Several lines of evidence demonstrated that vitamin A is involved in the regulation of adiposity and energy balance. Hence, it was reported that administration of vitamin A to mice leads to weight loss (11
), that genetic manipulation of enzymes that mediate vitamin A metabolism in mice result in alterations in adiposity (45
), and that treatment of rodents with vitamin A or RA can change the expression levels of adipose genes involved in energy homeostasis (11
). However, the molecular mechanisms that underlie these effects remained incompletely understood.
The observation that, in addition to activating its classical nuclear receptors, RARs, RA can function as a ligand for PPARβ/δ (32
) suggests that the spectrum of the biological functions of this hormone is wider than previously suspected. An important function of the alternate RA receptor, PPARβ/δ, is its ability to regulate lipid and sugar homeostasis in various tissues, including the adipose tissue (2
). We show here that the expression levels of RARs and their cognate lipid-binding protein, CRABP-II, are downregulated and that the expression levels of proteins that mediate the alternate RA-induced pathway, i.e., PPARβ/δ and FABP5, markedly increase upon adipocyte differentiation. Consequently, RA can activate both RAR and PPARβ/δ in both preadipocytes and in differentiated adipose cells, but the balance of RA signaling shifts upon adipocyte differentiation, reducing CRABP-II/RAR activities and enabling efficient FABP5/PPARβ/δ activation. It is interesting in regard to these observations that it has been reported that activation of RARs in the early stages of adipogenesis inhibits differentiation (34
). Hence, downregulating RAR activity and diverting RA to PPARβ/δ appears to be a critical component of the differentiation process. In accordance with this conclusion, we show that knocking down FABP5 expression resulted in incomplete adipocyte differentiation (Fig. ). These observations are in agreement with the reports that the receptor associated with FABP5, PPARβ/δ, is a necessary component for adipocyte differentiation, as demonstrated by the observations that PPARβ/δ-null mice display reduced adipose tissue (2
Using cultured cells and a high-fat/high-carbohydrate diet-induced mouse model of obesity, we demonstrate that RA evokes multiple aspects of the program known to be triggered upon activation of PPARβ/δ. In adipocytes, RA induced the expression of PPARβ/δ target genes, including genes involved in lipid metabolism—e.g., genes encoding uncoupling proteins, ALDH9, required for fatty acid oxidation, and ANGPTL4, an adipokine that regulates plasma lipoprotein metabolism (20
)—and genes encoding proteins such as PDK1 and GLUT4, which are involved in insulin responses. In vivo, RA treatment upregulated the expression of lipid- and sugar-processing PPARβ/δ target genes in adipose tissue and liver, and it recapitulated the reported activity of PPARβ/δ in increasing skeletal muscle mitochondrial content (42
). Remarkably, RA treatment of obese mice led to weight loss and to an improved glucose tolerance despite the larger food intake of the treated mice. Taken together, with the higher body temperature of RA-treated mice, these observations indicate that the weight loss originated from enhanced energy utilization.
In further support of the notion that the beneficial effects of RA were to a large extent mediated by PPARβ/δ, the in vivo effects of the hormone were similar to those of a synthetic selective ligand for this receptor. Thus, both RA and GW0742 induced the expression of adipose PPARβ/δ target genes, enhanced food intake, raised the body temperature, increased the plasma concentrations of glycerol without affecting levels of free fatty acids, reduced the blood levels of triglycerides and insulin, and upregulated the expression of adipose PPARβ/δ and FABP5. Interestingly, although similar trends were observed, RA was more effective than GW0742 in inducing weight loss and in reducing insulin and triglyceride levels. These findings may be understood in view of the observations that some genes involved in lipid hydrolysis and fatty acid oxidation, such as ALDH9
, and HSL
(see Fig. , , and ), are regulated by both PPARβ/δ and RARs. Hence, while a major component of the protective activities of RA against adiposity and insulin resistance stems from activation PPARβ/δ, the heightened efficacy of this ligand likely originates from its ability to also activate RARs. RA-induced activation of both RARs and PPARβ/δ also underlies the observations that treatment of obese mice with GW0742 but not with RA results in elevated plasma cholesterol. Hence, while PPARβ/δ induces the expression of apo A1, RARs inhibit the effect (Fig. ). Consequently, administration of a PPARβ/δ-selective ligand leads to higher blood levels of HDL cholesterol (19
), but activation of both RARs and PPARβ/δ by RA does not.
The expression levels of both FABP5 and PPARβ/δ were found to be markedly lower in obese mice (Fig. ), and they were restored to levels similar to those observed in lean mice after RA treatment. In accordance with these observations, it has been reported that FABP5 levels are lower in obese than in lean or weight-reduced human subjects (12
). Deregulation of FABP5/PPARβ/δ signaling thus appears to be associated with the onset of obesity, and weight loss is correlated with reactivation of the pathway. The data indicate that the recovery of expression of the two proteins is a later indirect outcome of RA treatment. The mechanisms by which obesity downregulates these proteins and by which RA induces their recovery remain to be clarified. In an apparent discrepancy with the conclusion that downregulation of FABP5 is associated with obesity, it has been reported that FABP5-null mice are protected from diet-induced obesity and insulin resistance (22
). It should be noted, however, that in this mouse model FABP5 was lacking throughout development. Since FABP5 and PPARβ/δ are closely involved in adipocyte differentiation, it is likely that the observed phenotype in the mice stems from impaired adipogenesis and thus reflects reduced or altered adipose tissue rather than reports on the functions of FABP5 in adipocytes that differentiate under normal conditions. Such an interpretation is supported by the observations that reducing the expression of FABP5 during adipogenesis resulted in incomplete differentiation (Fig. ).
The results of the present study show that RA treatment of obese mice leads to depletion of adipose lipid stores, induction of weight loss, reversal of hepatic steatosis, increase of muscle mitochondrial content, improvement of glucose tolerance, and reactivation of the FABP5/PPARβ/δ pathway. These data provide a rationale for the long-noted but poorly understood function of vitamin A in regulating energy balance, and they suggest that RA may be an efficacious agent in suppressing obesity and insulin resistance.