Non-receptor-mediated actions are responsible for the lipid-lowering effects of iodothyronines in FaO rat hepatoma cells Elena Grasselli 1 , Adriana Voci 1 , Laura Canesi 1,3 , Fernando Goglia 2 , Silvia Ravera 1 , Isabella Panfoli 1 , Gabriella Gallo 1 and Laura Vergani 1,3 1 Dipartimento di Biologia, Universita ` di Genova, Corso Europa 26, Genova 16132, Italy 2 Dipartimento di Scienze Biologiche ed Ambientali, Universita ` del Sannio, Benevento 82100, Italy 3 Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma 00136, Italy (Correspondence should be addressed to L Vergani at Dipartimento di Biologia, Universita ` di Genova; Email: laura.vergani@unige.it) Abstract Iodothyronines influence lipid metabolism and energy homeostasis. Previous studies demonstrated that 3,5- L-diiodothyronine (T 2 ), as well as 3,3 0 ,5-L-triiodothyronine (T 3 ), was able to both prevent and reverse hepatic steatosis in rats fed a high-fat diet, and this effect depends on a direct action of iodothyronines on the hepatocyte. However, the involvement of thyroid hormone receptors (TRs) in mediating the lipid-lowering effect of iodothyronines was not elucidated. In this study, we investigated the ability of T 2 and T 3 to reduce the lipid overloading using the rat hepatoma FaO cells defective for functional TRs. The absence of constitutive mRNA expression of both TRa1 and TRb1 in FaO cells was verified by RT-qPCR. To mimic the fatty liver condition, FaO cells were treated with a fatty acid mixture and then exposed to pharmacological doses of T 2 or T 3 for 24 h. Lipid accumulation, mRNA expression of the peroxisome proliferator-activated receptors (PPAR-a,-g,-d) the acyl-CoA oxidase (AOX), and the stearoyl CoA desaturase (SCD1), as well as fuel-stimulated O 2 consumption in intact cells, were evaluated. Lipid accumulation was associated with an increase in triacylglycerol content, PPARg mRNA expression, and a decrease in PPARd and SCD1 mRNA expression. The addition of T 2 or T 3 to lipid-overloaded cells resulted in i) reduction in lipid content; ii) downregulation of PPARa, PPARg, and AOX expression; iii) increase in PPARd expression; and iv) stimulation of mitochondrial uncoupling. These data demonstrate, for the first time, that in the hepatocyte, the lipid-lowering actions of both T 2 and T 3 are not mediated by TRs. Journal of Endocrinology (2011) 210, 59–69 Introduction Thyroid hormones (THs) play a major role in lipid metabolism, with the liver representing one of their main target tissues. Other iodothyronines display some thyro- mimetic activities; among them, 3,5-L-diiodothyronine (T 2 ) mimics several effects of 3,3 0 ,5-L-triiodothyronine (T 3 ) on energy metabolism without inducing thyrotoxic effects (Cimmino et al. 1996, Lanni et al. 2005). Also T 2 , as T 3 , is able to stimulate both resting metabolic rate and mitochondrial activity in hypothyroid rats (Moreno et al. 1997). Moreover, when administered to rats receiving a high-fat diet, T 2 was able to both prevent (Lanni et al. 2005, Grasselli et al. 2008) and reduce (Mollica et al. 2009) the development of liver steatosis. Once entering the cell, THs mostly bind to specific nuclear receptors (TRs) acting as ligand-dependent transcription factors (‘receptor-mediated’ effects). In humans and rodents, TRs are encoded by two genes, Thra and Thrb, each encoding different isoforms (Izumo & Mahdavi 1988, Lazar 1993). Actions of iodothyronines that are not initiated by binding to TRs are termed ‘non-receptor-mediated’ mechanisms (Cheng et al. 2010), and could involve specific membrane- associated binding sites coupled with activation of rapid signaling pathways, involving MAP kinases, phosphoinositol- 3-kinase (PI3K), or Ca 2C mobilization (Bergh et al. 2005). Similar to THs, the peroxisome proliferator-activated receptors (PPARs) play important roles in lipid homeostasis. In humans and rodents, PPARs are encoded under three isoforms a, g, and d that bind to free fatty acids (FFAs; Viswakarma et al. 2010), but only PPARa binds to saturated FFAs (Xu et al. 1999). In the liver, PPARa is the master regulator of FFA oxidation, regulates the expression of acyl- CoA oxidase (AOX), and decreases FFA efflux (Martin et al. 1997). Also PPARd is highly expressed in the liver, where it enhances FFA catabolism (Barish et al. 2006) and reduces steatosis (Shan et al. 2008). PPARg is mainly expressed in adipose tissue (Schoonjans et al. 1996) but promotes lipid accumulation also in hepatocytes (Yu et al. 2003). Overall, PPARa and PPARd function as catabolic regulators of energy, while PPARg plays anabolic functions on lipid metabolism, even though an overlapping in their expression has been reported (Moreno et al. 1997). 59 Journal of Endocrinology (2011) 210, 59–69 DOI: 10.1530/JOE-11-0074 0022–0795/11/0210–059 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 12/24/2021 09:57:31AM via Massachusetts Inst of Technology