149 3,5-Diiodo-L-thyronine modulates the expression of genes of lipid metabolism in a rat model of fatty liver Elena Grasselli1, Adriana Voci1, Ilaria Demori1, Laura Canesi1,5, Rita De Matteis2, Fernando Goglia3, Antonia Lanni4, Gabriella Gallo1 and Laura Vergani1,5 1DIPTERIS, Universita` di Genova, Genova, Italy 2Dipartimento di Scienze Biomolecolari, Universita` di Urbino, Urbino, Italy 3Dipartimento di Scienze Biologiche ed Ambientali, Universita` del Sannio, Benevento, Italy 4Dipartimento di Scienze della Vita, Seconda Universita` di Napoli, Caserta, Italy 5Istituto Nazionale Biostrutture e Biosistemi (INBB), Rome, Italy (Correspondence should be addressed to G Gallo; Email: [email protected]) Abstract Recent reports demonstrated that 3,5-diiodo-L-thyronine protein component of very low-density lipoproteins (VLDLs) (T2) was able to prevent lipid accumulation in the liver of rats were analysed. Overall, our data demonstrated that T2 fed a high-fat diet (HFD). In this study, we investigated how administration to HFD rats counteracts most of the hepatic the rat liver responds to HFD and T2 treatment by assessing transcriptional changes that occurred in response to the excess the transcription profiles of some genes involved in the exogenous fat. In particular, our results suggest that T2 may pathways of lipid metabolism: oxidation, storage and prevent the pathways leading to lipid storage in LDs, promote secretion. The mRNA levels of the peroxisome prolifera- the processes of lipid mobilisation from LDs and secretion as tor-activated receptors (PPARa,PPARg and PPARd), and of VLDL, in addition to the stimulation of pathways of lipid their target enzymes acyl-CoA oxidase and stearoyl-CoA oxidation. In conclusion, our findings might give an insight desaturase were evaluated by real-time RT-PCR. Moreover, into the mechanisms underlying the anti-steatotic ability of the expression of the adipose triglyceride lipase involved in T2 and help to define the potential therapeutic role of T2 for lipid mobilisation, of the main PAT proteins acting in lipid preventing or treating liver steatosis. droplet (LD) turnover, and of apoprotein B (apo B), the major Journal of Endocrinology (2012) 212, 149–158 Introduction 2007, Kapadia et al. 2008). In summary, PPARa and PPARd mainly participate in energy burning, whereas PPARg is Hepatic steatosis refers to an excess accumulation of lipids, critical in regulating energy storage (Musso et al. 2009), primarily triglycerides (TAGs) and it is a major element of even though an overlapping in their function has been widely non-alcoholic fatty liver disease (NAFLD; Unger et al. 2010). reported. Lipid metabolism is mainly regulated by the peroxisome Excess TAGs are stored under form of cytosolic lipid proliferator-activated receptors (PPARs), a subfamily of droplets (LDs) regulating storage and traffic of lipids (for a lipid-activated transcription factors, consisting of three review see Ducharme & Bickel (2008) and Olofsson et al. members, PPARa,PPARg and PPARd, with distinct (2009)). Typically, LDs exhibit a core of TAGs and cholesteryl functional roles (Viswakarma et al. 2010, Wanders et al. esters surrounded by a phospholipid monolayer that contains 2010). In the liver, PPARa is the master regulator of free fatty numerous proteins including the ‘PAT proteins’ with acid (FFA) oxidation systems, and decreases FFA efflux structural, regulatory or enzymatic functions (Bickel et al. (Reddy 2001, Pyper et al. 2010). Also PPARd induces 2009). A previous study of our group (Grasselli et al. 2010) expression of genes required for FFA oxidation (Evans et al. showed that, in rat hepatocytes, lipid overload was associated 2004, Pyper et al. 2010). Conversely, PPARg is mainly with altered expression of three PAT proteins: the adipocyte expressed in adipose tissue, but it promotes lipid accumulation differentiation-related protein (ADRP, also called PLIN2), also in hepatocytes where it plays anti-inflammatory effects by the oxidative tissue-enriched PAT protein (OXPAT or both reducing inflammatory cytokine and chemokine PLIN5) and the tail-interacting protein (TIP47 or PLIN3; expression and preventing the activation of pro-inflammatory Kimmel et al. 2010), which are thought to be involved in LD transcription factors (Delerive et al. 2001, Straus & Glass turnover together with the endoplasmic reticulum (ER) Journal of Endocrinology (2012) 212, 149–158 DOI: 10.1530/JOE-11-0288 0022–0795/12/0212–149 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/26/2021 05:52:04PM via free access 150 E GRASSELLI and others . Anti-steatotic effects of T2 resident protein fat-inducing transcript 2 (FIT2; Kadereit et al. Materials and Methods 2008, Sun et al. 2010). In