FIGURE 2.

Regulation of glycine and glycine derivatives by the hepatic glycine cleavage system. A, Gene‐trapping of Gldc at the Gldc ^GT1 allele is reversible by cre‐mediated recombination of the inverted loxP sites, generating a ‘conditional rescue’ allele in which the splice acceptor (SA) of the trap is in an inactive orientation. B, mRNA expression is restored in liver of Gldc ^GT1/GT1 mice following hepatocyte‐specific expression of cre recombinase (under control of the albumin and alpha‐fetoprotein regulatory elements). C, Immunoblot confirms that Gldc protein expression is restored in liver but not in brain of Aflp ^cre;Gldc ^GT1/GT1 mice, as compared with Gldc ^GT1/GT1 mice in which Gldc is below the limit of detection (Gapdh was used as a loading control). D, Glycine abundance is increased in liver of adult Gldc ^GT1/GT1 mice and normalised by reinstatement of hepatic Gldc expression (**significant difference from Gldc ^+/+; P < .01, ANOVA). E, Heatmap shows relative abundance of glycine and glycine derivatives in liver of adult wild‐type (n = 6), Gldc‐deficient (n = 4) and hepatic Gldc‐rescued (n = 4) mice. Each column represents an individual mouse. Fold‐changes show relative abundance in Gldc ^GT1/GT1 vs Gldc ^+/+ (left side of panel) and Aflp ^cre;Gldc ^GT1/GT1 vs Gldc ^GT1/GT1 (right side of panel). All fold‐changes within brackets show significant difference between genotypes (*P < .05; paired t test) except where indicated (#P < .1). F‐K, Abundance of glycine and selected glycine derivatives (arbitrary scale, representing raw counts from mass spectrometry); ** different from Gldc ^+/+, *different to constitutive Gldc ^GT1/GT1; P < .05, ANOVA). K, Clustering of samples based on relative abundance of the glycine metabolites in panel E groups hepatic‐rescued mice with wild‐types and distinct from constitutive Gldc ^GT1/GT1 mice. L,M, Reinstatement of Gldc expression in liver is associated with normalisation of plasma glycine and guanidinoacetate concentrations in Aflp ^cre;Gldc ^GT1/GT1 mice