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. 2019 Mar 12;510(3):409-415.
doi: 10.1016/j.bbrc.2019.01.115. Epub 2019 Jan 31.

Exosomal release through TRPML1-mediated lysosomal exocytosis is required for adipogenesis

Mi Seong Kim  1 , Shmuel Muallem  2 , Sung Hyun Kim  3 , Kang Beom Kwon  4 , Min Seuk Kim  5
Affiliations

Exosomal release through TRPML1-mediated lysosomal exocytosis is required for adipogenesis

Mi Seong Kim et al. Biochem Biophys Res Commun. .

Abstract

The lysosomal Ca2+ permeable channel TRPML1 (MCOLN1) plays key roles in lysosomal membrane trafficking, including the fusion of late endosomes to lysosomes and lysosomal exocytosis, both of which are essential for release of exosomes into the extracellular milieu. Multiple lines of evidence indicate that the contents of adipocyte-derived exosomes mediate diverse cellular responses, including adipogenic differentiation. In this study, we aimed to define the potential roles of TRPML1 in lysosomal membrane trafficking during adipogenesis and in exosomal release. In response to adipogenic stimuli, the endogenous TRPML1 expression in OP9 pre-adipocytes was increased in a time-dependent manner, and the acute deletion of TRPML1 reduced lipid synthesis and expression of differentiation-related marker genes. Notably, mature adipocyte-derived exosomes were found to be necessary for adipogenesis and were dependent on TRPML1-mediated lysosomal exocytosis. Taken together, our findings indicate that TRPML1 mediates diverse roles in adipocyte differentiation and exosomal release. Further, we propose that TRPML1 should be considered as a regulator of obesity-related diseases.

Keywords: Adipogenesis; Exosome; Lysosomal exocytosis; TRPML1.

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Figures

Fig. 1.
Fig. 1.. Expression of endogenous TRPML1 is up-regulated by adipogenic stimuli.
Cells were induced to adipogenic differentiation for indicated days. (A) Quantitative real-time PCR of TRPML1 and adipogenic marker gene expression (C/EBPβ and PPARγ) during adipogenesis. (B) Western blot analysis of TRPML1 and PPARγ expression in response to adipogenic stimuli. Data are expressed as relative fold change of mRNA and protein expression. Graphs represent mean ± SEM from 3 independent experiments. **P < 0.05 compared with control group (0 D).
Fig. 2.
Fig. 2.. Effects of TRPML1 depletion on the formation of lipid droplets and adipogenic marker gene expression.
(A) OP9 pre-adipocytes were transfected with mock (transfection reagent only), scrambled siRNA (Scr), and siTRPML1 (siML1) in a dose-dependent manner. Following adipogenic induction, cells were stained with oil red O. (Scale bar, 100 μm). The data in Fig. 2A are expressed as relative fold change of O.D value compared with control group (uninduced mock). (B) Real-time qPCR of TRPML1 and adipogenic marker genes (C/EBPα, aP2 and PPARγ) in each group. (C) Western blot analysis of TRPML1, PPARγ, and C/EBPα expression in each group. Data are expressed as relative fold change of mRNA and protein expression. Graphs represent mean ± SEM from 3 independent experiments. **P < 0.05 between the indicated groups.
Fig. 3.
Fig. 3.. Regulation of lipid formation by adipocyte-derived exosomes.
(A) Image of exosomes released from mature adipocytes, taken through TEM (Scale bar, 200 nm). Exosomes are marked with white arrows. (B) Adipocyte-derived exosomes (ADEs) suspended in PBS, and 10 μg ADE was incubated with OP9 pre-adipocytes for 2 days. Cells were then stained with oil red O. (Scale bar, 100 μm). (C) OP9 pre-adipocytes were pretreated with DMA in indicated concentrations, and adipogenic differentiation was induced for 4 days, after which cells were stained with oil red O. Data are expressed as fold change of OD value compared with control group (PBS only). **P < 0.05 compared with each control group.
Fig. 4.
Fig. 4.. Exosomal release is regulated by TRPML1-mediated lysosomal exocytosis.
(A) Following induction of adipogenic differentiation, expression of exosomal marker genes (CD9, CD81, CD63, and HSP70) in TRPML1-depleted cells (scramble siRNA; Scr, siTRPML1; siML1). (B) Effects of TRPML1 depletion on localization of LAMP1 in plasma membrane. Plasma membrane (Plasma M) was separated from total membrane (Total M) and used for LAMP1 expression analysis by western blot. (C) Mature adipocytes were immunostained with anti-LAMP1 antibody (Red) and DAPI (Blue), and fluorescence of each image were measured. Data represent mean ± SEM from 3 independent experiments. **P < 0.05 compared between indicated groups.
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