START domain-mediated nuclear translocation of Them1 regulates the expression of metabolic genes relative to thermogenesis in brown adipocytes

Abstract

Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue (BAT), where it limits energy expenditure. Them1 is comprised of tandem enzymatic domains and a C-terminal lipid-binding steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and increases activity, whereas lysophosphatidylcholine (LPC) acts in the opposite manner. In brown adipocytes, Them1 localizes to puncta within the cytoplasm, but diffuses in response to β-adrenergic stimulation after which it is also found in the nucleus. The aim of this study was to determine the mechanism of Them1 nuclear trafficking and the metabolic consequences. METHODS: Identification of a putative nuclear localization signal (NLS) was determined by computational analysis. Full-length, NLS-deleted, or truncated Them1 cDNA constructs were cloned and linked to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal imaging via EGFP and with NucSpot nuclear staining. Signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated in the presence or absence of norepinephrine (NE), leptomycin B, cycloheximide, or lipids that bind the START domain. Them1 nuclear localization was also quantified in BAT from mice treated with a β3-adrenergic receptor agonist, CL316,243. mRNA expression in iBAs expressing full-length or NLS-mutant Them1 in response to NE was determined by RNA-Seq. RESULTS: In the diffuse state, Them1 localized to the nucleus in a time-dependent manner. The NLS resides in a hinge region between the thioesterase domains and the START domain. Deletion of either the NLS or the START domain blocked nuclear targeting. Conversely, truncated Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus and new protein synthesis was not required, as evidenced by using leptomycin B to block nuclear efflux and cycloheximide to block protein synthesis, respectively. Incubation of NE-stimulated iBAs with LPC promoted nuclear Them1 translocation. Them1 protein was also detected in the nucleus of BAT from wild-type mice in response to CL administration. Nuclear Them1 localization in iBAs was associated with down-regulated mRNA expression of genes that control lipid and glucose metabolism during cold adaptation. CONCLUSIONS: Our data suggest that LPC generated during thermogenesis binds and destabilizes the START domain, leading to exposure of the NLS and nuclear trafficking of Them1 in response to β-adrenergic stimulation. Once inside the nucleus, Them1 regulates the transcription of lipid and glucose metabolic enzymes that are required for chronic cold adaptation. This work was supported by NIH DK103046 to DEC, SJH, and EAO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.