Metabolic responses to exhaustive exercise change markedly during the protracted non-trophic spawning migration of the lamprey Geotria australis.

Adults of the Southern hemisphere lamprey Geotria australis were subjected to an exercise/recovery regime at the commencement and end of their 12-15 month non-trophic, upstream spawning migration. In early (immature) migrants and pre-spawning females, muscle glycogen was markedly depleted during exercise, but became rapidly replenished. As muscle lactate rose during exercise and peaked 1-1.5 h into the recovery period, and therefore after muscle glycogen had become replenished, it cannot be the direct source for that replenishment. However, both plasma lactate and glycerol (but not muscle glycerol and glucose) rose sharply during exercise and then declined markedly during the first 0.5 h of recovery and thus exhibited the opposite trend to that of muscle glycogen, implying that these limited pools of glycogenic precursors contribute to glycogen replenishment. Although plasma glucose rose following exercise, and consequently could also be a precursor for muscle glycogen replenishment, it remained elevated even after muscle glycogen had become replenished. While resting pre-spawning females and mature males retained high muscle glycogen concentrations, this energy store became permanently depleted in females during spawning. In mature males, muscle glycogen remained high and lactate low during the exercise/recovery regime, whereas muscle glycerol declined precipitously during exercise and then rose rapidly. In summary, vigorous activity by G. australis is fuelled extensively by anaerobic metabolism of glycogen early in the spawning run and by pre-spawning females, but by aerobic metabolism of its energy reserves in mature males.

Muscle glycogen, lactate and glycerol-3-phosphate concentrations of larval and young adult lampreys in response to exercise.

When stimulated, the ammocoetes (larvae) of Geotria australis swim continuously at a moderate rate for only approximately 20 min, whereas the downstream migrants (young adults) of this species did not become exhausted following similar swimming activity over the same period. Mean concentrations of muscle glycogen in ammocoetes declined during exercise, but returned to resting levels within 30 min of recovery, whereas those in young adults changed little during the corresponding periods. Moreover, muscle lactate concentrations of ammocoetes rose markedly during exercise and the first 30 min of recovery, before declining significantly, while those of young adults remained similar during and immediately after exercise. Calculations, using the glycogen and lactate concentrations immediately after exercise, suggest that during exercise glycogen is, to some extent, utilised anaerobically (approx. 24%) by ammocoetes, but only aerobically by young adults. Furthermore, since young adults used only a small amount of glycogen, they presumably metabolised triacylglycerol aerobically to produce energy. Muscle glycerol-3-phosphate levels were far higher prior to and immediately after exercise in downstream migrants than in ammocoetes and then declined precipitously. The above trends in muscle glycogen and lactate of larval G. australis parallels, to some degree, those recorded by other workers for upstream migrant Petromyzon marinus that had been exercised to exhaustion.

The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I.

The affinity of recombinant rat acyl-CoA binding protein (ACBP) towards acyl-CoAs was investigated using both fluorimetric analysis and isothermal titration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (K(d)). The displacement of 11-(dansylamino)undecanoyl-CoA (DAUDA-CoA) from ACBP yielded binding parameters for the competing acyl-CoAs that compared favourably with those obtained using ultra-sensitive microcalorimetric titration. The K(d) values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 microM, respectively. Under identical experimental conditions, carnitine palmitoyltransferase I (CPT I) of purified rat liver mitochondria has K(d) values of 2.4 and 22.7 microM for oleoyl-CoA and docosahexaenoyl-CoA, respectively. Given that CPT I was not only present at a much lower concentration but also has an appreciably lower affinity for acyl-CoAs than ACBP, it is proposed that CPT I is capable of interacting directly with ACBP-acyl-CoA binary complexes. This is supported by the fact that the enzyme activity correlated with the concentration of ACBP-bound acyl-CoA but not the free acyl-CoA. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I could be a result of the enzyme inducing a conformational alteration in the ACBP leading to the release of acyl-CoA.

Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases.

