Vacuolar protein sorting 13C is a novel lipid droplet protein that inhibits lipolysis in brown adipocytes

Abstract

Objective: Brown adipose tissue (BAT) thermogenesis depends on the mobilization and oxidation of fatty acids from intracellular lipid droplets (LD) within brown adipocytes (BAs); however, the identity and function of LD proteins that control BAT lipolysis remain incomplete. Proteomic analysis of mouse BAT subcellular fractions identified vacuolar protein sorting 13C (VPS13C) as a novel LD protein. The aim of this work was to investigate the role of VPS13C on BA LDs.

Methods: Biochemical fractionation and high resolution confocal and immuno-transmission electron microscopy (TEM) were used to determine the subcellular distribution of VPS13C in mouse BAT, white adipose tissue, and BA cell culture. Lentivirus-delivered shRNA was used to determine the role of VPS13C in regulating lipolysis and gene expression in cultured BA cells.

Results: We found that VPS13C is highly expressed in mouse BAT where it is targeted to multilocular LDs in a subspherical subdomain. In inguinal white adipocytes, VPS13C was mainly observed on small LDs and β3-adrenergic stimulation increased VPS13C in this depot. Silencing of VPS13C in cultured BAs decreased LD size and triglyceride content, increased basal free fatty acid release, augmented the expression of thermogenic genes, and enhanced the lipolytic potency and efficacy of isoproterenol. Mechanistically, we found that BA lipolysis required activation of adipose tissue triglyceride lipase (ATGL) and that loss of VPS13C greatly increased the association of ATGL to LDs.

Conclusions: VPS13C is present on BA LDs where is targeted to a distinct subdomain. VPS13C limits the access of ATGL to LD and loss of VPS13C elevates lipolysis and promotes oxidative gene expression.

Keywords: ATGL; Brown adipose tissue; Free fatty acids; Oxidative genes; Perilipin 1; Thermogenesis.

Figure 1

VPS13C is highly expressed in mouse BAT and concentrates in LDs. A) Mass spectrometry results from subcellular BAT fractions were analyzed using hierarchical clustering analysis. BAT from mice treated with vehicle (ctrl) or 0.75 nmol/h CL 316,243 for 24 h (CL) were used to collect total lysates (total) and LD fractions. Colors in the heatmap represent row Z score calculated from normalized spectral counts that were converted to a log scale. Insert shows that the cluster where VPS13C is present includes well established LD proteins. B) VPS13C tissue distribution was assessed by Western blot, left panel: representative Western blot for VPS13C, tubulin and UCP1 (BAT marker); right panel: cumulative data (n = 4; *p < 0.05 vs BAT, ANOVA one way followed by Dunnett's multiple comparison test). C) VPS13C subcellular distribution, left panel: Western blot for VPS13C, perilipin 1 (PLIN1), GAPDH and prohibitin of BAT subcellular fractions; right panel: correlation between VPS13C and PLIN1, VPS13C and GAPDH and VPS13C and prohibitin expression levels (AU = arbitrary units).

Figure 2

VPS13C is targeted to a unique LD subdomain. Immunofluorescence images were obtained by confocal microscopy. Arrows show VPS13C subspherical distribution, arrowheads show VPS13C homogeneous distribution in small LDs. From left to right: composite image, magnified section and separate fluorescence channel images from magnified section. A) Mouse BAT paraffin sections immunostained for VPS13C and perilipin 1 (PLIN1). B) Mouse BAs from cell cultures were stained for VPS13C and lipids were stained with LipidTOX deep red (3D projection). C) Mouse inguinal white adipose tissue (IWAT) and D) Mouse gonadal white adipose tissue (GWAT) paraffin sections immunostained for VPS13C and perilipin 1 (PLIN1). E) Transmission electron micrographs of mouse BAT LR-White sections stained with VPS13C primary and anti-rabbit FluoroNanogold secondary antibodies; a- VPS13C concentrates in subspherical domain; b- VPS13C is homogeneously distributed in smaller LDs; c and d-sections enhanced with HQ Silver. M = mitochondria, LD = lipid droplets; black arrows show VPS13C on LDs; red arrows show VPS13C at LD-mitochondria interface; red arrowheads show VPS at LD–LD interface.

Figure 3

VPS13C levels increase during BA differentiation. Brown pre-adipocytes were grown in cell culture and harvested at 1–6 days after differentiation. A) protein levels (n = 4) and B) VPS13C mRNA (n = 3) were measured by Western blot and qt-PCR respectively (ND = non-differentiated). C) BAs Postnuclear (PN), cytosol (cyto), LD, membrane (memb), mitochondrial (mito) and nuclear (nuclei) fractions were collected at 2 and 4 days after differentiation. VPS13C was detected by Western Blot. D) Immunofluorescence staining of VPS13C in BAs at 0–4 days after induction of differentiation. VPS13C = green, lipids stained with LipidTOX deep red.

Figure 4

VPS13C increases in response to sustained adrenergic stimulation in inguinal white adipose tissue. A) Western blot and immunofluorescence analysis of total VPS13C levels in inguinal adipose tissue (IWAT) in control and mice infused with CL 316-243 (0.75 nmol/h) for 3 days (CL 3d). B) Inguinal white adipose tissue (IWAT), BAT, BAT-WAT intersection, sections from UCP-1-Cre td-tomato mice were stained for VPS13C. VPS13C is found in cells with (arrows) and without (arrowheads) a history of UCP-1 indicating that VPS13C is not restricted to brown adipocytes.

Figure 5

VPS13C knock down decreases LD size and triglyceride content. Characterization of control and cells silenced for VPS13C: A) Representative Western blot for VPS13C at different differentiation stages (ND = non-differentiated, 1d-6d = 1–6 days after induction). B) Cumulative data at day 4 after differentiation (n = 3). C) Immunostaining for VPS13C at day 4 after differentiation, green: VPS13C, red: lipids stained with lipidTOX deep red. D) LD size distribution as percentage of total, quantified using confocal images in cells stained with LipidTOX and DAPI (n = 3). Insert shows magnified areas of the graph. E) Triglyceride (TG) content (n = 4).

Figure 6

VPS13C knock down increases free fatty acid release in BA. A) Free fatty acid release in media (n = 5), B) Glycerol release in media (n = 4) C) Free fatty acid/glycerol ratio (n = 4). D) Free fatty acid release in response to isoproterenol. From left to right: representative experiment, sensitivity (EC50) and maximal response to isoproterenol (n = 4). E) Free fatty acid release in response to 1 nM isoproterenol in the presence and absence of the ATGL inhibitor atglistatin (n = 4).

Figure 7

VPS13C knock down increases targeting of ATGL to LDs. A) Western blot for ATGL in BA cell fractions. Left panel: representative Western blot; right panel: cumulative data (n = 4). KD = BA knocked down for VPS13C; PN = post-nuclear; cyto = cytoplasm; LD = lipid droplets. B) Western blot for phospho-HSL and HSL in total lysates from cells treated with control shRNA (ctrl) or shRNA against VPS13C (KD). Left panel: representative Western blot; right panel cumulative data (n = 4) C) Immunostaining for ATGL in control and VPS13C-silenced BAs.

Figure 8

VPS13C knock down increases oxidative gene expression in BAs. Analysis of thermogenic gene expression by qRT-PCR in control and VPS13C knockdown cells 4 days after differentiation.