No abstract

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT genetrapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P ¼ 0.02) and longer time-to-pregnancy (P ¼ 0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P ¼ 0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (−74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT2 Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 minutes, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.