Alterations in adipose-tissue pyruvate dehydrogenase activity in starved, fat-fed and diabetic rats.

The pyruvate dehydrogenase complex in mammalian tissues exists in a phosphorylated (inactive) and in a non-phosphorylated (active) form. These forms are interconverted by an ATP-requiring kinase, which is inhibited by pyruvate, ADP and thiamin pyrophosphate, and a phosphatase, which is activated both by Mg2+ and by Ca2+ ions (Linn et al., 1969; for recent review, see Denton et al., 1975). Brief exposure of rat epididymal adipose tissue to insulin results in a marked increase in the proportion of the complex in its active form (Coore et al., 1971; Jungas, 1971; Weiss et al., 1971). This increase, and the parallel activation of acetyl-CoA carboxylase, explains the preferential incorporation of pyruvate carbon atoms into fatty acids that is observed in the fat-pads incubated in the presence of insulin (Denton, 1974). The effect of insulin on pyruvate dehydrogenase can be observed within lOmin in vitro and in vivo, and does not involve a change in the total amount of the complex. The mechanism has not been clearly established, but presumably involves inhibition of the kinase, or activation of phosphatase (perhaps by Ca2+), or both (Denton et al., 1975). In the present communication, we present results of studies on the long-term regulation of the pyruvate dehydrogenase complex in adipose tissue. We have investigated the effects of alloxan-diabetes and starvation, which are both conditions associated with marked decreases in plasma insulin concentrations and with greatly decreased rates of fatty acid synthesis from glucose both in vivo and in vitro. We have also studied the effect of fat-feeding, which results in greatly decreased rates of fatty acid synthesis but not necessarily in a large diminution in plasma insulin concentration (Hausberger & Milstein, 1955; Malaisse et al., 1969; Saggerson & Greenbaum, 1970; Zaragoza & Felber, 1970; Zaragoza-Hermans & Felber, 1972; D. Stansbie & R. M. Denton, unpublished work). All three conditions have been shown previously to result in a marked repression of many other enzymes involved in fatty acid synthesis in adipose tissue. Rat epididymal fat-pads from male Wistar rats (6-7 weeks old) were used throughout, and maintained on diets described in the legend to Table 1. The pads were frozen in liquid N2 and extracted with 100mM-potassium phosphate buffer, pH7.0, containing ~ ~ M E D T A , by using a Polytron homogenizer. Pyruvate dehydrogenase was assayed in samples of the extract immediately (initial activity), or after treatment with pyruvate dehydrogenase phosphate phosphatase, in the presence of Mg2+ and Ca2+ (total activity). Further details are given in Severson et al. (1974). Measurements were also made of glutamate dehydrogenase activity (as an index of recovery of mitochondria1 enzymes), protein and DNA. Alloxan-diabetes and starvation have very similar effects on pyruvate dehydrogenase activity in vivo (Table 1). The initial activity is very greatly diminished to 25-30% of that found in tissues from control animals. On the other hand, only a small decrease in total pyruvate dehydrogenase activity was evident whether the results were expressed in terms of wet weight, or pair, of fat-pads (as in Table l), or as an activity ratio with glutamate dehydrogenase, or in terms of tissue protein or DNA (not shown). Thus the major effect of alloxan-diabetes and starvation is to decrease greatly the proportion of the enzyme in its active non-phosphorylated form (from about 50% to between 10 and 20 % of the total activity). Similar results for the effects of starvation have been reported previously (Wieland et al., 1973). The changes could be related to the differences in plasma insulin concentrations in these animals, and it was decided to ascertain the extent to which the differences persisted in pads incubated in vitro under standard conditions, with or without insulin. It was found that in the absence of insulin, the proportion of pyruvate dehydrogenase in its