Ernest Bailey

Intracellular distribution of enzymes associated with lipogenesis and gluconeogenesis in fat body of the adult cockroach, Periplaneta

Abstract The intracellular distribution and maximal activities of some enzymes associated with lipogenesis, gluconeogenesis and fatty acid oxidation have been determined in the fat body of the adult male cockroach Periplaneta americana . Of the enzymes of lipogenesis, acetyl-CoA synthase, acetyl-CoA carboxylase, glucose 6-phosphate dehydrogenase, and ATP citrate lyase are located entirely in the cytosol. Of the other enzymes of citrate metabolism studied, citrate synthase and NAD-dependent isocitrate dehydrogenase are almost exclusively mitochondrial, whereas NADP-dependent isocitrate dehydrogenase and aconitase are predominantly cytosolic although significant mitochondrial activity is also present. The latter subcellular distribution is also observed for ‘malic enzyme’ and NAD and NADP dependent malate dehydrogenase. The enzyme of fatty acid oxidation studied, 3-hydroxyacyl-CoA dehydrogenase is entirely mitochondrial. Of the enzymes possibly involved in gluconeogenesis, glucose 6-phosphate is microsomal, fructose 1,6-diphosphatase cytosolic, pyruvate carboxylase mitochondrial and phosphoenolpyruvate carboxykinase predominantly cytosolic. Of the enzymes of amino acid metabolism studied, glutamate dehydrogenase is NAD-dependent and located in the mitochondria whereas glutamate/oxalacetate and glutamate/pyruvate transaminases are predominantly cytosolic but with significant activity in the mitochondria. Glycerol kinase and sorbitol dehydrogenase are cytosolic and the glyoxylate cycle enzymes malate synthetase and isocitrate lyase are not detected in the fat body. The distribution and relative activities of the enzymes are discussed in relation to fat body biosynthesis and in comparison to mammalian liver and adipose tissue.

The intracellular distribution of enzymes of carbohydrate degradation in the fat body of the adult male cockroach

Abstract The intracellular distribution and maximal activities of phosphorylase, trehalase, all the enzymes of glycolysis, NAD and FAD-linked glycerol 3-phosphate dehydrogenase, arginine kinase, and adenylate kinase have been determined in the fat body of the adult male cockroach Periplaneta americana . Phosphorylase activity is predominantly in the soluble form but some can be sedimented. Both forms of the enzyme exhibit large increases in activity due to the presence of AMP. Only low activities of trehalase are detected and the enzyme appears to exist in both active and inactive forms. FAD-linked glycerol 3-phosphate dehydrogenase is only found in the mitochondria whereas the NAD-linked enzyme and the glycolytic enzymes are located entirely in the cytosol. Of the glycolytic enzymes, phosphoglucomutase, phosphofructokinase and aldolase exhibit unusually low activities. The results suggest that conversion of hexose to triose phosphate may occur predominantly via the pentose phosphate pathway and not via glycolysis. Whereas considerable activities of both types of glycerol-3-phosphate dehydrogenase are present in the fat body only low activities of lactate dehydrogenase are detected in the tissue.

Changes in hepatic fatty acid degradation and blood lipid and ketone body content during development of the rat.

The following have been measured during the development of the laboratory rat: the rate of oxidation of palmitoylcarnitine, decanoylcarnitine, and 14C-palmitate by liver mitochondria; the concentrations of ketone bodies in the blood; the plasma concentrations of non-esterified fatty acids, glycerol, and triglyceride. In each case, a rise after birth and a fall at weaning were observed. These changes can be correlated with the dietary changes which occur at these times. However, during the suckling period, when a constant high fat content diet is consumed, further marked changes in the parameters measured were observed which cannot be related to nutritional factors.

Hepatic mitochondrial fatty acid oxidation during the perinatal period in the rat

1. The activity of hepatic mitochondrial carnitine acyltransferase I increases rapidly after birth, is high during the suckling period and falls after weaning. In contrast, carnitine acyltransferase II and acyl-CpA dehydrogenase exhibit few developmental changes. 2. These and previous studies indicate that outer mitochondrial membrane acyl-CoA synthetase and inner membrane carnitine acyltransferase I increase in activity after birth much more rapidly than to any other enzymes of fatty acid oxidation. 3. Studies of the 18 hr after caesarian delivery indicate that whereas the major increase in the activity of acyl-CoA synthetase occurs within 3 hr of birth the change in carnitine acyltransferase I activity is less rapid. 4. Prolonged pregnancy, starvation of the mother or feeding the mother a high polyunsaturated fat content diet resulted in increased activities of acyl-CoA synthetase and carnitine acyltransferase I in the fetal liver.

