All-trans-retinoic acid (RA) inhibits adipogenesis in established preadipocyte cell lines. Dosing pharmacological amounts of RA reduces weight gain in mice fed a high-fat diet, i.e. counteracts diet-induced obesity (DIO). The aldehyde dehydrogenase Raldh1 (Aldh1a1) functions as one of three enzymes that converts the retinol metabolite retinal into RA, and one of many proteins that contribute to RA homeostasis. Female Raldh1-ablated mice resist DIO. This phenotype contrasts with ablations of other enzymes and binding-proteins that maintain RA homeostasis, which gain adiposity. The phenotype observed prompted the conclusion that loss of Raldh1 causes an increase in adipose tissue retinal, and therefore, retinal functions independently of RA to prevent DIO. A second deduction proposed that low nM concentrations of RA stimulate adipogenesis, in contrast to higher concentrations. Using peer-reviewed LC/MS/MS assays developed and validated for quantifying tissue RA and retinal, we show that endogenous retinal and RA concentrations in adipose tissues from Raldh1-null mice do not correlate with the phenotype. Moreover, male Raldh1-null mice resist weight gain regardless of dietary fat content. Resistance to weight gain occurs during adolescence in both sexes. We show that RA concentrations as low as 1 nM, i.e. in the sub-physiological range, impair adipogenesis of embryonic fibroblasts from wild-type mice. Embryonic fibroblasts from Raldh1-null mice resist differentiating into adipocytes, but retain ability to generate RA. These fibroblasts remain sensitive to an RA receptor pan-agonist, and are not affected by an RA receptor pan-antagonist. Thus, the data do not support the hypothesis that retinal itself represses weight gain and adipogenesis independently of RA. Instead, the data indicate that Raldh1 functions as a retinal and atRA-independent promoter of adiposity during adolescence, and enhances adiposity through pre-adipocyte cell autonomous actions.

RDH1 is one of several enzymes that catalyze the first of two reactions to convert retinol into alltrans-retinoic acid (atRA). Here we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20 weeks old, despite not consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develops following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express 3

We report an UHPLC-MS/MS to quantify all-trans-retinal in biological samples of limited size (15-35 mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5 fmol lower limit of detection and a linear range from 5 fmol to 1 pmol. This assay revealed that extra-ocular concentrations of retinal range from ~2 to 40 pmol/g in multiple tissues: the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (kd ≤ 0.4 nM) for its nuclear receptors (RARα, β, γ), whereas retinal has low, if any affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2 to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro, and therefore provides an efficient method to study metabolism of retinol in vivo and in vitro.