Sustained hepatic and renal glucose-6-phosphatase expression corrects glycogen storage disease type Ia in mice.

Deficiency of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, causes glycogen storage disease type Ia (GSD-Ia), an autosomal recessive disorder characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. G6Pase is an endoplasmic reticulum-associated transmembrane protein expressed primarily in the liver and the kidney. Therefore, enzyme replacement therapy is not feasible using current strategies, but somatic gene therapy, targeting G6Pase to the liver and the kidney, is an attractive possibility. Previously, we reported the development of a mouse model of G6Pase deficiency that closely mimics human GSD-Ia. Using neonatal GSD-Ia mice, we now demonstrate that a combined adeno virus and adeno-associated virus vector-mediated gene transfer leads to sustained G6Pase expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. Our results suggest that human GSD-Ia would be treatable by gene therapy.

The catalytic center of glucose-6-phosphatase. HIS176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis.

Glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, is anchored to the endoplasmic reticulum by nine transmembrane helices. The amino acids comprising the catalytic center of G6Pase include Lys(76), Arg(83), His(119), Arg(170), and His(176). During catalysis, a His residue in G6Pase becomes phosphorylated generating an enzyme-phosphate intermediate. It was predicted that His(176) would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate, and His(119) would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently bound the phosphate moiety, we generated recombinant adenoviruses carrying G6Pase wild type and active site mutants. A 40-kDa [(32)P]phosphate-G6Pase intermediate was identified after incubating [(32)P]glucose 6-phosphate with microsomes expressing wild type but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His(119) is predicted to be within a 116-amino acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6-121), and His(176) is predicted to be within a 149-amino acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131-279). We show that after digestion of a non-glycosylated [(32)P]phosphate-G6Pase intermediate by cyanogen bromide, the [(32)P]phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His(176) is the phosphate acceptor in G6Pase.

A mouse model of renal tubular injury of tyrosinemia type 1: development of de Toni Fanconi syndrome and apoptosis of renal tubular cells in Fah/Hpd double mutant mice.

Hereditary tyrosinemia type 1 (HT1) (McKusick 276700), a severe autosomal recessive disorder of tyrosine metabolism, is caused by mutations in the fumarylacetoacetate hydrolase gene Fah (EC 3.7.1.2), which encodes the last enzyme in the tyrosine catabolic pathway. HT1 is characterized by severe progressive liver disease and renal tubular dysfunction. Homozygous disruption of the gene encoding Fah in mice causes neonatal lethality (e.g., lethal Albino deletion c14CoS mice), an event that limits use of this animal as a model for HT1. A new mouse model was developed with two genetic defects, Fah and 4-hydroxyphenylpyruvate dioxygenase (Hpd). The Fah-/- Hpd-/- mice grew normally without evidence of liver and renal disease, and the phenotype is similar to that in Fah+/+ Hpd-/- mice. The renal tubular cells of Fah-/- Hpd-/- mice, particularly proximal tubular cells, underwent rapid apoptosis when homogentisate, the intermediate metabolite between HPD and FAH, was administered to the Fah-/- Hpd-/- mice. Simultaneously, renal tubular function was impaired and Fanconi syndrome occurred. Apoptotic death of renal tubular cells, but not renal dysfunction, was prevented by pretreatment of the animals with YVAD, a specific inhibitor of caspases. In the homogentisate-treated Fah-/- Hpd-/- mice, massive amounts of succinylacetone were excreted into the urine, regardless of treatment with inhibitors. It is suggested that apoptotic death of renal tubular cells, as induced by administration of homogentisate to Fah-/- Hpd-/- mice, was caused by an intrinsic process, and that renal apoptosis and tubular dysfunctions in tubular cells occurred through different pathways. These observations shed light on the pathogenesis of renal tubular injury in subjects with FAH deficiency. These Fah-/- Hpd-/- mice can serve as a model in experiments related to renal tubular damage.