Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...

Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity.

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45Ca2+ and the incorporation of 45Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions was essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium. 2. Incorporation of 45Ca into mitochondrial fractions of both fat-cells and fat-pads was found to be complete within 2-5 min, suggesting that mitochondria contain a pool of calcium in rapid isotopic exchange with extracellular Ca2+. This pool was about 20 times larger in mitochondria within fat-cells than within fat-pads. In fat-cells, 45Ca incorporation into the mitochondrial fraction accounted for about 34% of the total 45Ca incorporation into cells after 20 min and about 50% of the total mitochondrial calcium content measured by atomic absorption; values in fat-pads were about 7 and 20% respectively.

Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.

1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by Ca2+, but perhaps less sensitive than the mitochondrial activity.

Regulation of mammalian pyruvate dehydrogenase.

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase. Evidence is presented that all components of the pyruvate dehydrogenase complex including the phosphatase and kinase are located within the inner mitochondrial membrane. Direct measurements of the matrix concentration of substrates and effectors is not possible by techniques presently available. This is the key problem in the identification of the mechansims involved in the alterations in pyruvate dehydrogenase activity observed in adipose tissue and muscle. A number of indirect approaches have been used and these are reviewed. Most hopeful is the recent finding in this laboratory that in both adipose tissue and heart muscle, differences in activity of pyruvate dehydrogenase in the intact tissue persist during preparation and subsequent incubation of mitochondria.