HEXOKINASE ISOZYME EXPRESSION IN DEVELOPING RAT LUNG: A QUANTITATIVE APPROACH † 1509

Hexokinase (HK) is a rate-limiting enzyme for glucose metabolism in mammalian lung. HK phosphorylates glucose to glucose 6-phosphate, thereby committing the substrate to cellular metabolism. HK exists as four distinct isozymes and we have previously shown by protein and enzyme activity studies that HK1 and HK3 are differentially expressed in rat lung at various ages. The purpose of this study was to determine the relative expression of HK1, HK2 and HK3 mRNAs in rat lung from fetal through adult stages. We studied lungs of fetal (E20), newborn (P1-P25) and mature (P30-P45) Sprague-Dawley rats. RNA samples were reverse transcribed with isozyme-specific oligonucleotide primers for HK1, HK2 and HK3. An aliquot of each reaction was amplified by polymerase chain reaction (PCR) using a γ-³²P labeled antisense primer and unlabeled sense primer. Primers for all three isozymes were included in the multiplex PCR. In separate reactions, the amount of total HK and isozyme specific mRNAs were normalized to PGK mRNA, a gene with consistent developmental expression. Amplification products were separated by polyacrylamide gel electrophoresis which was analyzed and quantified on a PhosphorImager. We found that HK1 was the predominant isozyme at all ages studied, contributing 55% before birth, then increasing to 73% at P15 and decreasing at P45 to 45% of the total HK mRNA. In contrast, HK2 showed maximal expression just before birth (40%) and at maturity (35%); expression from birth to P30 was 10-20% of the total expression. HK3 contributed the least to total mRNA: 5% before birth, increasing to 20% on P1 and remaining at 20-25% into adulthood. This approach not only confirmed our previous data from protein and enzyme activity studies, but also allowed us to quantitate the relative contributions of the three HK isozymes present in lung. We conclude that HK1 and HK2 are the predominant enzymes throughout the developmental ages studied, with HK3 present in lesser amounts. We speculate that each isozyme plays a distinct role in the development of the mammalian lung, perhaps by involvement in different metabolic and synthetic pathways as the lung matures to become the organ of respiration.