FLCN mediates an alternate mTOR pathway to regulate browning of fat

Birt-Hogg-Dubé syndrome is caused by mutations in the FLCN gene. FLCN interacts with mTOR and is expressed in most tissues, however, until recently its role in adipose tissue has been unknown. Earlier this year Yan et al. (2016) showed that loss of FLCN regulates browning of adipose tissue via AMPK.  Now, Wada et al. (2016) support this by showing that FLCN regulates the browning of adipose tissue via mTOR, that adipose-specific deletion of FLCN allows TFE3 to migrate to the nucleus where it induces PGC-1, which drives mitochondrial biogenesis and the browning program.

White adipose tissue (WAT) stores energy, and its overload can lead to obesity and diabetes (Harms and Seale 2013). Brown adipose tissue (BAT) burns energy via thermogenesis and therefore activation of mitochondrial biogenesis and browning in WAT could be used as a therapeutic approach for obesity and diabetes.

To investigate the role of FLCN in adipose tissue, the authors generated mice with adipose specific deletion of FLCN (FLCN adipKO). WAT from FLCN adipKO mice was browner than wildtype mice, showed features typical of beige adipocytes, and showed higher expression of brown/beige fat genes. Gene expression studies demonstrated that the three gene sets most induced by deletion of FLCN in WAT were oxidative phosphorylation (OXPHOS), fatty acid metabolism, and adipogenesis. The authors also found a higher O2 consumption in FLCN-deficient WAT and significantly more mitochondrial DNA supporting the model that loss of FLCN in WAT leads to mitochondrial biogenesis.

FLCN is known to retain TFE3 in the cytoplasm cytoplasm (Hong et al., 2010).  Wada et al. deleted FLCN in immortalized adipocytes and saw, as expected, a dramatic nuclear translocalization of TFE3 and increased expression of the TFE3 target genes GPNMB and PGC-1α and β. TFE3 is homologous to TFEB, which is known to be phosphorylated by mTOR (Martina et al. 2012), so the authors considered whether mTOR phosphorylates TFE3, thus retaining it within the cytoplasm.  They found that an mTOR inhibitor relocalised TFE3 to the nucleus, and that alanine substitution at the putative mTOR phosphorylation site on TFE3 led to TFE3 nuclear localization. The authors concluded that FLCN regulates the mTOR phosphorylation of TFE3.

The authors then considered whether the mTOR–TFE3 signalling pathway is separate from the canonical TSC–mTOR–S6K pathway.  The authors found that in the absence of TSC the mTOR pathway was active even in the absence of amino acids: however, TFE3 remained nuclear in these cells and only became cytoplasmic after addition of amino acids and in a FLCN-dependent manner.

The authors looked at double knockout mice to try to confirm their findings.  In mice lacking FLCN and either TFE3 or PGC-1β in fat, beige adipocytes in WAT appeared much less frequent and expression levels of several genes (including PGC-1 coactivators, mitochondria-encoded genes, creatine futile cycle components, and  brown/beige fat genes that had been induced in FLCN single knockout mice) were normalized, compared with the FLCN adipKO mice.  This demonstrated in vivo that FLCN in adipose tissue regulates browning and mitochondrial biogenesis via TFE3 and PGC-1β.

Authors also showed that TFE3 directly regulates PGC 1β using ChIP experiments. PGC-1β overexpression strongly induced expression of brown/beige fat genes, mitochondrial-encoded genes, genes of the creatine futile cycle and respiratory chain proteins. The TFE3 target gene GPNMB was not induced by PGC-1β, supporting the idea that PGC-1β acts downstream from TFE3. This data demonstrates that PGC-1β is in part sufficient to activate the browning program that occurs upon FLCN deletion.  This work supports Yan et al. findings that the FLCN–AMPK pathway activates a PGC-1α/ERRα complex.

In summary, the authors show a FLCN mediated browning of WAT via a non-canonical mTOR pathway. FLCN regulates subcellular localization of TFE3 via mTOR phosphorylation, TFE3 directly regulates PGC-1β gene expression, and PGC-1β is necessary and sufficient for beige/mitochondrial gene induction in the absence of FLCN. The discovery of this FLCN–mTOR–TFE3 pathway that regulates the browning program has interesting medical relevance in treatments for obesity and diabetes.  It will also increase understanding of mTOR signalling, thus helping clarify the role of FLCN in mTOR signalling.

  •  Wada S, Neinast M, Jang C, Ibrahim YH, Lee G, Babu A, Li J, Hoshino A, Rowe GC, Rhee J, Martina JA, Puertollano R, Blenis J, Morley M, Baur JA, Seale P, & Arany Z (2016). The tumor suppressor FLCN mediates an alternate mTOR pathway to regulate browning of adipose tissue. Genes & development PMID: 27913603

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