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. 2017 Feb 13:8:45.
doi: 10.3389/fphys.2017.00045. eCollection 2017.

Icariin Inhibits AMPK-Dependent Autophagy and Adipogenesis in Adipocytes In vitro and in a Model of Graves' Orbitopathy In vivo

Hong Li  1 , Yifei Yuan  2 , Yali Zhang  3 , Xia Zhang  1 , Long Gao  1 , Rongjuan Xu  1
Affiliations

Icariin Inhibits AMPK-Dependent Autophagy and Adipogenesis in Adipocytes In vitro and in a Model of Graves' Orbitopathy In vivo

Hong Li et al. Front Physiol. .

Abstract

Graves' orbitopathy (GO), an extrathyroidal manifestation of Graves' disease, is an inflammatory autoimmune disorder of the orbit that involves the differentiation of precursor cells into mature adipocytes and retro-orbital adipose tissue accumulation. Here, we examined the involvement of autophagy in adipogenesis and explored the effects of icariin, a flavonoid isolated from the genus Epimedium with a wide range of biological and pharmacological effects, on autophagy and adipogenesis in 3T3-L1 preadipocytes and in a mouse model of GO. Microscopic examination of autophagosome formation and lipid droplet accumulation by Oil Red O staining, and western blot assessment of autophagic markers in the presence of the autophagy inhibitors Asn and 3-MA showed that autophagy is essential for adipogenesis. Icariin inhibited the differentiation of preadipocytes into mature adipocytes by suppressing autophagy, and these effects were mediated by the inhibition of AMPK/mTOR pathway activation. In a mouse model of thyroid stimulating hormone receptor induced GO, icariin reduced orbital muscle adipose tissue expansion and lipid droplet accumulation by inhibiting AMPK/mTOR mediated autophagy. Collectively, these results reveal a potential mechanism underlying the protective effects of icariin against autophagy induced adipogenesis and suggest that icariin could be developed as a new therapeutic candidate for the prevention and treatment of GO. VSports手机版.

Keywords: Graves' orbitopathy; adipogenesis; autophagy; icariin; thyroid stimulating hormone receptor. V体育安卓版.

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Figures

Figure 1
Figure 1
Autophagy is necessary for adipocyte differentiation. 3T3-L1 cells were exposed to control medium (FM) or adipocyte differentiation medium (DM) in the presence or absence of the autophagy inhibitors Asn and 3-MA for 14 days. (A) Oil Red O staining shows the effect of autophagy on lipid droplet formation. (B) Quantification of intracellular lipids of 3T3-L1 cells by spectrophotometry. Data represent the mean ± SD. ***P < 0.001 vs. the untreated control (n = 5). (C) Western blot analysis of the expression of the autophagy markers LC3-I, LC3-II and PPARγ with β-actin as the loading control. (D,E) The LC3-II/LC3-I ratio and relative PPARγ expression were quantified by densitometric scanning and graphed. Data indicate the mean ± SD (n = 5). ***P < 0.001 vs. control. (F) Representative fluorescence microscopy images of GFP-LC3 transfected cells treated as indicated. (G) The number of autophagosomes was counted in 10 random fields. Data represent the mean ± SD. ***P < 0.001 vs. the untreated control (n = 10). FM, control medium; DM, differentiation medium.
Figure 2
Figure 2
Icariin treatment inhibits adipogenesis of 3T3-L1 cells by suppressing autophagy. 3T3-L1 preadipocytes were incubated in adipocyte DM in the presence or absence of 5 μM icariin. (A) Oil Red O staining showing the effect of icariin on lipid droplet accumulation. (B) Quantification of intracellular lipids of 3T3-L1 cells by spectrophotometry. Data represent the mean ± SD. ***P < 0.001 vs. the untreated control (n = 5). (C) Western blot analysis the expression of the autophagy markers LC3-I, LC3-II, and PPARγ with β-actin as the loading control. (D,E) The LC3-II/LC3-I ratio and relative PPARγ expression were quantified by densitometric scanning and graphed. Data indicate the mean ± SD (n = 5). ***p < 0.001 vs. FM. (F) Representative fluorescence microscopy images of GFP-LC3 transfected cells treated as indicated (magnification: 400×). (G) The number of autophagosomes was counted in 10 random fields. The data represent the mean ± SD. (n = 10), *P < 0.05, ***P < 0.001. (H) Electron microscopy images (5000×) show the inhibition of autophagy in response to icariin treatment. Arrows, autophagic vacuoles. FM, control medium; DM, differentiation medium.
Figure 3
Figure 3
Icariin suppressed autophagy by inhibiting AMPK activation. 3T3-L1 preadipocytes were incubated in adipocyte DM in the presence or absence of 5 μM icariin. (A) The expression of autophagy related proteins and the activation of the AMPK/mTOR pathway were analyzed by western blotting. (B–F) The levels of phosphorylated mTOR (B) and AMPK (C) (normalized to the levels of mTOR and AMPK, respectively), and the expression of Beclin-1 (E), p62 (D), and LC3 (F) were determined by densitometric scanning and graphed. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5).
Figure 4
Figure 4
Icariin inhibits the expansion of adipose tissue and lipid droplet accumulation in retrobulbar fat in a mouse model of Graves' orbitopathy (GO). (A) Hematoxylin and eosin staining showing adipose tissue widely separating the orbital muscle fiber bundles in the GO group and the suppression of adipogenesis by icariin. Scale bar: 20 μm. (B) Oil Red O staining of orbital tissue sections showing icariin suppression of lipid droplet accumulation in TSHR induced GO mice. Scale bar: 20 μm. (C) Western blot analysis of the expression of the adipose differentiation marker PPARγ with β-actin as the loading control. (D) The relative PPARγ expression was quantified by densitometric scanning and graphed. Data indicate the mean ± SD (n = 5). *P < 0.05, ***P < 0.001 vs. Normal control. ###P < 0.001 vs. GO group. (E) The expression of autophagy related proteins and the activation of the AMPK/mTOR pathway were analyzed by western blotting of retrobulbar adipose tissue lysates. (F–J) Densitometric quantification of western blot data in (E). Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Normal control. ##P < 0.01, ###P < 0.001 vs. GO group. (n = 5).
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