Pathways of adenosine metabolism. Adenosine is formed either by hydrolysis of AMP or by hydrolysis of SAH, which arises from the action of methyltransferases. Adenosine can be metabolized by ADA or ADK into inosine and AMP, respectively. Note the AMP/adenosine futile cycle, which is catalyzed by ADK and 5′-nucleotidase. SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; X = methyl-group acceptor; X-CH₃ = methylated compound X.

Growth kinetic of offspring of Adk^+/− intercrosses. The body weight of Adk^+/+, Adk^+/−, and Adk^−/− pups was followed during a 14-day period. The population size at the time of birth (P0; n = 17 for each genotype) dropped to n = 2 at P14 for homozygous mutants (Table 1). Note that heterozygous offspring did not differ from wild-type littermates in their growth curve. Points are given as means ± SD.

Overview of livers from Adk^+/+ (Left) and Adk^−/− (Right) mice at P7. (Scale bar, 1 cm.)

Homologous recombination into the murine Adk locus. (a) Restriction enzyme maps of the murine Adk wild-type allele, targeting vector pAdk-, and targeted allele. The thick lines indicate genomic sequences of the murine Adk gene homologous to the targeting vector. The thin lines in the wild type and targeted alleles indicate external Adk sequences not present in the targeting vector. In the wild-type sequence a checkered line marks the region with homology to mouse long interspersed nucleotide elements. Exon sequences, as well as the insertion cassette for the neomycin resistance gene (PGK-neo), are outlined as open boxes, loxP sites are outlined as vertical bars. PGK-neo is transcribed in the direction indicated by the arrow. The bicistronic expression cassette (hatched box) for the enhanced green fluorescent protein, the internal ribosomal entry site, and the tetracycline-dependent transactivator is fused in-frame to the Adk-specific exon, thus causing a disruption of the Adk gene. The hatched lines indicate the 5′ and 3′ flanking probes external to pAdk− and the neo-probe inside pAdk−. (b) Southern blot analysis of the murine Adk locus. Genomic DNA was digested with the restriction enzymes EcoRV (Left) or BglII + SspI (Right) and hybridized with the 5′ probe and 3′ probe, respectively. Hybridizing fragments are 4,959 bp (+, wild-type allele) and 3,389 bp (−, mutant allele) with EcoRV digestion, and are 3,549 bp (+) and 4,131 bp (−) with BglII/SspI digestion. The DNA size marker shown is a 1-kb ladder (Life Technologies). (c) Western blot analysis of liver extracts from E17.5 embryos from an Adk^+/− × Adk^+/− intercross. The 8- or 16-μg liver extracts from wild type (+/−), heterozygous (+/−), and homozygous (−/−) mutant embryos were probed with a polyclonal rabbit antiserum raised against recombinant mouse ADK.

Hematoxylin and eosin staining of livers during the perinatal development from E17.5 to P4. In wild-type mice the liver developed normally with hepatocytes displaying a normal size with centrally located nuclei. A compact and homogeneous tissue mass was formed at P4. In ADK-deficient mice (ADK^−/−) the livers are morphologically normal at E17.5 but develop micro- and macrovesicular diffuse panlobular steatosis (intracellular lipid accumulation) after birth, which appears as cytoplasmic pallor. Postnatal liver cells contained fine lipid vesicles, which increased in size and became confluent during the following days (P2, P4). There was no inflammation, no development of cirrhosis, and no evidence of liver cell necrosis. Mallory bodies were not encountered. Original magnification (objective): first and third column, ×10; second and fourth column, ×40. Original microscopical magnification: first and third column, ×100; second and fourth column, ×400. Bars in first and third column, 200 μm; bars in second and fourth column, 50 μm.

Adenosine-related metabolites in livers. The concentrations of SAM and SAH (a), and of the sum of AMP and ADP (b) as well as the concentration of ATP were determined in liver extracts from Adk^+/+(+/+), Adk^+/− (+/−), and Adk^−/− (−/−) mice at P2. ***, P < 0.0001, Student's t test.