TG68, a Novel Thyroid Hormone Receptor-β Agonist for the Treatment of NAFLD

Abstract

Activation of thyroid hormone receptor β (THRβ) has shown beneficial effects on metabolic alterations, including non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effect of TG68, a novel THRβ agonist, on fatty liver accumulation and liver injury in mice fed a high-fat diet (HFD). C57BL/6 mice fed HFD for 17 or 18 weeks, a time when all mice developed massive steatohepatitis, were then given TG68 at a dose of 9.35 or 2.8 mg/kg for 2 or 3 weeks, respectively. As a reference compound, the same treatment was adopted using equimolar doses of MGL-3196, a selective THRβ agonist currently in clinical phase III. The results showed that treatment with TG68 led to a reduction in liver weight, hepatic steatosis, serum transaminases, and circulating triglycerides. qRT-PCR analyses demonstrated activation of THRβ, as confirmed by increased mRNA levels of Deiodinase-1 and Malic enzyme-1, and changes in lipid metabolism, as revealed by increased expression of Acyl-CoA Oxidase-1 and Carnitine palmitoyltransferase-1. The present results showed that this novel THRβ agonist exerts an anti-steatogenic effect coupled with amelioration of liver injury in the absence of extra-hepatic side effects, suggesting that TG68 may represent a useful tool for the treatment of NAFLD.

Keywords: MAFLD; NASH; Resmetirom; steatosis; thyromimetics; triglycerides.

Figure 1

Effect of two-week treatment with TG68 or MGL-3196 on liver weight. (A) Experimental design and timeline of the in vivo experiments; (B) body weight; (C) liver weight; (D) liver weight/body weight ratio. Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values represent mean ± standard deviation (n ≥ 3). * p < 0.05, ** p < 0.01. Abbreviations: BD, basal rodent diet; HFD, high fat diet.

Figure 2

Two-week treatment with MGL-3196 or TG68 specifically reduced hepatic neutral fat accumulation. (A) Representative images of liver sections stained with hematoxylin and eosin (5×, upper panel) or Oil Red O (10×, lower panel) at 10 mg/kg (MGL-3196) or 9.35 mg/kg (TG68); (B) gene expression analysis of hepatic Dio1, Me1 and (C) cardiac Myh6. All measurements were normalized to β-actin. Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values represent mean ± standard deviation. * p < 0.05, **** p < 0.0001. Abbreviations: BD, basal rodent diet; HFD, high fat diet.

Figure 3

Three-week treatment with TG68 (2.8 mg/kg) or MGL-3196 (3 mg/kg) reduced body and liver weight. (A) Experimental design and timeline of the in vivo experiments. (B) Body weight B; (C) liver weight; (D) liver weight/body weight ratio; (E) heart weight; (F) kidney weight. Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values are shown as mean ± standard deviation (n ≥ 3). * p < 0.05, *** p < 0.001, **** p < 0.0001. Abbreviations: BD, basal rodent diet; HFD, high fat diet.

Figure 4

Three-week treatment with TG68 or MGL-3196 reduced hepatic neutral fat accumulation. (A) Representative images of liver sections stained with H&E (20×, upper) or Oil Red O (10 ×, lower); (B) Oil Red O staining positive area quantification by using ImageJ. Data were normalized to controls (BD). Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values are shown as mean ± standard deviation. * p < 0.05, ** p < 0.01. Abbreviations: BD, basal rodent diet; HFD, high fat diet; H&E, hematoxylin and eosin; ORO, Oil Red O.

Figure 5

Three-week treatment with TG68 reduced liver damage, hepatocyte proliferation, and serum triacylglycerides and cholesterol levels. (A,B) Effect of TG68 and MGL-3196 on serum alanine aminotransferases (ALT) and aspartate aminotransferases (AST); (C) representative images of liver sections immunostained for BrdU detection, (D) BrdU Labeling Index. Labeling index was calculated as the number of BrdU-positive hepatocyte nuclei/per field at 40× magnification. Additionally, 50 fields per rat liver were scored; (E) representative images of liver sections immunostained for alpha-smooth-muscle actin (α-SMA) detection (20×). Mice treated with carbon tetrachloride (CCl₄) were used as positive controls; (F) gene expression analysis of Actin alpha 2 (Acta2) and Galectin 3 (Lgals3) in mice liver. All measurements were normalized to β-actin; (G,H) effect of TG68 and MGL-3196 on serum triacylglycerides and cholesterol levels. Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values are shown as mean ± standard deviation. * p < 0.05, **** p < 0.0001. Abbreviations: BD, basal rodent diet; HFD, high fat diet; AST, aspartate aminotransferases; ALT, alanine aminotransferases; BrdU, 5-Bromo-2′-deoxyuridine; CCl₄, carbon tetrachloride; TAGs, triacylglycerides.

Figure 6

Gene expression analysis of hepatic genes involved in thyroid hormone, lipid, and glucose metabolism following a three-week treatment with TG68 or MGL-3196. Total hepatic RNA was extracted from snap-frozen tissues using Qiazol reagent and reverse-transcribed using high-capacity cDNA reverse transcription kit. All measurements were normalized to β-actin. Groups were compared using unpaired two-tailed Student’s t-test and non-parametric Mann–Whitney U Test, or one-way ANOVA followed by Tukey post hoc analysis. Values are shown as mean ± standard deviation (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: Acta2, Actin alpha 2; Acox1, Acyl-CoA Oxidase-1; BD, basal rodent diet; Cpt1, Carnitine palmitoyltransferase-1; Dio1, Deiodinase-1; Fasn, Fatty acid synthase; HFD, high fat diet; Lgals3, Galectin 3; Me1, Malic enzyme-1; Myh6, Myosin heavy chain 6; Pnpla2, Patatin-like phospholipase domain containing 2; Srebf1, Sterol regulatory element binding transcription factor 1; Thrsp, Thyroid hormone responsive.

Figure 7

Three-week treatment with TG68 or MGL-3196 did not cause injury to extra-hepatic tissues. Representative images of kidney and heart sections immunostained for BrdU detection (20×). Abbreviations: BD, basal rodent diet; HFD, high fat diet.