Them1 was initially named brown fat inducible thioesterase (BFIT) because it is selectively enriched expressed in brown adipose tissue (BAT) and because it was markedly upregulated when mice were exposed to cold ambient temperatures. When taken together with the observation that Them1 gene expression was higher in BAT of mouse strains that were resistant to diet-induced obesity, it was predicted that Them1 might function to promote energy expenditure. Although not systematically characterized as an enzyme, the same investigators commented that recombinant Them1 functioned as a medium- to long-chain fatty acyl-CoA thioesterase. Them1 was subsequently incorporated into the Acot family as Acot11 and into the START domain family as StarD14.
Our own attention was drawn to Them1 because of its ties to both the Acot and START domain gene families. In an effort to understand the regulatory effects of another START domain protein, phosphatidylcholine transfer protein (PC-TP; synonym, StarD2) in hepatic glucose and lipid metabolism and energy homeostasis, we performed a yeast two-hybrid screen that identified Them2 as a PC-TP-binding protein. The motivation for seeking a binding partner was that PC-TP is an example of a START domain minimal protein that comprises a lipid binding pocket, but contains no other apparent functional domains. It was curious to unearth an interaction between a START minimal domain protein and a hotdog fold thioesterase, when two related proteins (i.e. Them1 and Acot12) each contained tandem hotdog fold thioesterase domains plus a START domain within the same multidomain protein.
To test its own role in nutrient metabolism and energy homeostasis, we created Them1-/- mice. Unexpectedly, Them1-/- mice exhibited increased energy expenditure and were resistant to diet-induced obesity, as well as associated metabolic disorders. Increased concentrations of fatty acyl-CoAs in BAT and decreased thioesterase activities of BAT homogenates suggested that Them1 functions as an Acot in vivo. Consistent with increased rates of energy expenditure, rates of fatty acid oxidation in BAT were increased in the absence of Them1 expression.
Them1-/- mice also displayed resistance to diet induced inflammation in white adipose tissue (WAT) and to hepatic steatosis and insulin resistance. Whereas these effects may have been ascribed to increased energy consumption by BAT, the data indicated that Them1 expression in WAT and liver per se played important roles in the development of obesity associated metabolic disorders. In WAT, Them1 expression was upregulated several-fold in response to high fat feeding. Using white adipocytes from chow fed animals, we demonstrated a reduced capacity of conditioned media to activate cultured wild type macrophages. This suggested the possibility that Them1 activity in wild type adipocytes led to the release of one or more pro-inflammatory molecules, possibly including free fatty acids. Along similar lines, endoplasmic reticulum (ER) stress led to upregulation of Them1 expression in mouse liver, and livers of Them1-/- mice exhibited reduced response to ER stress. This effect appeared to be cell autonomous, as evidence by reduced ER stress responses in cultured hepatocytes and MEFs prepared from Them1-/- mice. Collectively, these observations suggest a pathogenic role for Them1 in the metabolic abnormalities that accompany overnutrition, potentially by liberating excess free fatty acids intracellularly.
Although the molecular mechanism by which Them1 reduces energy consumption is not yet known, a key regulatory role for this enzyme is in line with evidence that interconversion of free fatty acids and fatty acyl-CoAs is important for the control of thermogenesis in BAT. Studies in mice with adipose-specific deletion of long-chain acyl-CoA synthetase 1 (ASCL1), which catalyzes the formation of fatty acyl-CoA thioesterases from free fatty acids and CoASH, have revealed that activation of free fatty acids to form fatty acyl-CoAs facilitates their mitochondrial oxidation in BAT. By reversing this reaction, Them1 could play an important role in reducing rates of fatty acid oxidation. We also provided evidence in Them1-/- mice for the differential regulation of genes that are under the control of fatty acid-activated nuclear hormone receptors that transcriptionally regulate BAT differentiation and thermogenesis (e.g. PPARγ and PPARα). This suggests that Them1 activity may regulate intracellular concentrations of selected ligands, which activate these nuclear hormone receptors.
Whereas our paper suggests that Them1 functions in vivo as an acyl-CoA thioesterase, additional mechanistic insights into metabolic regulation and energy homeostasis will no doubt emerge from a deeper understanding of both the cellular biology and the structure-function relationships of the protein. Using subcellular fractionation techniques, we found that Them1 was mainly concentrated in the ER and to a lesser extent mitochondria and cytosol. In liver, the protein was mainly concentrated in cytosol. However, unlike the other Them proteins discussed below, Them1 was not identified as a component of the mitochondrial proteome. A clearer understanding of the cellular localization and its determinants would contribute to our understanding of Them1’s biological function.
At present, neither the enzymatic characteristics of Them1 nor the lipid ligand of the START domain have been elucidated. Whereas the crystal structure of the unliganded Them1 START domain revealed a hydrophobic binding pocket, it did not provide firm clues as to the identity of the endogenous ligand. A number of lines of indirect evidence led the authors to propose that fatty acids ranging up to 18 carbons could constitute the natural ligands. Once a ligand is identified, it will be important to discern whether its binding to the START domain regulates the enzymatic activity of Them1. Knowledge of the enzymatic characteristics and endogenous ligand will should also help to determine the feasibility of targeting Them1 for the management of obesity and its associated metabolic disorders.