Introduction: Mitochondrial dysfunction and Toll-like receptors (TLRs) are commonly associated with oxidative stress and apoptotic cell death in metabolic disorders; however, their roles in Classical Galactosemia remain largely unexplored. This study examined the activities of key mitochondrial enzymes, as well as the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and Toll-like receptor-2, under hypergalactosemic or hyperglycemic conditions in fibroblast cultures.

Methods: Confluent fibroblast cultures were established from the post-circumcision skin of 2–3-day-old healthy neonates. Next, these fibroblast cultures were treated with D-Galactose (D-Gal, 0–20 mM), galactose-1-phosphate (Gal-1-P, 0–5 mM), or “high glucose” (HG, 25 mM). Peripheral blood mononuclear cells (PBMCs) were isolated from galactosemia patients and control subjects. Activities of medium-chain acyl-CoA dehydrogenase (MCAD), cytochrome c oxidase (CcO), and carnitine palmitoyltransferase-1 (CPT-1) were analyzed in cell homogenates and in PBMCs of both patients and controls. ATP production and malondialdehyde (MDA) levels in fibroblasts were measured using commercial kits. In addition, PGC-1α and TLR-2 were examined in fibroblast cultures by western blot analysis.

Results: D-Gal and Gal-1-P significantly (p < 0.05) reduced the enzyme activities of CPT-1, MCAD, and CcO, with Gal-1-P causing a more pronounced (p < 0.01) effect on CPT-1 compared to the control group. HG had no significant impact on these mitochondrial enzymes. Moreover, mitochondrial enzyme activities were markedly lower in PBMCs from galactosemia patients than from control subjects. Galactose-treated fibroblasts showed elevated TLR-2 levels but unchanged PGC-1α expression compared to control cells. Only Gal-1-P significantly diminished PGC-1α levels, whereas HG, D-Gal, and Gal-1-P significantly upregulated the expression of TLR-2.

Conclusion: These findings suggest that galactosemia pathogenesis involves Gal-1-P–mediated mitochondrial dysfunction and elevated TLR-2 gene expression.