My move to Oxford coincided with a major development in research on PDH phosphorylation. This was the demonstration that increasing concentration ratios of [ATP]/[ADP], [NADH]/ [NAD+], and [acetylCoA]/[CoA] activate PDH kinase - both in rat heart mitochondria and in association with purified pig heart PDH complex [37,40,41]. Thus products of the PDH complex reaction and of ^-oxidation of fatty acids had been shown to inhibit the PDH complex reaction directly (end product inhibition) and indirectly (inactivation by protein phosphorylation).
However, comparative mitochondrial studies showed that additional mechanism(s) were involved in the effects of diabetes or starvation. In studies with Drs. Kerbey Sugden and Hutson, three phosphorylation sites were identified in porcine and rat PDH complexes, and amino acid sequences around the sites were determined in porcine complex [44,45]. Inactivation was shown to require phosphorylation of only one of three sites (site 1) [43,44]. Later studies with ATPyS showed that complexes thiophosphory-lated in sites 2 plus 3 only are also inactive; i.e. inactivation is not site 1 specific . The eventual conclusion was that phosphoryla-tion of sites 1 or 2, but not of site 3 are inactivating. Phosphoryla-tion of sites 2 and 3 which occurs largely over the range of 70-100 percent phosphorylation of site 1 was shown to inhibit reactivation by PDH phosphatase i.e. multisite phosphorylation provides a locking mechanism retarding reactivation [46,47]. This was shown to involve an increase in apparent Km for Ca2+ . Multisite phosphorylation was shown to be ordered in pig and rat complexes. Relative rates were site 1 > site 2 > site 3 and inhibitors and activators of PDH kinase were effective with respect to phosphorylation at each of the sites; evidence for operation of this locking mechanism in vivo was summarized . It was considered to be of particular importance for glucose conservation in starvation and dietary carbohydrate deprivation; and for impaired glucose oxidation in diabetes. In these conditions the proportion of complex in the active dephosphorylated form fell by 75-85% in rat heart, kidney, adipose tissue, and liver (summarized in ). After some initial doubts these mechanisms were also shown to be involved in regulation of the skeletal muscle complex [51-53].
Longer term regulation of the PDH phosphorylation/depho-sphorylation cycle was discovered when it was shown that the effect of starvation or alloxan diabetes to lower percent PDHa in rat tissues persists into isolated heart muscle mitochondria incubated with respiratory substrates in vitro. These and subsequent investigations with Denyer, Fatania, Stace, Jackson, Marchington, Jones, Priestman, Mistry, and Halsall led to the further discovery that 24-48 h of starvation or diabetes effects a stable 2-3-fold increase in the activity of PDH kinase in isolated rat heart, skeletal muscle, and liver mitochondria [54-56]. Complete reversal of the effect of starvation required 4h of refeeding for liver and 24-48 h for muscle. In order to investigate these phenomena in more detail, studies were made in tissue culture of rat hepatocytes, cardiac myocytes, and soleus muscle strips [57-63]. These studies showed unequivocally that culture of these cell or tissue samples from fed rats with agents which increase cAMP (glucagon or dibutyrylcAMP) or with NEFA (n-octanoate or albumin bound palmitate) increased PDH kinase activity 2-3 -fold within 24 h. and that the effect of glucagon was blocked by insulin. Culture of hepatocytes from starved rats reversed the effect of starvation by 60% in 24 h; this reversal was blocked by n-octanoate, dibutyrylcAMP, glucagon, or a combination. These studies thus provided strong evidence that the effects of diabetes and starvation to inhibit glucose oxidation are mediated by an increase in PDH kinase activity, thereby enhancing phosphorylation and inactivation of PDH complex.
I gave up research in 1995 after two years of retirement (so-called). Since then, the further studies of R.A. Harris and of M.C. Sugden and their colleagues have identified four isozymes of PDH kinase and their respective roles in the regulation of PDH complex activity [64-70].
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