The impact of Thiosulfate sulfurtransferase (TST) on metabolic dysfunction-associated steatotic liver disease (MASLD) and the metabolic benefits of calorie restriction

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects 20-30% of adults in western countries and is closely linked to obesity and type 2 diabetes. Hydrogen sulfide (H₂S), once solely perceived as toxic, is now recognised for its role in various physiological and pathological processes. H₂S donors have shown promise in treating fatty liver disease and reducing blood pressure in animal models, but their therapeutic use is hindered by challenges in H₂S pharmacokinetics. The sulphur oxidation pathway (SOP), which regulates H₂S levels through its disposal, has been underexplored as a potential route to therapeutic H₂S elevation. Thiosulfate sulfurtransferase (TST), a mitochondrial enzyme, is part of the SOP and metabolises H2S, indirectly, to prevent toxicity. Previous work leading up to this thesis showed that TST mRNA levels were upregulated during the early steatosis stage of MASLD in humans. Given the previously identified metabolic protective effect of adipose tissue TST elevation, I hypothesised that elevation of hepatic TST in early MASLD was a protective mechanism, counteracting declining liver function in MASLD. Improved metabolic health following calorie restriction (CR) is mediated in part through increased hepatic production of H₂S. Tst⁻/⁻ mice exhibited elevated systemic H₂S levels, therefore I hypothesised they may have an enhanced response to CR. In chapters 3 and 4, I tested the hypothesis that elevated hepatic TST expression in MASLD offered protection against MASLD development using a liver-specific overexpression mouse model (Liv_hTST). Male and female C57BL/6J and Liv_hTST mice were fed either a control diet or MASLD-inducing GAN diet for 20 weeks. Systemic and hepatic sulfide levels were measured, fat and lean mass assessed, and glucose tolerance evaluated. In vitro, HepaRG cells with TST overexpression were tested for lipid accumulation, oxidative stress, and mitochondrial function. Results showed sex-specific effects on sulfide levels and glucose tolerance, with protective effects against fibrosis in male mice in vivo, and a worsening of the impaired lipid metabolism and mitochondrial function in vitro. This research addresses the gap in understanding of the protective role ascribed to elevated TST expression against steatogenic liver changes in MASLD and revealed novel sex-specific effects on systemic sulfide levels, glucose tolerance, and fibrosis. In chapter 5, I investigated whether Tst⁻/⁻ mice experienced enhanced metabolic benefits from CR due to their elevated systemic sulfide. Ten-week-old male and female C57BL/6J and Tst⁻/⁻ mice underwent 4-week 30% CR. Indirect calorimetry, glucose tolerance, H₂S production, and disposal (SOP) enzyme levels were assessed. Tst⁻/⁻ males had higher systemic but similar hepatic sulfide levels compared to C57BL/6J males, confirming previous work. CR did not affect sulfide levels but improved glucose tolerance in Tst⁻/⁻ males, despite their resistance to fat mass loss. Energy expenditure and substrate utilisation were similar between genotypes. Females were unaffected by the lack of TST and had lower levels of hepatic H₂S metabolism enzymes. Our findings suggested mechanisms beyond hepatic sulfide modulation mediate CR benefits. Understanding the novel role of elevated systemic H₂S and TST deficiency in maintaining fat mass and concurrent metabolic benefits with CR may inform H₂S -targeted therapeutic strategies in the future