A new and highly sensitive fluorimetric assay for hexokinase (HK) isoenzymes which requires only 5 μg. of tissue protein is described. This assay has been used to examine the alterations in the HK isoenzymes of rat skeletal muscle and adipose tissue under a variety of physiologic conditions. In both tissues, HK-II declined progressively with increasing age, reaching plateau levels in animals larger than 300 gm., whereas HK-I remained constant. HK-II activity decreased markedly in both tissues of young rats after a twenty-four to forty-eight hour fast and was restored after two days of refeeding. HK-I was not affected by the fasting-refeeding regimen. In older rats (300 gm.), adipose tissue HK-II did not fall during fasting but exhibited a significant rise when refeeding was instituted. “Pulsing” the fasted rat with periodic episodes of hyperglycemia (0.5 gm. glucose intraperitoneally every eight hours) did not prevent the fall in HK-II during fasting or restore HK-II activity in the two day fasted animal.

Approximately 50 per cent of HK-I, but little or none of HK-II, is bound to subcellular organelles in skeletal muscle. In adipose tissue, however, about one-third of the HK-II and insignificant amounts of HK-I are in the bound form. Ratios of bound to free HK are not altered during aging, but HK-II binding in adipose tissue is decreased during refeeding.

Normal mice exhibited a decrease in both adipose tissue HK isoenzymes with aging. Adipose tissue of sixteen week old obese hyperglycemic mice (obob) has lower HK-II and higher HK-I activity than that of normal litter mates, but no differences were detected in obob and normal mice at five weeks of age.

The changes in muscle and adipose tissue HK-II activity associated with aging and fasting-refeeding parallel the known changes in insulin sensitivity of these tissues under comparable conditions.

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