Genetic Deletion of RHAMM Alleviates Hepatic Oxidative Stress, Reversing Thyroid Stimulating Hormone Elevation in Male Obese Mice

Abstract

Objective: Obesity induces hypothyroidism with unknown mechanisms. This study investigates the role of (Receptor for Hyaluronan-Mediated Motility (RHAMM) in obesity-associated thyroid dysfunction, focusing on hepatic oxidative stress. Methods: Global RHAMM-deficient mice and their wildtype littermate controls were fed a normal chow diet or high-fat diet (HFD) for 16 weeks. Thyroid function was evaluated by measuring plasma thyroid-stimulating hormone (TSH) levels. The hepatic oxidative response was assessed by measuring signaling pathways associated with nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Results: HFD feeding increased plasma TSH levels in male mice but not in female mice. RHAMM deletion in male mice mitigated HFD-induced TSH elevation, which was associated with enhanced hepatic antioxidant defenses and reduced inflammation. This was evidenced by elevated expression of the Nrf2 target gene NAD(P)H: quinone oxidoreductase 1 (Nqo1), reduced protein carbonylation and nitration levels, and reduced expression of the pro-inflammatory cytokines IL-1β and TNF-α in livers of male RHAMM-deficient mice. Mechanistically, RHAMM deletion decreased AKT/ERK signaling, increased GSK3 signaling, increased CD44 protein expression, and increased Nqo1 levels in the liver. Conclusions: RHAMM promotes obesity-induced thyroid dysfunction by regulating oxidative stress and inflammation in male mice. Targeting RHAMM may provide a novel therapeutic strategy for mitigating obesity-related endocrine and metabolic disorders.

Keywords: Nrf2; RHAMM; obesity; oxidative stress; thyroid dysfunction.

Figure 1

Global Hmmr gene deletion attenuates obesity-induced elevation of TSH in male mice. (A) Plasma TSH levels were measured in male and female mice fed a high-fat diet (HFD) or a standard chow diet (CHOW). (B) TSH levels were measured in male Hmmr^+/+ and Hmmr^−/− mice under both dietary conditions. The same samples were used for male Hmmr^+/+ mice in both panels (A,B). (C,D) Western blot analysis and quantification of CD44 protein expression levels in Hmmr^+/+ and Hmmr^−/− mice under different dietary conditions. Samples from heterozygous Hmmr^+/− mice were included for reference. (E) Hepatic triglyceride (TG) levels were measured in Hmmr^+/+ and Hmmr^−/− mice under different dietary conditions. Sample sizes were presented as the number of data points. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 2

Global Hmmr gene deletion modulates AKT, ERK, and GSK3 signaling pathways in the liver under chow and HFD conditions. (A) Representative Western blot images show phosphorylated AKT (p-AKT), total AKT, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated GSK3α/β (p-GSK3α/β), and total GSK3α/β in liver samples from Hmmr^+/+ and Hmmr^−/− mice. (B–D) Protein levels were normalized to GAPDH. Sample sizes were presented as the number of data points. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons. p < 0.05 (*) and p < 0.01 (**).

Figure 3

Global Hmmr gene deletion increases Nrf2 target gene Nqo1 expression in liver tissues. (A–C) Representative Western blot images and quantification of Nrf2 and Nqo1 protein expression in liver from Hmmr^+/+ and Hmmr^−/− mice. β-actin or GAPDH served as loading controls. Protein levels were normalized to the Hmmr^+/+ chow group. (D–G) Relative mRNA expression levels of Nrf2, Nqo1, HO1, and GCLM, normalized to Hmmr^+/+ CHOW, assessed by qRT-PCR. Sample sizes were presented as the number of data points. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons. p < 0.05 (*) and p < 0.01 (**).

Figure 4

Genetic hmmr deletion ameliorates hepatic oxidative stress and inflammation. (A) A representative OxyBlot image shows the levels of oxidized proteins. Lanes: 1, molecular weight standard; 2, 4, 6, 8, negative controls; 3, CHOW-Hmmr^+/+; 5, HFD-Hmmr^+/+; 7, CHOW-Hmmr^−/−; 9, HFD-Hmmr^−/−. The graph shows a densitometric analysis of lanes 3, 5, 7, and 9 normalized to CHOW-Hmmr^+/+. (B) Quantification of protein carbonyl levels in liver tissue. (C) Measurement of malondialdehyde (MDA) levels as an indicator of lipid peroxidation in liver tissues. (D) Representative Western blot images and quantification of nitrotyrosine protein expression in liver of Hmmr^+/+ and Hmmr^−/− mice. β-actin served as loading control. Protein levels were normalized to the Hmmr^+/+ chow group. (E–H) Relative mRNA expression of IL-1β, IL-6, TNF-α, and IL-10, normalized to CHOW-Hmmr^+/+, analyzed by qRT-PCR. Sample sizes were presented as the number of data points. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons. p < 0.05 (*) and p < 0.01 (**).

Figure 5

Nrf2 activation decreases CD44 and RHAMM expression and suppresses plasma TSH concentrations in mice. mRNA expression levels of Nqo1 (A), CD44 (D), and RHAMM (E) were measured in the liver tissues of WT, Nrf2 KO, and Keap1 KD mice. Representative Western blot images (B,F) and quantification of Nqo1 (C) and CD44 (G) protein expression in liver from WT, Nrf2 KO, and Keap1 KD mice. β-actin or GAPDH served as loading controls. (H) Plasma TSH concentrations. Sample sizes were presented as the number of data points. Data were analyzed using one-way ANOVA. Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Figure 6

A working model by which RHAMM regulates thyroid function via modulating Nqo1-mediated hepatic oxidative stress and inflammation. Genetic deletion of RHAMM decreases AKT/ERK signaling, increases GSK3 activity, and increases CD44 expression. These changes collectively increase Nqo1 expression and lead to reduced hepatic oxidative stress and inflammation, potentially reversing obesity-induced TSH elevation. ↓ indicates a decrease, ↑ indicates an increase, ↔ indicates no change, and ? indicates unknown mechanisms.