Lipogenesis at the suckling-weaning transition in liver and brown adipose tissue of the rat

The responses of rat hepatic and brown adipose tissue in vivo lipogenesis to premature (15 days) and normal (21 days) weaning have been correlated to changes in the activities of acetyl-CoA carboxylase and two NADPH-producing enzymes, malic enzyme and glucose-6-phosphate dehydrogenase. Both tissues show an induction of lipogenesis in response to weaning. In the liver, lipogenic flux is closely linked to the activity of acetyl-CoA carboxylase, but not necessarily that of malic enzyme or glucose-6-phosphate dehydrogenase, whereas no such dissociation between enzyme activity and flux rate occurs in brown adipose tissue. Thyroid hormones, implicated in many physiological changes around weaning, do not seem to play a primary role in the adaptation of lipogenesis to the dietary change at this time, although a permissive role in both tissues is possible.

Perinatal lipogenesis in the liver and brown adipose tissue of the rat

1. Hepatic lipogenesis falls during late foetal life, reaching low levels soon after birth. 2. Of the lipogenic enzymes studied only changes in foetal liver acetyl-CoA carboxylase activities correlate well with the changes in lipogenic flux. 3. In contrast to liver, brown adipose tissue lipogenesis increases during late foetal life. 4. A similar developmental pattern in foetal brown adipose tissue was observed for the activities of a number of enzymes normally associated with lipogenesis. 5. The studies suggest the existence of different controls over the development of lipogenesis in the two tissues investigated during the perinatal period.

Factors involved in the control of the activity of enzymes of hepatic ketogenesis during development of the rat

Abstract 1. 1. The activities of rat liver paniculate and cytoplasmic hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA lyase have been measured from late foetal to adult life. 2. 2. Most pronounced changes occur in the 24 hours following birth, during which time there are large increases in the activities of the paniculate synthase and lyase and the cytoplasmic synthase but not the cytoplasmic lyase. 3. 3. The postnatal increase in the activity of the mitochondrial hydroxymethylglutaryl-CoA synthase (the rate-limiting enzyme in ketogenesis) is prevented by injection of puromycin or by starvation, whereas the simultaneous increase in the activity of the cytoplasmic synthase is unaffected by such treatment.

Crystallization of an NADP+-dependent malic enzyme from rat liver

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.

Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach

Abstract The ketone bodies acetoacetate and D-3-hydroxybutyrate are present in the haemolymph of the adult male cockroach, Periplaneta americana . Starvation increases the acetoacetate but not the D-3-hydroxybutyrate content of the haemolymph. The activities of two key enzymes of ketogenesis, hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA lyase, have been determined in various cell fractions of the fat body and thoracic muscle. Highest activities of both enzymes are found in the fat body. Whereas in the fat body most of the activity of the synthase and the lyase is detected in the particulate cellular fraction and little in the cytosol, the reverse is true for the muscle. Starvation has little effect on the activity of either of the enzymes studied.

Changes in activities of some enzymes associated with hepatic lipogenesis in the rat from weaning to old age and the effect of sucrose feeding

Abstract 1. 1. Hepatic pyruvate kinase, aldolase and ATP citrate lyase exhibit similar developmental patterns with peaks of activity at 25–35 and 55–65 days of age whereas cytosolic aconitate hydratase and isocitrate dehydrogenase have troughs of activity at these stages. 2. 2. Glucose 6-phosphate dehydrogenase and “malic enzyme” have peaks of activity at 35–45 days of age and in mature rats the activities are higher in females than males. 3. 3. The activity of phosphofructokinase changes little during development. 4. 4. At all ages sucrose feeding increased the activities of each of the enzymes other than phospho-fructokinase which was unaffected by diet and cytosolic aconitate hydratase and isocitrate dehydrogenase which decreased in activity.