Michael R. Munday

Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases

We have reported previously that cyclic AMP‐dependent protein kinase phosphorylates two sites on acetyl‐CoA carboxylase (site 1: Arg‐Met‐Ser(P)‐Phe, and site 2: Ser‐Ser(P)‐Met‐Ser‐Gly‐Leu), while the AMP‐activated protein kinase also phosphorylates site 1, plus site 3 (Ser‐Ser‐Met‐Ser(P)‐Gly‐Leu), the latter being two residues C‐terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP‐dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in V max produced by the AMP‐activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP‐activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP‐dependent protein kinase.

MECHANISMS INVOLVED IN THE COORDINATE REGULATION OF STRATEGIC ENZYMES OF GLUCOSE-METABOLISM

In this review, we evaluate the relative regulatory importance of specific strategic enzymes (in particular glycogen synthase, acetyl-CoA carboxylase [ACC] and the pyruvate dehydrogenase complex [PDH]) for carbohydrate utilization as an anabolic precursor and as an energy substrate during the nutritional transitions between the fed and fasted states. The involvement of the specific protein kinases contributing to the inactivation of these enzymes by phosphorylation [cyclic AMP-dependent protein kinase, AMP-activated protein kinase and PDH kinase] in achieving each regulatory response is also assessed. We demonstrate a striking temporal correlation between hepatic glycogen mobilization and PDH and ACC inactivation by phosphorylation during the immediate postabsorptive period; in contrast, rates of hepatic glycogen synthesis and PDH and ACC expressed activities do not change in parallel during refeeding. The results are consistent with shifting of the primary sites of control for overall hepatic carbon flux during the fed-to-starved and starved-to-fed nutritional transitions achieved, at least in part, by a complex pattern of regulation by protein phosphorylation and metabolites which is critically dependent on the precise nutritional status. Data are also presented that demonstrate asynchronous suppression of glucose uptake/phosphorylation and pyruvate oxidation in cardiac and skeletal muscle during progressive starvation. Analogous asynchrony is observed in the reactivation of these processes in cardiac and skeletal muscle during refeeding after starvation. We provide evidence in support of the concept that selective suppression of pyruvate oxidation in oxidative muscles during early starvation and during the initial phase of refeeding is achieved because of differential sensitivity of glucose uptake/phosphorylation and pyruvate oxidation to lipid-fuel utilization. We discuss the relative importance of regulatory events governing local fatty acid production and utilization (via lipoprotein lipase and carnitine palmitoyltransferase 1, respectively) or overall fatty acid supply (dictated by events at the adipocyte) for fuel utilization by muscle during nutritional transitions. Finally, we assess the regulatory importance of glycogen synthesis in determining overall rates of glucose clearance by skeletal muscle during alimentary hyperglycemia and hyperinsulinemia.

Short-term dietary regulation of lipogenesis in the lactating mammary gland of the rat

Short-term (6 hr) withdrawal of chow diet from lactating rats decreases the rate of lipogenesis in mammary gland by 87%. This inhibition is in part explained by a 60% decrease in the extraction of glucose (the major lipogenic precursor) by the mammary tissue. These changes are not accompanied by any significant alteration in the arterial concentrations of glucose, lactate or insulin; the concentration of acetoacetate did increase by about 30%. Removal of food for 6 hr did not alter the activation state of acetyl-CoA carboxylase or the total activity of the enzyme. Glucose utilization by mammary gland acini from short-term starved rats was not depressed although a higher proportion of the glucose appeared as lactate in the medium and consequently less glucose was converted to lipid. Insulin was able to reverse these changes. Glucagon, adrenaline or cAMP did not inhibit glucose utilization or lipogenesis in isolated acini. It is concluded that the inhibition of lipogenesis in mammary gland after short-term withdrawal of food is mainly due to decreased extraction of glucose. The signal for this change does not appear to be an alteration in plasma insulin and it is postulated that there may be an intestinal factor(s) which acts synergistically with insulin.

Effects of starvation, insulin or prolactin deficiency on the activity of acetyl-CoA carboxylase in mammary gland and liver of lactating rats

During lactation in the rat the rate of lipogenesis in vivo in mammary gland is several-fold higher than that in liver and adipose tissue and it alters with the nutritional and hormonal state of the animal [1 ]. Starvation (24 h) decreases the rate in the lactating gland by 98% and this is reversed by refeeding for 2 h [1,2] or by injection of insulin [2]. Conversely, shortterm insulin deficiency induced with streptozotocin [3] inhibits mammary gland lipogenesis [1 ]. These changes in the rates of mammary gland lipogenesis correlate with the alterations in the activation state of pyruvate dehydrogenase in vivo [4-7]. In starvation, inactivation of pyruvate dehydrogenase appears to be the major factor in the control of mammary gland lipogenesis, but there is evidence for an insulin-sensitive step which is after the formation of acetyl-CoA [21. In the other lipogenic tissues (adipose tissue and liver) it is now well established that acetyl-CoA carboxylase is a regulatory enzyme and that its activity can be altered by hormones [8]. In epididymal fat pads, unlike liver, there appears to be coordinate control between the activation state of pyruvate dehydrogenase and acetyl-CoA carboxylase in response to alterations of the plasma insulin concentration [9]. Phosphorylation and inactivation of purified acetyl-Co A carboxylase from lactating rabbit [ 10] and rat mammary gland [11] by endogenous cAMPdependent and independent protein kinases has been demonstrated. Dephosphorylation of the acetyl-CoA

Role of Protein Phosphorylation in the Regulation of Fatty Acid Synthesis in the Mammary Gland of the Lactating Rat

of Thesis The mammary gland of the lactating rat uses 30 mmol of glucose a day. 70% of this is used for the synthesis of fatty acids which is therefore stringently regulated. This thesis describes investigations designed to elucidate some of the mechanisms of this regulation. Acetyl-CoA carboxylase (ACC) and pyruvate dehydrogenase (PDH) are key regulatory enzymes in the pathway from glucose to fatty acid. Pyruvate dehydrogenase catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA and ACC catalyses the committed step in the synthesis of fatty acids from acetyl-CoA. In lactating rat mammary gland the activities of both enzymes are profoundly inhibited by starvation and rapidly re-activated (within 3 hours) upon refeeding. Unexpectedly during these dietary manipulations the rate of fatty acid synthesis can be more closely correlated with PDH than ACC even though the latter is considered to be the major rate determining enzyme. Perfusion of the mammary gland in situ was performed using known inhibitors of fatty acid synthesis in the perfusate. Palmitic acid inhibited PDH but not ACC; acetoacetate had no effect on either enzyme. In vitro experiments suggest differential effects of palmitate (16:0) and oleate (18:1), palmitate being a less potent inhibitor of fatty acid synthesis and ACC than oleate. ACC in lactating rat mammary gland is phosphorylated and inactivated in response to 24hr starvation. One possible candidate for such phosphorylation is cAMP-dependent protein kinase. This had been purified from lactating mammary gland and its unusual tissue specific properties are described and discussed in relation to the results of in vivo experiments showing that in mammary gland the activity of this kinase does not correlate with the rate of fatty acid synthesis or ACC activity. The partial purification of a kinase capable of phosphorylating and inhibiting ACC in a manner identical to that which occurs during starvation in vivo is described. Its characteristics, regulation and physiological significance are discussed.