In this study a pathway for the synthesis of triacylglycerol (TAG) within the lumen of the endoplasmic reticulum has been identified, using microsomes that had been preconditioned by depleting their endogenous substrates and then fusing them with biotinylated phosphatidylserine liposomes containing CoASH and Mg(2+). Incubating these fused microsomes with tri[(3)H] oleoylglycerol and [(14)C]oleoyl-CoA yielded microsome-associated triacylglycerol, which resisted extensive washing and had a [(3)H]:[(14)C] ratio close to 2:1. The data suggest that the precursor tri[(3)H]oleoylglycerol was hydrolyzed by microsomal lipase to membrane-bound di[(3)H]oleoylglycerol and subsequently re-esterified with luminal [(14)C]oleoyl-CoA. The accumulation of TAG within the microsomes, even when overt diacylglycerol acyltransferase (DGAT I) was inactive, is consistent with the existence of a latent diacylglycerol acyltransferase (DGAT II) within the microsomal lumen. Moreover, because luminal synthesis of TAG was carnitine-dependent and markedly reduced by glybenclamide, a potent carnitine acyltransferase inhibitor, microsomal carnitine acyltransferase appears to be essential for trafficking the [(14)C]oleoyl-CoA into the microsomal lumen for subsequent incorporation into newly synthesized TAG. This study thus provides the first direct demonstration of an enzymatic process leading to the synthesis of luminal triacylglycerol, which is a major component of very low density lipoproteins.

Liver mitochondria, confirmed as intact by complete suppression of succinate uptake and oxidation, possess a carnitine palmitoyltransferase I that is totally inhibited by malonyl CoA.

Succinate dehydrogenase activity in mitochondria, which were isolated by centrifuging partially purified mitochondria through 1. 315 M sucrose, was completely suppressed when [14C]succinate uptake was abolished by prior incubation of the mitochondria with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin. The conclusion that these mitochondria were intact was confirmed by the fact that, when these mitochondria were broken by a freeze-thaw cycle followed by sonication, such inhibition was totally abolished. The yield of mitochondria, microsomes, and peroxisomes from the initial homogenate was 17.8, <0.1, and 0%, respectively, indicating that the mitochondria were not only intact but also essentially free of contamination from microsomes and peroxisomes. The overt form of carnitine palmitoyltransferase (CPT I) in these intact and pure mitochondria was totally inhibited by malonyl CoA, indicating that previous reports of incomplete inhibition in mitochondrial preparations resulted from interference from CPT activity in the inner mitochondrial membrane (CPT II), microsomes, or peroxisomes.

Evaluation of the affinity and turnover number of both hepatic mitochondrial and microsomal carnitine acyltransferases: relevance to intracellular partitioning of acyl-CoAs.

Mitochondrial carnitine palmitoyltransferase I (CPT I) and microsomal carnitine acyltransferase I (CAT I) regulate the entry of fatty acyl moieties into their respective organelles. Thus, CPT I and CAT I occupy prominent positions in the pathways responsible for energy generation in mitochondria and the assembly of VLDL in the endoplasmic reticulum, respectively. Previous attempts to determine the intrinsic kinetic properties of CPT I and CAT I have been hampered by the occurrence of sigmoidal velocity curves. This was overcome, in this study, by the inclusion of recombinant acyl-CoA binding protein in the assay medium. For the first time, we have determined the concentrations of total functional enzyme (E(t)) by specific radiolabeling of the active site, the dissociation constants (K(d)) and the turnover numbers of CPT I and CAT I toward the CoA esters of oleic acid (C18:1) and docosahexaenoic acid (C22:6). The data show that carnitine inhibits CAT I at physiological concentrations which are not inhibitory to CPT I. Thus, carnitine concentration is likely to be a significant factor in determining the partitioning of acyl-CoAs between mitochondria and microsomes, a role which has not been previously recognized. Moreover, the finding that CAT I elicits a lower turnover toward the CoA ester of C22:6 (25 s(-)(1)) than toward that of C18:1 (111 s(-)(1)), while having similar K(d) values, suggests the use of this polyunsaturated fatty acid to inhibit VLDL biosynthesis.

Influence of diet on the kinetic behavior of hepatic carnitine palmitoyltransferase I toward different acyl CoA esters.

