The tumor suppressor FLCN mediates an alternate mTOR pathway to regulate browning of adipose tissue

Abstract

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1β. Conversely, inducible expression of PGC-1β in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1β pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.

Keywords: FLCN; TFE3; adipose tissue; beige fat; mTOR; mitochondria.

Figure 1.

Adipocyte-specific deletion of FLCN induces browning of WAT. (A) Schematic of an animal model for FLCN adipKO. (B, left) Efficiency of FLCN protein deletion in BAT and iWAT. (Right) Relative mRNA expression of FLCN in the indicated fat depots. (C) Gross images of iWAT from FLCN^lox/lox and FLCN adipKO mice. Bar, 1 cm. (D) Low-power (left) and high-power (right) images of H&E staining of iWAT from FLCN^lox/lox or FLCN adipKO mice. Bars: left, 200 µm; right, 50 µm. (E–I) Relative mRNA expression of the indicated genes in iWAT from FLCN^lox/lox or FLCN adipKO mice. (J) Low-power (top) and high-power (bottom) transmission electron microscopy images of iWAT from FLCN^lox/lox and FLCN adipKO mice. Bars, 500 nm. (K) Relative O₂ consumption of iWAT isolated from FLCN^lox/lox and FLCN adipKO mice. (L) Relative mitochondrial DNA content in iWAT from the respective genotypes. (M) Whole-body respiration of FLCN^lox/lox and FLCN adipKO animals upon subcutaneous injection of 1 mg/kg norepinephrine (NE) after induction of anesthesia by pentobarbital. (*) P < 0.05 versus FLCN^lox/lox. n = 6–8 per group. Two-way analysis of variance (ANOVA) was used for M. Values are represented as mean ± SEM.

Figure 2.

FLCN deletion and mTOR inhibition promote TFE3 nuclear localization to induce TFE3 target genes in preadipocytes. (A) Western blot of FLCN in nontarget control (control) or FLCN guide RNA (gRNA) plus CRISPR/Cas9-expressing stromal vascular fraction (SVF) cells. (B) Immunofluorescence staining of TFE3 in control or FLCN-deleted SVF cells. (C) Quantification results of TFE3 nuclear localization shown in B. (D) Relative mRNA expression of TFE3 target genes in the control or FLCN-deleted SVF cell line. (E) Western blot of FLCN in vehicle (veh) or 4OHT-treated (10 μM for 48 h) CreERt2-expressing SVF cells isolated from FLCN^lox/lox iWAT. (F–H) As in B–D but with FLCN^lox/lox CreERt2 cells treated with vehicle versus 4OHT. (I) Schematic of the mTOR phosphorylation site in TFEB and its conservation in TFE3. (J–L) As in B–D but with cells treated with vehicle versus 250 nM mTOR inhibitor Torin1 for 1 h (J,K) or 4 h (L). (M) Western blot of stably expressed HA-tagged wild-type TFE3 or mTOR site-mutated TFE3 (S320A). Cells were treated with vehicle (veh) or Torin1 for 2 h. (N–P) As in B–D but with cells expressing TFE3 wild type or TFE3 S320A. (Q) Western blot of FLCN, phospho-TFE3 (Ser320), TFE3, phospho-S6K (Thr 389), and total S6K in FLCN^lox/lox cells infected with adenovirus harboring Cre versus GFP (mock) and 250 nM Torin1 for 2 h versus vehicle. (R) In vitro mTORC1 kinase assay of TFE3: GST-fused TFE3 wild-type and S320A mutant were incubated with purified mTORC1, and phosphorylated Ser320 was detected by anti-pTFE3 (S320) antibody. (*) P < 0.05 by Student's t-test; (#) P < 0.05 versus mock; ($) P < 0.05 versus TFE3 wild type. n = 3 or 4 per group. Values are represented as mean ± SEM. Bars, 50 µm.

Figure 3.

The constitutively active Rag complex rescues TFE3 cytoplasmic retention in the absence of FLCN. (A) Schematic of mTORC1 activation by FLCN and subsequent phosphorylation of TFE3 by mTORC1. (B) Immunofluorescence staining of TFE3. Constitutively active or inactive RagB and RagD (HA-tagged) were overexpressed in FLCN-deficient preadipocytes. (Green) RagB/D (HA); (red) TFE3; (blue) DAPI. (C) Quantification result of B. (D) Constitutively active (CA) RagB or RagD was individually overexpressed in FLCN knockout preadipocytes. (Green) RagB or RagD (HA); (red) TFE3; (blue) DAPI. Arrowheads indicate nuclei of RagB/D transfected cells. (*) P < 0.05 versus nontransfected control. Values are represented as mean ± SEM. Bars, 50 µm.

