Novel Thyroid Hormone Receptor-β Agonist TG68 Exerts Anti-Inflammatory, Lipid-Lowering and Anxiolytic Effects in a High-Fat Diet (HFD) Mouse Model of Obesity

Abstract

Recent advances in drug development allowed for the identification of THRβ-selective thyromimetic TG68 as a very promising lipid lowering and anti-amyloid agent. In the current study, we first investigated the neuroprotective effects of TG68 on in vitro human models of neuroinflammation and β-amyloid neurotoxicity in order to expand our knowledge of the therapeutic potential of this novel thyromimetic. Subsequently, we examined metabolic and inflammatory profiles, along with cognitive changes, using a high-fat diet (HFD) mouse model of obesity. Our data demonstrated that TG68 was able to prevent either LPS/TNFα-induced inflammatory response or β-amyloid-induced cytotoxicity in human microglial (HMC3) cells. Next, we demonstrated that in HFD-fed mice, treatment with TG68 (10 mg/kg/day; 2 weeks) significantly reduced anxiety-like behavior in stretch-attend posture (SAP) tests while producing a 12% BW loss and a significant decrease in blood glucose and lipid levels. Notably, these data highlight a close relationship between improved serum metabolic parameters and a reduction of anxious behavior. Moreover, TG68 administration was observed to efficiently counteract HFD-altered central and peripheral expressions in mice with selected biomarkers of metabolic dysfunction, inflammation, and neurotoxicity, revealing promising neuroprotective effects. In conclusion, our work provides preliminary evidence that TG68 may represent a novel therapeutic opportunity for the treatment of interlinked diseases such as obesity and neurodegenerative diseases.

Keywords: anxiety; microglia; neurodegeneration; neuroinflammation; obesity; thyromimetics.

Figure 1

Novel Synthetic Thyroid Hormone Receptor-β Agonists. Chemical structures of resmetirom, the first synthetic thyromimetic approved for clinical use, and TG68.

Figure 2

Effects of pretreatment with TG68 on inflammatory response in LPS/TNF⍺-stimulated HMC3 cells. Selected concentrations of TG68 (0.1, 1, and 10 µM) decreased the levels of pro-inflammatory interleukin IL-6 (A), induced the release of anti-inflammatory interleukin IL-10 (B), and did not interfere with cell viability (C). Data represent means ± SEM from three independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05 and *** p < 0.005 compared to LPS/TNF⍺ treated cells; ### p < 0.005 compared to vehicle-treated cells used as control.

Figure 3

Effects of TG68 on Aβ_25–35 treated HMC3 cells. HMC3 cells were treated with 10 µM Aβ_25–35 for 24 h in the presence of different concentrations of TG68 observing a dose-dependent cytoprotective effect (A). Aβ_25-35 exposure induced an increased release of pro-inflammatory cytokines TNF-α (B) and IL-6 (C), with no effect on the release of anti-inflammatory IL-10 (D). The most effective TG68 concentration (10 µM) was also able to reduce the levels of pro-inflammatory cytokines IL-6 (B) and TNF-⍺ (C) and to induce the release of the anti-inflammatory IL-10 (D). Data represent means ± SEM from three independent experiments performed in duplicate. Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. ** p < 0.01 and *** p < 0.005 compared to Aβ25–35 exposed cells; ### p < 0.005 compared to vehicle-treated cells used as control.

Figure 4

In HFD mice, TG68 induces a significant body weight loss and modulates the circulating levels of metabolic parameters. Administration of TG68 (10 mg/kg/day) for two weeks to HFD mice (10 weeks) led to 12% body-weight loss (A) and a significant decrease in serum cholesterol, triglycerides, and glucose (B–D). Each assay was carried out in technical duplicates, and values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s (A) or Dunnett’s (B–D) multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.005).

Figure 5

Effect of TG68 on transcriptional expression of hepatic genes. qPCR analysis revealed that TG68 counteracted HFD-induced changes in the hepatic expression of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase (A) and of THRβ -target genes, namely DIO1 (B) and DIO3 (C). Each experiment was performed in technical triplicate, and values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (*** p < 0.005; **** p < 0.001).

Figure 6

Effects of TG68 on transcriptional expression of metabolism-related genes in adipose tissue and hypothalamus. qPCR analysis revealed that TG68 counteracted HFD-induced changes in the expression of genes that regulate lipid metabolism and mobilization, glucose uptake, and metabolism. Specifically, TG68 treatment increased the expression of SIRT6 (A), PPARγ (B), and ADIPOQ (C), while decreasing leptin (D) and APOD (E) expression in adipose tissue. Additionally, in the HFD+TG68 group, increased expression of hypothalamic glucose transporters, namely GLUT1, GLUT3, and GLUT5, was observed compared to HFD mice (F–H). Each experiment was performed in technical triplicate, and values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 7

Chronic treatment with TG68 counteracts the inflammatory response in HFD mice. Chronic treatment with TG68 (10 mg/kg/die) significantly reduced the serum levels of pro-inflammatory cytokines TNFα (A) and IL-6 (B) in HFD mice. Accordingly, the HFD-induced increased expression of TNFα (C,E) in both adipose tissue and hypothalamus, as well as IL-1β (D) in adipose tissue and IL6 (F) in hypothalamus, was totally abolished by TG68. In HFD mice hypothalamus, the significant increase in NF-kB expression (G) was effectively reversed by TG68 treatment. Each experiment was performed in technical triplicate, and values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s test (* p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 8

TG68 induces a neuroprotective effect on the hypothalamus of HFD mice. In HFD mice hypothalamus, TG68 counteracts a significant decrease in BDNF, either at protein (A) and transcriptional (B) levels. In hypothalamus of HFD mice, TG68 treatment increases the transcriptional expression of TREM2 (C), IGF1(D), FGF2 (E), NGF (F), IL4 (G), and IL10 (H). Each experiment was performed in triplicate, and values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01; **** p < 0.0001.

Figure 9

TG68 improves anxiety-like behavior at open field test (A,B). In HFD mice, TG68 significantly reduces the latency to start movement and latency to reach the inner zone of the arena, suggesting an improvement of anxiety-like behavior (SD: mice fed with standard diet n = 15, HFD: mice fed with HFD n = 15, HFD+TG68: mice fed with HFD and treated with TG68 n = 15). (C,F) Example trajectories for mice during open field test. (D,E) Latency to the inner zone is positively correlated with serum triglycerides and glucose (in the whole HFD group n = 30). Values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s test; linear regression; *** p < 0.005.

Figure 10

TG68 improves anxiety-like behavior at elevated plus maze test (A). In HFD mice, TG68 significantly reduces the SAP, suggesting an improvement of anxiety-like behavior (SD: mice fed with standard diet n = 15, HFD: mice fed with HFD n = 15, HFD+TG68: mice fed with HFD and treated with TG68 n = 15) (B). In the whole groups, there was an inverse correlation between the time spent in the open arms and serum glucose (rho = −0.40; p = 0.009, n = 35 available samples) and serum TNFα (rho = −0.43; p = 0.04, n = 20 available samples) (C). Inverse correlation between the total number of overlooking and serum cholesterol (rho = −0.31, p = 0.04, n = 35) and serum glucose (rho = −0.44, p = 0.005, n = 35) (D). Apparatus used for elevated plus maze test. Values represent the mean ± SEM. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s test; linear regression; *** p < 0.005.