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Objective: Retinol saturase (RetSat) is an endoplasmic reticulum-localized oxidoreductase highly expressed in organs involved in lipid metabolism such as white (WAT) and brown adipose tissue (BAT). Cold exposure was shown to increase RETSAT protein in BAT but its relevance for non-shivering thermogenesis, a process with beneficial effects on metabolic health, is unknown. Methods: We analyzed the regulation of RetSat expression in white and brown adipocytes and different murine adipose tissue depots upon β-adrenergic stimulation and cold exposure. RetSat function during the differentiation and β-adrenergic stimulation of brown adipocytes was dissected by loss-of-function experiments. Mice with BAT-specific deletion of RetSat were generated and exposed to cold. Gene expression in human WAT was analyzed and the effect of RetSat depletion on adipocyte lipolysis investigated. Results: We show that cold exposure induces RetSat expression in both WAT and BAT of mice via β-adrenergic signaling. In brown adipocytes, RetSat has minor effects on differentiation but is required for maximal thermogenic gene and protein expression upon β-adrenergic stimulation and mitochondrial respiration. In mice, BAT-specific deletion of RetSat impaired acute but not long-term adaptation to cold exposure. RetSat expression in subcutaneous WAT of humans correlates with the expression of genes related to mitochondrial function. Mechanistically, we found that RetSat depletion impaired β-agonist-induced lipolysis, a major regulator of thermogenic gene expression in adipocytes. Conclusions: Thus, RetSat expression is under β-adrenergic control and determines thermogenic capacity of brown adipocytes and acute cold tolerance in mice. Modulating RetSat activity may allow for therapeutic interventions towards pathologies with inadequate metabolic activity.

Summary: The ability to feed on a sugar-containing diet depends on a gene regulatory network controlled by the intracellular sugar sensor Mondo/ChREBP-Mlx, which remains insufficiently characterized. Here, we present a genome-wide temporal clustering of sugar-responsive gene expression in Drosophila larvae. We identify gene expression programs responding to sugar feeding, including downregulation of ribosome biogenesis genes, known targets of Myc. Clockwork orange (CWO), a component of the circadian clock, is found to be a mediator of this repressive response and to be necessary for survival on a high-sugar diet. CWO expression is directly activated by Mondo-Mlx, and it counteracts Myc through repression of its gene expression and through binding to overlapping genomic regions. CWO mouse ortholog BHLHE41 has a conserved role in repressing ribosome biogenesis genes in primary hepatocytes. Collectively, our data uncover a cross-talk between conserved gene regulatory circuits balancing the activities of anabolic pathways to maintain homeostasis during sugar feeding.

Hepatocytes secrete retinol-binding protein 4 (RBP4) into circulation, thereby mobilizing vitamin A from the liver to provide retinol for extrahepatic tissues. Obesity and insulin resistance are associated with elevated RBP4 levels in the blood. However, in a previous study, we observed that chronically increased RBP4 by forced Rbp4 expression in the liver does not impair glucose homeostasis in mice. Here, we investigated the effects of an acute mobilization of hepatic vitamin A stores by hepatic overexpression of RBP4 in mice. We show that hepatic retinol mobilization decreases body fat content and enhances fat turnover. Mechanistically, we found that acute retinol mobilization increases hepatic expression and serum levels of fibroblast growth factor 21 (FGF21), which is regulated by retinol mobilization and retinoic acid in primary hepatocytes. Moreover, we provide evidence that the insulin-sensitizing effect of FGF21 is associated with organ-specific adaptations in retinoid homeostasis. Taken together, our findings identify a novel crosstalk between retinoid homeostasis and FGF21 in mice with acute RBP4-mediated retinol mobilization from the liver.