the liver, TAGs stored in LDs may be mobilised by Chemicals lipases that trigger FFAs either for oxidation or for secretion as All chemicals, unless otherwise indicated, were of analytical very low-density lipoproteins (VLDLs). The relative abun- grade and were obtained from Sigma–Aldrich Corp. dance of PAT proteins on LDs is decisive for the regulation of the action of lipases (Zimmermann et al. 2009). Recently, discovered adipose triglyceride lipase (ATGL) selectively Animals and treatments performs the first step in TAG hydrolysis (Duncan et al. 2007, Animal maintenance and treatment were carried out Zechner et al. 2009), and is constitutively expressed in the according to the guidelines of the European Community liver of mammals (Reid et al. 2008). Council for animal care and use, as described elsewhere Tobe secreted by the liver through exocytosis, TAGs must be (Grasselli et al. 2008). Briefly, 24 male Wistar rats (Harlan, packaged together with apoprotein B (apo B), the major protein San Pietro al Natisone, Italy), housed in individual cages, component of VLDL (Vance & Vance 1990). Recent studies were randomly divided into four groups (each group, have pointed out that hepatic secretion of VLDL is increased six animals). In each group, BW was normally distributed, during NAFLD, even though it seems to be inadequate to and BW means were similar for all animals. The first group (C) match the increased TAG availability (Zivkovic et al.2007). received a standard diet (15.88 kJ gross energy/g); the second Thyroid hormones (THs) are major modulators of energy group (D) was fed a HFD (19.85 kJ gross energy/g); the third homeostasis, and their role in lipid metabolism has supported group (DT) received the same HFD and a simultaneous daily their potential use as drugs to promote weight loss. Several i.p. injection of T2 (25 mg/100 g BW); and the fourth group evidences suggested that 3,5-diiodo-L-thyronine (T2), a (CT) received a standard diet and a simultaneous daily i.p. naturally occurring iodothyronine, mimics several effects of 0 injection of T2 (25 mg/100 g BW). C and D rats were daily i.p. 3,3 ,5-L-triiodothyronine (T3) on energy metabolism with- injected with the same volume of physiological saline solution. out inducing thyrotoxic effects (Lanni et al. 1994, Cimmino After 30 days of treatment, rats were anesthetised and killed by et al. 1996, Lombardi et al. 1998). Also T2, like T3, is able to cervical dislocation. Before blood and tissue sampling, rats stimulate both resting metabolic rate and mitochondrial were not fasted to avoid possible interference with the effects activity in hypothyroid rats (Moreno et al. 1997). However, of HFD. Blood was collected and serum concentrations of although it is likely that iodothyronines regulate energy TAGs, cholesterol and glucose were measured using a Hitachi metabolism by both short- and long-term actions, the 7170 clinical analyser (Hitachi) by following standard molecular mechanisms are still unclear (Goglia 2005). procedures. Immediately after death, livers were rapidly Moreover, long-term administration of T2 to rats fed high- dissected, weighed, cut into small pieces, quickly frozen in fat diet (HFD) was able to counteract the body weight (BW) liquid nitrogen and stored at K80 8C until use. and fat mass gain, as well as lipid accumulation and oxidative stress in the liver (Lanni et al. 2005, Grasselli et al. 2008). T2 affects liver metabolism by increasing mitochondrial FFA Liver morphological analysis and lipid staining oxidation and mitochondrial uncoupling (Lanni et al. 2005, To detect fat accumulation in the livers, frozen tissue sections Grasselli et al. 2008), but exerts metabolic effects also in were stained with Oil Red O (ORO) according to the skeletal muscle by increasing mitochondrial oxidative capacity method described by Koopman et al. (2001). Briefly, liver (Lombardi et al. 2007). In a recent study (Grasselli et al. cryosections were fixed in 10% formalin at room temperature 2011b), we demonstrated that the lipid-lowering effect of for 15 min, then dipped in 70% isopropanol for 3 min. both T2 and T3 occurs via non-receptor-mediated Afterwards, slides were immersed in 1% ORO solution for mechanisms that seem to involve both a short-term action 15 min, washed first in 70% isopropanol then in distilled by stimulation of mitochondrial O2 consumption, and a long- water, and mounted using aqueous mounting medium. term action by differential transcriptional effects on PPARs. Haematoxylin–eosin staining of liver sections was done In the attempt to clarify the mechanisms underlying the using standard protocols (Grasselli et al. 2011a). Livers were anti-steatotic ability of T2 better, in this study we analysed