Them1 Regulates the Subcellular Localization of Glycogen in Brown Adipocytes: Implications for Energy Homeostasis

Molecular mechanisms that integrate lipid and glycogen metabolism are not well understood. In brown adipose tissue (BAT), cold exposure increases energy expenditure by increasing mitochondrial β‐oxidation of lipid droplet‐derived fatty acids. This process is suppressed by thioesterase superfamily member 1 (Them1), a fatty acyl‐CoA thioesterase that is also upregulated by cold ambient temperatures. Them1 limits thermogenesis by reducing the availability of fatty acid for β‐oxidation in mitochondria. Previous studies suggested that the effects of Them1 can be blocked by PKCβ‐mediated phosphorylation at S15, but the cellular mechanism remains unclear. Here, we demonstrate that Them1 associates with glycogen and controls its 3‐D physical state in a phosphorylation‐dependent manner. Methods The full‐length cDNA for THEM1 and a series of mutants were cloned and linked to tandem dimer tomato (tdTomato) fluorescent protein or EGFP that included an engineered ascorbate peroxidase ( APEX2 ) fusion protein at the C‐terminus. The plasmids were transfected into a differentiated brown adipocyte cell line that does not express Them1. The localization of Them1 was visualized by confocal microscopy (EFGP or tdTomato; living cells) or by electron microscopy (EM; APEX2). Volocity software was used to produce 3‐D images from confocal z‐stacks. Them1 localization was also evaluated in the presence or absence of inhibitors and activators of key effectors of the lipolysis pathway. Immunoprecipitation, using isolated lipid droplets from BAT and anti‐Them1, was used to evaluate potential binding partners by proteomics from Them1‐expressing vs deficient cold‐exposed mice. Results In unstimulated cells, Them1 formed small (100–200 nm in diameter), punctate structures that were localized near lipid droplets and mitochondria and also circumferentially surrounded lipid droplets. EM studies confirmed that Them1 was associated with glycogen in both locations. In response to stimulation by norepinephrine (β3‐adrenergic receptor agonist), PKA, or adipose triglyceride lipase, Them1 was redistributed from a punctate to a diffuse localization. By contrast, inhibiting PKA or PKC retained Them1 in a punctate localization. The punctate form was also observed when the first 36 amino acids of Them1 were deleted or when the S15 phosphorylation site was mutated from S15 to A15. In the diffuse distribution, Them1 localized to individual β‐glycogen particles by EM. Proteomic analysis revealed glycogen phosphorylase as a likely binding partner for Them1. Conclusions Our results show that Them1 is associated with glycogen in BAT cells, most likely via its interactions with glycogen phosphorylase. Them1 phosphorylation induces a major conformational rearrangement of glycogen, which in turn redistributes Them1 away from lipid droplets. This presumably prevents Them1 from suppressing lipolysis during a period of energy demand. We speculate that the Them1‐mediated glycogen rearrangement may also function to coordinate lipid and glucose metabolism within BAT. Support or Funding Information This work was supported by NIH DK103046 to DEC and SJH. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .