Four-week-old male Fischer-344 (F-344) rats were purchased from Charles River Italy (Calco, Italy). Rats were housed for two weeks at 22°C with free access to basal rodent diet (Mucedola s.r.l., Settimo Milanese, Italy) and drinking water, and with a 12-hours light/dark daily cycle before starting the experiments. All animal procedures were approved by the Italian Ministry of Health (the authorization codes are 1247/15-PR and 560/2019-PR), complied with national ethical guidelines for animal experimentation and were conducted in accordance with the guidelines of the local ethical committee for in vivo experimentation.

Three experimental protocols were adopted:

Experimental Protocol 1. Eleven rats given a single intraperitoneal dose of diethylnitrosamine (DEN, 150 mg/kg body weight) were fed ad libitum a high fat diet (HFD, 42% kcal/fat diet containing sucrose and 1,25% cholesterol, (Mucedola s.r.l.) for 30 weeks. HFD-fed rats were then split into two groups: group 1 (N=5) was maintained on HFD for further three weeks; group 2 (N=6) was fed a HFD plus TG68 (9.35 mg/kg in drinking water for 3 weeks). The dose 9.35 mg/kg was selected based on the dose-response to MGL-3196 on cholesterol lowering in DIO mice (30) and on our previous experiments (32).

Experimental Protocol 2. Twenty-three rats given a single dose of DEN as in Experimental Protocol 1 were fed ad libitum a HFD for 30 weeks and then split into three groups: group 1 (N=6) was maintained on HFD for further two weeks; group 2 (N=6) was fed a HFD plus TG68 (2.8 mg/kg, in drinking water) while a third group of rats (N=6) was fed a HFD plus TG68 (1.4 mg/kg in drinking water). Animals fed a basal diet were used as control group (N=5). Animals given TG68 were sacrificed 2 weeks later.

Experimental Protocol 3. Thirteen rats given DEN as in Experimental Protocol 1 and 2 were fed ad libitum a HFD for 39 weeks and then split into three groups: group 1 (N=4) was maintained on HFD for further two weeks; group 2 (N=5) was fed a HFD plus TG68 (2.8 mg/kg, in drinking water). A third group (N=4) was fed HFD plus Resmetirom (MGL-3196, 3 mg/kg in drinking water, MedChemExpress), for the last two weeks. All animals were sacrificed under isoflurane anaesthesia. Blood and tissues, including liver, heart and kidney, were collected.

Blood samples were collected from abdominal aorta. Serum was separated by centrifugation and tested for triglycerides (TGs), cholesterol (CH), glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using a commercially available kit from Boehringer (Mannheim, Germany).

Lipid extraction and measurement of TGs were performed according to Schroeder-Gloeckler et al. (34). Briefly, liver samples were homogenized in 8 volumes of deionized water and in 1 volume of 5 M NaCl. Subsequently, a 200 µl aliquot of the homogenate was mixed with 500 µl of methanol and 250 µl of chloroform. Following centrifugation, the organic phase was collected. Complete extraction of any residual lipids was achieved by re-extracting with 250 µl chloroform:methanol (9:1). The organic phase was separated by centrifugation and samples were dried at room temperature (RT). The lipids were dissolved in a solution of 90% isopropanol:10% Triton X-100 (2%) to disperse the TGs for assay. Hepatic TG content was measured colorimetrically using a kit from Sigma-Aldrich (TR0100; Sigma-Aldrich, Milan, Italy).

Immediately after sacrifice, liver, heart and kidney were weighted; sections were fixed in 10% buffered formalin and processed for histological analysis (hematoxylin and eosin, H&E) or immunohistochemistry (IHC). The remaining tissues were snap-frozen in prechilled 2-methylbutane in liquid nitrogen and stored at -80°C until use. To determine the hepatic neutral lipid content, frozen liver sections were stained with Oil Red O (ORO, Sigma Aldrich) for 15 min, rinsed with 60% isopropanol, and stained with Mayer hematoxylin. The ORO staining positive area for each sample was quantified by using ImageJ analysis software (National Institute of Mental Health, Bethesda, Maryland, USA). To investigate glucose-6-phosphatase (G6Pase) activity, 15 μm serial frozen sections were cut in a cryostat (Leica LMD6000), and stained for G6Pase and Glutathione S-transferase Placental form (GSTP) as previously described (27).

Paraffin-embedded tissues were cut into 4 μm sections, dewaxed, and hydrated. Slides were microwaved in citrate buffer pH 6.0 and incubated overnight with the primary antibodies: GSTP (#311, MBL International); Glucose-6-Phosphate Dehydrogenase (G6PD, ab87230, Abcam). Sections were incubated with the appropriate polymer DAKO Envision secondary antibody at RT. Signal was detected using the VECTOR^® NovaRED™ Peroxidase (HRP) Substrate Kit (Vector Laboratories). Sections were counterstained with Harris hematoxylin solution (Bio-Optica).

The area of GSTP-positive preneoplastic lesions was measured with ImageJ according to Abramoff et al. (35).

Total RNA was extracted from snap-frozen rat liver tissues by using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) followed by RNeasy extraction kit (Qiagen). Extracted RNA was reverse-transcribed by using High-Capacity cDNA Reverse Transcription kit with RNase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). RNA was quantified by NanoDrop ND1000 (Thermo Fisher Scientific), while RNA integrity was assessed by Agilent Bioanalyzer 2100. Gene expression analysis was performed using TaqMan Gene expression Master Mix (Thermo Fisher Scientific) and the following specific TaqMan probes: Dio1 Rn00572183_m1, Myh7 Rn00568328_m1, Myh6 Rn00568304_m1, Cpt1a Rn00580702_m1, Acox1 Rn01460628_m1, Pnpla2 Rn01479968_g1, Fasn Rn00569117_m1, Dgat1 Rn00584870_m1, Thrsp Rn01511034_m1, Klf9 Rn00589498_m1, G6pd Rn01529640_g1, Gstp1 Rn00821792_g1, Gapdh 4351317. Each sample was run in triplicate and all measurements were normalized to Gapdh. Relative mRNA expression analysis for each gene was calculated by using the 2^-ΔΔCt method.

All data were expressed as the mean ± standard deviation (SD). Differences between groups were compared by student’s t-test or by using one-way ANOVA followed by Tukey post hoc analysis using the GraphPad software (Prism 9) (La Jolla, California). P-values were considered significant at p < 0.05.

To investigate the effect of TG68 on hepatic fat accumulation, in the first set of our experiments, we administered 9.35 mg/kg of the drug - dissolved in drinking water - to HFD-fed rats for the last 3 weeks (See experimental protocol in Figure 1A). TG68 caused a reduction of body weight, albeit not significant, compared to HFD alone, despite the fact that the food intake was similar in both the groups (Figure 1B). While an increase of heart and kidney weight was observed following TG68 treatment (Figure 1C), a significant reduction of liver weight and liver weight/body weight ratio compared to HFD-fed untreated rats was detected. (Figure 1D).

To investigate whether the observed reduction in liver weight was due to an amelioration of the hepatic steatosis, liver samples from both groups were subjected to comparative pathological analysis. The microscopic analysis of hematoxylin and eosin (H&E) stained sections confirmed the presence of steatosis in all the HFD livers from untreated mice (Figure 1E). ORO staining for neutral lipid content supported the histological observation highlighting the impressive reduction of hepatic fat content caused by TG68 (Figures 1E, F).

The reduction of liver fat accumulation was accompanied by a significant up-regulation of Phospholipase Domain Containing 2 (Pnpla2), and Carnitine Palmitoyltransferase-1 (Cpt1a), highlighting the effect of TG68 in decreasing the content of TGs on the one hand, and improving mitochondrial fatty acid oxidation in rats subjected to HFD on the other hand (Figure 2A). TG68 did not modify the mRNA levels of Acyl-CoA oxidase1 (Acox1), Fatty acid synthase (Fasn) and Diacylglycerol O-acyltransferase1 (Dgat1) (Figure 2A). Taken together, these results suggested that TG68 increased lipolysis in steatotic liver without a major impact on lipogenesis. In addition to the effect on hepatic fat accumulation, TG68 caused a striking reduction of circulating TGs and CH accompanied by a significant reduction of blood glucose levels, compared with HFD rats (Figure 2B).

To verify whether the observed effects were associated with TG68-induced activation of THRs, the expression of deiodinase 1 (Dio1) and thyroid hormone responsive (Thrsp), two well-known THR target genes, were investigated. As shown in Figure 2C, the expression of Dio1 and Thrsp was significantly increased following treatment with TG68.

Notably, unlike what was observed in the liver of HFD fed mice (33), microscopic analysis of rat liver did not exhibit any major sign of cell damage typically associated with NAFLD, such as cell swelling, Mallory-Denk bodies, acidophilic bodies or spotty necrosis. No detectable sign of liver cell injury following TG68 treatment was observed at light microscopic examination, as also shown by serum levels of ALT (Figure 2D). On the other hand, an increased cholangiocyte proliferation was occasionally observed in the liver of rats fed a HFD and co-treated with TG68.

Experimental evidence has shown that T3 exerts an anti-tumorigenic effect at early and late stages of the process (27, 28). To investigate whether TG68 could exert a similar effect, we scored the presence of preneoplastic lesions immuno-stained against GSTP, the best marker for the identification of preneoplastic rat liver foci/nodules (36). As shown in Figure 3A, a single treatment with DEN followed by HFD feeding for 30 weeks resulted in a high number of GSTP+ foci (50.9/cm²), with 0.66% of the liver being positive for GSTP staining. Co-administration of TG68 for the last three weeks caused a significant reduction in the number (4.3/cm², respectively) and the percentage of liver area occupied by GSTP-positive lesions (0.09%) (Figure 3A). Interestingly, while the vast majority of the GSTP-positive foci present in the liver of rats fed a HFD displayed an intense and homogeneous staining (persistent foci), almost all the ones observed in TG68-treated rats showed only a faint and discontinuous staining (remodeling foci) (Figures 3B, C), suggesting their reversion to a more differentiated phenotype following treatment with the thyromimetic.

The observed loss of GSTP staining caused by TG68 was not the consequence of a general transcriptional repression of GSTP expression by the drug, but a specific effect of TG68 on preneoplastic lesions. Indeed, immunohistochemistry showed no inhibitory effect of the drug on GSTP protein levels of bile ducts. (Figure 3D), thus indicating that the loss of GSTP immunostaining was due to the reacquisition of a differentiated phenotype of preneoplastic lesions. Further support to this proposition comes from the finding that TG68 caused an almost complete disappearance of preneoplastic lesions also when they were identified by G6PD immunostaining. Indeed, while almost all preneoplastic GSTP⁺ lesions in animals fed the HFD alone were also G6PD⁺ (Figures 3E, F), in rats co-treated with TG68 they were losing also G6PD positivity (Figures 3E, F), in spite of the increased hepatic mRNA levels of G6pd (Figure 3G). Taken together, these findings support the concept that TG68 caused the regression of preneoplastic foci by inducing a differentiated biochemical phenotype, and support the notion that activation of THRs by TG68 exerts an antitumoral effect. Support to the pro-differentiating effect of TG68 comes also from the finding of the enhanced expression of Klf9 ( Figure 3H), a Kruppel-like factor that contains a thyroid hormone response element and is implicated in the regulation of the balance between pluripotency, self-renewal differentiation, and metabolism.

T3 and other thyromimetics have been shown to exert their effects on several organs, including heart and kidney (37). Indeed, as shown in Figure 1C, treatment with TG68 for three weeks caused a significant increase in both heart and kidney weight.

Searching for experimental conditions that could avoid any possible impact of TG68 on extrahepatic organs, we adopted a protocol wherein rats given DEN and fed HFD were exposed to 1.4 or 2.8 mg/kg of TG68 for only 2 weeks (Experimental Protocol 2, Figure 4A). As shown in Figure 4B, no changes in food and water intake or body weight were detected at both the doses of the drug compared to HFD alone. Notably, heart and kidney weight were not affected compared to animals fed HFD alone (Figure 4C). Although no evidence of tissue damage could be observed by histological analysis (Figure 4D), to further investigate whether TG68 could cause damage to the heart through activation of THRs, we determined the mRNA levels of Myosin heavy chain 6 (Myh6) and Myosin heavy chain 7 (Myh7), two genes under TH control (38). As shown in Figure 4E, qRT-PCR analysis did not reveal any significant change in the cardiac expression of Myh6 and Myh7 in rats treated with both doses of TG68 compared to control rats. Notably, Dio1 mRNA levels were not modified following TG68 administration (Figure 4E) suggesting a low delivery of the drug to cardiomyocytes, at least at the doses used in this experimental protocol.

On the other hand, both doses of TG68 caused a significant reduction of liver weight and liver weight/body ratio compared to HFD-fed animals (Figure 4F). These changes occurred in the absence of any significant sign of liver injury, as indicated by ALT serum levels as well as histologic analysis (Figures 4G, H). ORO staining confirmed that a massive reduction of fat accumulation accounted for the decreased liver weight observed with both the doses of TG68 (Figure 4H). Reduction of hepatic fat accumulation was accompanied by a significant decrease of the levels of TGs, CH and glucose in the blood (Figure 4I).

Interestingly, while no clear difference on the regression of steatosis was observed between the two doses of TG68, a remarkably different effect on the regression of preneoplastic lesions was observed only with the dose of 2.8 mg/kg. Indeed, as shown in Figures 5A, B 2.8 mg/kg of TG68 led to a striking reduction of the number of GSTP+ foci and percentage of liver occupied by GSTP positive area (25/cm² ± 4.8 and 0.4% in rats fed HFD alone vs. 4/cm² ± 2.3 and 0.1% in rats HFD vs. TG68 co-treated animals). On the other hand, only a small decrease of the number of GSTP⁺ foci and no difference in the percentage of the liver occupied by GSTP⁺ lesions were observed in rats treated with the dose of 1.4 mg (17/cm² ± 7.1 and 0.3%). Notably, while the intensely stained GSTP⁺ foci in the liver of rats fed HFD alone were virtually negative for G6Pase, an enzyme highly expressed by differentiated hepatocytes (Figure 5C), a faint staining of GSTP was observed in the preneoplastic lesions still present following TG68 treatment in concomitance with a re-expression of G6Pase (Figure 5C). This finding further supports the hypothesis that the anti-tumorigenic effect of TG68 is, at least in part, due to its ability to induce a switch of preneoplastic hepatocytes towards a differentiated phenotype.

Next, we investigated whether i) TG68 could exert its anti-tumorigenic effect also at later stages of the carcinogenic process, and ii) a similar effect could be exerted also by Resmetirom (MGL-3196), a THRβ agonist that provided significant reductions in liver fat, low-density lipoprotein cholesterol and other atherogenic lipids vs. placebo in a Phase II trial (30). As illustrated in the Experimental Protocol 3 (Figure 6A), the rats exposed to DEN and fed a HFD for 39 weeks were then given either TG68 (2.8 mg/kg) or MGL-3196 (3 mg/kg) for 2 weeks. As shown in Figure 6B, only TG68 caused a decrease of body weight as well as of liver weight and liver weight/body weight ratio. Notably, a slight decrease of heart and kidney weight was observed with TG68 that was more pronounced with MGL-3196, compared to HFD-fed rats (Figure 6C). Both drugs strongly reduced the lipid content in the liver as detected by histological analysis and ORO staining (Figure 6D). Furthermore, TG68 also led to a decline in the levels of TGs and CH, although only the treatment with TG68 caused a statistically significant reduction of these parameters compared to the HFD group (Figure 6E). On the other hand, while both the drugs did not modify the serum levels of ALT or GGT, they caused a strong reduction of total bilirubin (Figure 6F).

Subsequently, we investigated and compared the effect of TG68 and MGL-3196 on DEN-induced preneoplastic foci. As shown in Figure 6G, at 41 weeks after DEN treatment the number of GSTP⁺ foci and the percentage of area occupied by GSTP⁺ foci in rats treated with HFD alone were much higher than those observed at 33 weeks (43 vs. 25 and 2.1 vs. 0.6, respectively). Interestingly, TG68 exerted a striking anti-tumorigenic effect even at this later stage of hepatocarcinogenesis. Indeed, it caused a 50% decrease in the number of GSTP⁺ foci (23/cm² vs. 44/cm²) and an even stronger reduction in the % area occupied by preneoplastic lesions (0.8% vs. 2.1%). On the opposite, no significant change in the number of GSTP⁺ foci (42/cm²) or in the % area occupied by these lesions (1.5%) was induced by MGL-3196.