Keith Snell

Metabolic control analysis of mammalian serine metabolism

(1) Mammalian serine metabolism is discussed in relation to its synthesis and utilization in proliferating cells, particularly during the nonmalignant proliferation of lymphocytes. (2) An analysis of the control of serine biosynthesis de novo under conditions of high pathway flux has been carried out using metabolic control theory. (3) The important and novel conclusions are that control of pathway flux is localized exclusively at the final step of this biosynthetic pathway, phosphoserine phosphatase. This conclusion challenges the frequently stated maxim that control of biosynthetic pathways is always directed at the first pathway enzyme in a sequence. In the case of phosphoserine phosphatase, the enzyme is inhibited uncompetitively by its product serine, and this feedback control mechanism has the most significant controlling influence on overall pathway flux. Thus, the serine biosynthesis pathway, under these conditions, is controlled by product demand (serine utilization) and not by substrate supply (glycolytic provision of 3-phosphoglycerate), despite the high rate of glycolysis associated with cell proliferation. (4) The control structure of the pathway is not immutable. As has been observed with other pathways analyzed by metabolic control theory, the key points of control in the pathway can shift according to physiological circumstances. At low pathway flux, the control of serine biosynthesis is shared between all the component enzymes of the pathway, and the responsiveness of flux shifts from product demand to substrate supply. (5) Serine utilization has been studied in mitogenically-stimulated human peripheral lymphocytes. Cell proliferation and serine utilization for nucleic acid synthesis have been shown to be responsive to serine concentrations in the normal plasma range. (6) It is concluded that the maintenance of normal plasma serine concentrations is an important factor in the rate of lymphocyte proliferation and hence the effectiveness with which the body can mount an immune response to an antigenic challenge, such as in infection.

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes

It is shown that the time-course of incorporation of radioactivity from [3-14C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stim--ulation.

Alanine as a gluconeogenic carrier

Abstract Alanine can be synthesised de novo from amino acids in muscle and other tissues by a pathway recently described. It can therefore act as a common carrier of amino acid carbon for use in hepatic gluconeogenesis. The release of alanine from peripheral tissues is an important factor in maintaining glucose homeostasis in health and disease.

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.

Mitochondrial-cytosolic interrelationships involved in gluconeogenesis from serine in rat liver

In recent years evidence has accumulated suggesting the possibility that mammalian liver may be equipped with alternative pathways for the metabolism of serine towards glucose synthesis. There is good evidence that serine dehydratase (EC 4. 2 .1 .13) , which catalyses the formation of pyruvate from serine, is involved in initiating gluconeogenesis in the adult rat (see [1]). Earlier studies suggested the presence of an alternative pathway that involved hydroxypyruvate rattler than pyruvate as an intermediate [2] and more recent work with isolated rat liver cells supports this conclusion [3]. Rowsell et al. [4] suggested that this alternative pathway may play a significant role in the suckling neonatal rat and a possible sequence of enzymes to account for this was postulated. The pathway is initiated by serine-pyruvate aminotransferase (EC 2. 6 .1 .51) which catalyses the transamination of serine to form hydroxypyruvate. This enzyme sequence was also proposed by Lardy, et al. [5] to account for gluconeogenesis from serine in the adult rat when the normal pathway via pyruvate was blocked by use of quinolinic acid which inhibits phosphoenolpyruvate carboxykinase (see fig. 1). Further evidence for the proposed involvement of the aminotransferase pathway in gluconeogenesis in the neonatal rat was the relative insensitivity of perfused neonatal liver to inhibition of gluconeogenesis from serine by quinolinic acid [6]. For the process of gluconeogenesis in the rat, pyruvate formed by the action of serine dehydratase in the cytosol must be transported into the mitochondria for carboxylation to oxaloacetate to occur (see fig.l). In a recent paper use has been made of the specific inhibitor of pyruvate transport, a-cyano-4hydroxycinnamic acid, to inhibit the transport of pyruvate and so block the serine dehydratase-mediated pathway of gluconeogenesis from serine [7]. The considerable, though not complete, inhibition of gluconeogenesis from serine in adult rat liver cells in the presence of the inhibitor was taken as evidence for the obligatory participation of pyruvate as an intermediate in gluconeogenesis from serine [7]. The interpretation of of the transport inhibitor for the alternative pathway for serine gluconeogenesis, via serine aminotransferase, requires knowledge of the intracellular site of the initial transamination reaction. The subcellular site has previously been stated to be wholly particulate [8] but supporting data was not given. The present results suggest a predominantly mitochondrial location for the serine aminotransferase activity in adult and neonatal rat liver and it is suggested on this basis that cyanocinnamic acid may not be a suitable inhibitor for discriminating between the different pathways of gluconeogenesis from serine.

Alanine release by rat adipose tissue in vitro.

