Furthermore, our in vivo experiments in mice confirmed the upregulation of ZAG induced by T3 in the liver. By contrast, T3 did not regulate ZAG production either in primary human adipocyte cultures or in mouse adipocytes. Overall, these findings suggest a differential regulation of ZAG by T3 in liver and adipose tissue. This different tissue regulation of gene expression by thyroid hormone has been previously Evodiamine observed in other genes such as pigment epithelium-derived factor. In this regard, one could postulate it is could be possible the existence of an alternative promoter driving ZAG expression in adipose tissue, which might explain the lack of regulation by T3. In addition, it is worth recalling that although thyroid hormone receptors are expressed in both the liver and adipose tissue, it is possible that TR activation could differ due to the presence or absence of different co-activators or co-repressors. Moreover, the possibility that the presence of different deiodinases in these tissues also modulates thyroid hormone action should be taken into account. In the clinical setting we observed that the mean reduction of ZAG serum levels after successful treatment of hyperthyroidism was 15 mg/ml. Notably, this reduction was observed in all patients after treatment of hyperthyroidism. These results suggest that T3 exerts 
a subtle but consistent modulation of ZAG expression which is sufficient to significantly change its circulating levels. Since T3 has no effect on ZAG production in mature adipocytes, the T3 induced ZAG upregulation in the liver seems to be the main factor accounting for the increase of ZAG serum levels observed in patients with hyperthyroidism. This finding confirms the idea that, apart from adipose tissue, the liver is an important contributor to systemic levels of ZAG. The lack of Rosiridin relationship between circulating ZAG and weight changes deserves a specific comment. We did not find any relationship between the reduction of ZAG serum levels and the increase of either body weight or BMI after treatment of hyperthyroidism. This finding suggests that systemic levels of ZAG are not a significant factor involved in the weight changes induced by thyroid hormones. In fact, a recent study performed by Mracek et al showed that ZAG levels were not different between cachectic and weight stable cancer patients. In this regard, it should be noted the overexpression of ZAG in the adipose tissue rather than its serum levels is the main determinant of the lipolytic action of ZAG in the cancer-induced cachexia and also in end-stage-renal disease. Since we have found that T3 was unable to increase ZAG production by adipose tissue it seems reasonable to deduce that ZAG is not involved in weight loss associated with hyperthyroidism. Taken together, these findings suggest that the autocrine/paracrine action of ZAG in adipose tissue is more important than endocrine action through its systemic levels. It has recently been reported that iodothyronines induce a reduction of the excess of fat in primary cultures of rat hepatocytes. In addition, an inverse association between serum free thyroxine and hepatic steatosis has been found in a large population based study. In the present study we provide first evidence that ZAG exerts a lipolytic effect in the liver, and this effect was observed in a dose-dependent manner. Notably, the hepatic lipolytic effects were observed in mice using ZAG concentrations detected in hyperthyroid mice. Since T3 stimulates ZAG production in the liver it is possible that this is one of the mechanisms involved in fat storage regulation by thyroid hormones in the liver.