48, 72 and 96 h in differentiated mASCs, oil red‐O staining was carried out to investigate the adipogenic process. (b) Average area (pixels) of droplets per cell was measured at every checkpoint and adipogenic ability of each group was presented. This demonstrates that DAPT pre‐treatment promoted adipogenesis dose‐dependently between 5 and 10 μm (^#,*P < 0.05). ^#,*Significantly different from control and specified DAPT group respectively (P < 0.05 by ^#Dunnett t‐test, and *LSD t‐test, n = 10).

Figure 3

Three days after adipogenic induction, DAPT pre‐treatment increased transcription of PPAR‐γ2 (^# P < 0.05) and retained depression of Notch‐2, DLK‐1/Pref‐1 and Hes‐1 on lower levels than controls (^# P < 0.05). Surprisingly, Acrp mRNA level was not up‐regulated as had been predicted. Hey‐1 declined in the 10 μm DAPT group (^# P < 0.05). Inhibition effectiveness for DLK‐1/Pref‐1 and Hes‐1 was dose‐dependent on DAPT of 5–10 μm. ^#,*Significantly different from control and specified DAPT group respectively (P < 0.05 by ^#Dunnett t‐test, and *LSD t‐test, n = 9).

Figure 4

DAPT promoted protein expression of de‐PPAR‐γ, ph‐PPAR‐γ, and inhibited DLK‐1/Pref‐1 in differentiated mASCs, after 4 days of induction. (a) Western blot assay was performed to analyse protein expression. (b) Adj. Volume (INT*mm², mean ± SD) of each protein measured using Quantity One 4.6.2, presented above. (c) Relative expression level of protein was obtained by normalization with β‐actin. It demonstrates that de‐PPAR‐γ and ph‐PPAR‐γ proteins were up‐regulated by DAPT, dose‐dependently within 5–10 μμ (^#,*P < 0.05), while DLK‐1/Pref‐1 expression remained at lower levels than controls (^#,*P < 0.05). ^#,*Significantly different from control and specified DAPT group respectively (P < 0.05 by ^#Dunnett t‐test, and *LSD t‐test, n = 3).

Figure 5

Immunofluorescence staining of de‐PPAR‐γ and ph‐PPAR‐γ in differentiated mASCs after 4 days adipogenesis (magnification 10 × 40). (a) de‐PPAR‐γ proteins were localizing and condensed in nuclei of differentiated mASCs in two DAPT groups. Also, de‐PPAR‐γ was detected at higher density and intensity in the 10 μm cases than the 5 μm cases. (b) Accumulation patterns of ph‐PPAR‐γ in two DAPT groups were quite different from controls. In the control cases, ph‐PPAR‐γ was concentrated in nuclei, while in the 5 μm DAPT cases, it had accumulated in the cytoplasmic compartment; in 10 μm DAPT cases, it was positive in both nuclei and cytoplasm. (c) IOD of de‐PPAR‐γ and ph‐PPAR‐γ was significantly higher than control (^# P < 0.05). Furthermore, IOD of de‐PPAR‐γ positively responded to dose of DAPT (*P < 0.05). (c) ^#,*Significantly different from control and specified DAPT group respectively (P < 0.05, by ^#Dunnett t‐test, and *LSD t‐test, n = 6).