Summary Isolated rat fat pads incubated in vitro release alanine into the medium at increased rates in the presence of glucose, glutamate, leucine or valine. The increased release was inhibited by aminooxyacetate indicating that alanine release is due to intracellular formation by pyruvate transamination. Starvation and diabetes are associated with increased alanine release and evidence is given for the first time that amino acid carbon and nitrogen may be used for alanine formation in adipose tissue, so that this tissue is capable of making a net contribution to body glucogenic substrate provision.

Disturbances of perinatal carbohydrate metabolism in rats exposed to methylmercury in utero

Abstract Pregnant rats were given a single subcutaneous injection of methylmercuric chloride (at 4 or 8 mg/kg) on the ninth day of gestation. Foetal (2 days prenatal), newborn and postnatal (6 days post partum) animals from the methylmercury-treated mothers were investigated with respect to parameters of carbohydrate metabolism. In the absence of any physical abnormalities, foetal rats exposed to methylmercury in utero showed diminished concentrations of plasma glucose and liver glycogen concentrations and a lower hepatic glucose-6-phosphatase activity compared to control animals. Newborn rats from the methylmercury-treated mothers showed an impairment in glycogen mobilization in the first hours of extra-uterine life which was accompanied by a severe and protracted hypoglycaemic response. Postnatal rats exposed to methylmercury in utero exhibited higher liver glycogen concentration and decreased body weights compared to control rats. The results point to a derangement of perinatal carbohydrate metabolism in the offspring of pregnant rats exposed briefly to low doses of methylmercury during gestation (“metabolic teratogenesis”). The postnatal hypoglycaemic episode in exposed rats may contribute to the pathogenesis of the neurological disturbances revealed by these animals in later life.

Muscle phosphoenolpyruvate carboxykinase activity and alanine release in progressively starved rats.

Abstract 1. 1. Diaphragm muscle phosphoenolpyruvate carboxykinase activity is increased in starved rats. whereas malate dehydrogenase (decarboxylating) activity is decreased. 2. 2. Diaphragms removed from rats starved for varying periods (1–3 days) show a progressively increasing capacity for lactate and pyruvate release, whereas alanine release shows a delayed increase during starvation. 3. 3. The results indicate a role for muscle phosphoenolpyruvate carboxykinase in alanine formation from the carbon skeletons of other amino acids.

Characterization of rat liver β‐adrenoceptors during perinatal development as determined by [125I]‐iodopindolol radioligand binding assays

1 The subtype specificity of β‐adrenoceptors in foetal (20 days post coitum) rat liver membrane preparations has been determined by use of [125I]‐iodopindolol binding assays and the characteristics of radioligand binding have been resolved. 2 The kinetics of radioligand association and dissociation (in the presence of 5 × 10−4 m isoprenaline) showed an association rate constant of 1.5 × 107 m−1 s−1 and dissociation rate constant of 9.1 × 10−4 s−1, corresponding to a dissociation constant for [125I]‐iodopindolol of 60.7 pm. A similar dissociation constant (75 pm) was determined by saturation binding assays. 3 The rank order of potency for displacement of [125I]‐iodopindolol binding was consistent with binding to a predominantly β2‐adrenoceptor population (i.e. ICI 118551 > isoprenaline > adrenaline > noradrenaline > atenolol). Computer analysis of displacement curves in the presence of a β1‐subtype selective agent (atenolol) or a β2‐subtype selective agent (ICI 118551) revealed the presence of β2− and β1‐adrenoceptor subtypes in a ratio of about 80: 20%. 4 Saturation binding assays by use of [125I]‐iodopindolol were carried out at different perinatal ages to determine total β‐adrenoceptor concentrations and β2‐subtype (in the presence of 5 × 10‐7 m atenolol) adrenoceptor concentrations. Competition binding assays with atenolol confirmed that at all ages apparent β2‐adrenoceptor binding accounted for 84–95% of the total β‐adrenoceptor binding. The total β‐ and β2‐adrenoceptor binding capacity increased by 2.3 fold from 20 days post coitum to birth, and then decreased postnatally at 1 and 2 days post partum. The dissociation constant for [125I]‐iodopindolol binding did not show any change with age. 5 The change in β2‐adrenoceptor concentration with age is discussed in relation to the changing β‐adrenoceptor‐mediated responsiveness of glucose production by rat liver during perinatal development.

Regulation of hepatic glucose metabolism by insulin and counter-regulatory hormones

We have sought to define the role of the interplay between insulin and counter-regulatory hormones (such as glucagon, adrenaline, growth hormone or cortisol) in controlling hepatic metabolism in chronically altered insulin status. Situations involving chronic hyper- and hypoinsulinaemia have been studied: glucose homeostasis in the neonatal rat at birth and in the lactating rat.