Thioesterase Superfamily Member 1 Undergoes Stimulus‐coupled Reorganization to Regulate Metabolic Activity in Brown Adipose Tissue

Abstract

Thioesterase superfamily member 1 (Them1) is a long chain fatty acyl‐CoA thioesterase that is enriched in brown adipose tissue. Them1 is markedly upregulated by cold ambient temperatures, but functions to suppress energy expenditure. Consequently Them1−/− mice are highly resistant to diet‐induced obesity‐related disorders, most notably hepatic steatosis and insulin resistance. Although Them1 reduces fatty acid availability for b‐oxidation in mitochondria, how it limits thermogenesis remains undefined. Here we show that Them1 undergoes marked intracellular conformational changes after stimulation to regulate metabolic activity.MethodsCultured brown adipocytes, which do not constitutively express Them1, were transfected with full‐length Them1 fused to EGFP as well as an engineered ascorbate peroxidase (APEX2) fusion protein. EGFP was visualized by confocal microscopy in the presence or absence of norepinephrine (NE) stimulation to determine the 3‐D localization of Them1. APEX2 was used for high resolution correlative light and electron microscopy (CLEM). Cells were further transfected with Them1 phosphomimetic mutants at S15, 18, and 25 to evaluate the role of phosphorylation in Them1 organization. Rates of NE‐stimulated fatty acid oxidation were determined by using a Seahorse XFe96. The localization of Them1 in brown adipose tissue of mice was determined after injection of CL316,243, a β3 adrenoceptor agonist. The primary amino acid sequence of Them1 was subjected to multiple analyses to predict disordered regions, aggregation, and hydrophobicity.ResultsThem1 formed puncta that were localized near lipid droplets and mitochondria in cultured brown adipose cells. In response to NE, Them1 was phosphorylated, which resulted in a diffuse intracellular localization. This change in Them1 localization also occurred in brown adipose tissue after CL316,243 administration to mice. Puncta formation activated Them1, as evidenced by suppression of oxygen consumption. CLEM revealed that puncta are highly organized membraneless organelles, which typically form after a phase separation in cells. To support this possibility, Them1 contains one intrinsically disordered region at the N‐terminus that contains multiple interacting motifs, as is frequently found in phase‐separating proteins. Similarly, phosphorylation is known to disrupt aggregation after a phase transition, resulting in a diffuse localization.ConclusionsOur data establish that Them1 forms membraneless organelles (puncta) that function to suppress thermogenesis. Additionally, puncta are dispersed after phosphorylation at S15 to maximize fatty acid oxidation during a period of energy demand and to allow for the rapid suppression of thermogenesis once the demand no longer exists. We speculate that Them1 is an attractive molecular target in the management of non‐alcoholic fatty liver disease and other obesity‐related disorders.