Figure 4.

The FLCN–mTORC1–TFE3 pathway is separable from the canonical mTOR–S6K pathway. (A, top) Immunofluorescence staining of TFE3 in control (NTC) or FLCN knockout cells. Cells were starved in amino acid-free DMEM for 1 h or starved and restimulated with amino acids or 10% dFBS for 2 h, as indicated. (Bottom) The graph shows quantification of the percentage of cells with nuclear TFE3. (*) P < 0.05 versus NTC − amino acid/− serum. (B) Western blot to phospho-S6K Thr389. Preadipocytes isolated from FLCN^lox/lox mice were infected with adenovirus harboring GFP (mock) or Cre to knock out FLCN. Cells were then starved or starved and restimulated with amino acids, as indicated. Total S6K and actin were used as loading controls. (C) Western blot to phospho-rpS6 Ser240/244 in iWAT depots isolated from FLCN^lox/lox or FLCN adipKO mice. Animals were fasted overnight and refed ad lib for 4 h. Quantification of phospho-rpS6 is shown at the right, normalized to total rpS6. (*) P < 0.05 by Student's t-test. (D) Immunofluorescence staining of phospho-S6 (red) and TFE3 (green) in control (NTC) cells and cells lacking TSC1 or TSC2. Cells were starved or starved and restimulated with amino acids as indicated. TSC1 or TSC2 genes were deleted by the CRISPR/Cas9 system. (E) Western blot to phospho-S6K. Control and TSC1 or TSC2 knockout preadipocytes were starved or starved and restimulated with amino acids, as indicated (dashed lines indicate separation within the same gel). (F) Diagram of separable mTORC1 hubs. Values are represented as mean ± SEM. Bars, 50 μm.

Figure 5.

Codeletion of TFE3 with FLCN reverses the browning of white fat. (A) Breeding scheme to obtain FLCN/TFE3 double-deficient male mice. (B) H&E staining of iWAT from animals of the noted genotypes. Bar, 100 µm. (C–F) relative mRNA expression from brown/beige fat-specific genes (C), nuclear encoded mitochondrial genes (D), mitochondria-encoded mitochondrial genes (E), and creatine futile cycle genes (F). (*) P < 0.05; (**) P < 0.01 versus FLCN^lox/lox. n = 8 per group. Values are represented as mean ± SEM.

Figure 6.

PGC-1β is required for white fat browning upon FLCN deletion and is sufficient to induce browning gene expression in WAT in vivo. (A) H&E staining of iWAT from animals of the indicated genotypes. Bar, 100 µm. (B–E) Relative mRNA expression in iWAT from brown/beige fat-specific genes (B), nuclear-encoded mitochondrial genes (C), mitochondria-encoded mitochondrial genes (D), and creatine futile cycle genes (E). (*) P < 0.05 versus FLCN^lox/lox, PGC-1β^lox/lox. (F) Schematic of adipose tissue-specific doxycycline-inducible PGC-1β expression. (G–J) Relative mRNA expression as in B–E, respectively, in PGC-1β off versus PGC-1β on (2 wk) mice. (*) P < 0.05 versus PGC-1β off. n = 6–8 per group. Values are represented as mean ± SEM.

Figure 7.

Proposed models of two separable mTORC1 pathways. (A) Amino acid sensing in the presence of FLCN leads to activation of RagC/D, leading to recruitment of TFE3 to the Rag complex and subsequent phosphorylation by mTORC1. Phosphorylated TFE3 is bound by 14-3-3 and sequestered in the cytoplasm. In the absence of FLCN, TFE3 translocates to the nucleus and activates PGC-1β > PGC-1α and mitochondrial and brown/beige fat genes. (B) The canonical mTORC1–S6K pathway induced by amino acid or growth factor stimulation does not require FLCN, and this pathway is separable from TFE3 phosphorylation. Growth factors modulate mTORC1 activity via repression of TSC1/2, while β3 adrenergic signaling modulates mTORC1 activity via PKA-mediated phosphorylation of mTOR. The PKA–mTORC1 branch induces the classic thermogenic browning program and also leads to S6K phosphorylation but not cytoplasmic sequestration of TFE3.