The influence of diet on the kinetics of the overt form of rat liver mitochondrial carnitine palmitoyltransferase (CPT I; EC 2.3.1.21) was studied using rats fed either a low-fat diet (3% w/w fat), or diets which were supplemented with either olive oil (OO), safflower oil (SO) or menhaden (fish) oil (MO) to 20% w/w of fat (high fat diets). When animals were fed each of these four diets for 10 days, the order of the apparent maximal activity (Vmax) of CPT I toward various individual fatty acyl CoA, when measured under a fixed molar ratio of acyl CoA/albumin, was 16:1 n-7 > 18:1 n-9 > 18:2 n-6 > 16:0 > 22:6 n-3, and was thus not affected by the fat composition of the diet. However, in all but one case, the SO and MO diets elicited a higher Vmax for each substrate than either the LF diet or the high fat OO diet. The apparent K0.5 for the different acyl CoA esters was generally lowest in LF-fed animals, and highest in those fed the high-fat SO diet. Moreover, when compared with the situation of animals fed high-fat diets, the K0.5 values of CPT I in LF-fed animals for palmitoyl CoA and oleoyl CoA were low. This possession by CPT I of a high "affinity" toward these nonessential fatty acyl CoAs, but a lower "affinity" toward linoleoyl CoA, the ester of an essential fatty acid, may enable this latter fatty acid to be spared from oxidation when its concentration in the diet is low. The data also emphasize that palmitoleoyl CoA, if available in the diet, is likely to be utilized by CPT I at a high rate.

Stimulation of surfactant lipid secretion from fetal type II pneumocytes by gastrin-releasing peptide.

Gastrin-releasing peptide (GRP) and bombesin apparently enhance the rate of secretion of surfactant lipids from cultured fetal rat type II pneumocytes. This effect, evident within 1h of addition of the peptide, is concentration-dependent, with a maximal response at 3.0 nM. When the effect of GRP was assessed in comparison with other known secretagogues, it was found that, whereas GRP and isoproterenol were additive in their effect, there was no response to GRP in the presence of saturating concentrations of A23187 or phorbol 12-myristate 13-acetate. This suggests that the secretory response to GRP is via activation of Ca2+/calmodulin-dependent protein kinase and/or protein kinase C and is independent of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. This conclusion is supported by the observation that the GRP-induced secretion is inhibited by calphostin C, an inhibitor of protein kinase C, but not by H-89, an inhibitor of cAMP-dependent protein kinase. The fact that GRP regulates surfactant secretion from type II pneumocytes suggests that it and/or related peptides may play a significant role in the physiological maturation of the lung.

Hepatic molecular conversion and detoxification of ferritin iron in adult lampreys (Geotria australis), following natural and induced iron loading.

Weekly intramuscular injections of 3 mg of iron as horse spleen ferritin into adult Geotria australis over 10 weeks, resulted in a progressive increase in that form of iron in the serum. However, as with control animals, the ferritin in the liver of injected lampreys consisted of one subunit type, whose M(r) (20,300) differed from those of the two subunit types of horse spleen ferritin. Thus, lampreys had converted horse spleen ferritin iron into endogenous ferritin iron, presumably in their liver. Marked rises in hepatic non-haem iron during the first 2 weeks and between weeks 8 and 10 of iron injections were accompanied by pronounced increases in superoxide dismutase (SOD) activity. This rise, which parallels the rise in SOD activity that occurs as iron increases during the very protracted upstream migration of G. australis, is consistent with the view that SOD protects against iron-mediated damage by removing the superoxide radical, which facilitates the formation of the highly toxic hydroxyl radical. A levelling off of the iron concentration between weeks 2 and 8 was accompanied by a decline in SOD activity, even though nonhaem iron levels were well above those of control animals. Enhanced SOD activity may therefore only be required when there is an elevated flux of iron in the liver through low-molecular-mass intermediates. A small amount of ferritin iron was converted into the more inert haemosiderin iron.

Iron release from ferritin and its sensitivity to superoxide ions differs among vertebrates.

The influence of the superoxide-generating system, xanthine oxidase, on the release of iron from various vertebrate ferritins was determined both in the presence and absence of superoxide dismutase. The initial rate of iron release in the presence of this system was higher for ferritins from human, trout and rat liver than for those from lamprey liver and horse spleen. The proportion of this iron release that was superoxide-dependent in the case of rat, human and trout ferritins was 92, 86 and 84% respectively, whereas no such superoxide-dependent iron release occurred from the ferritins of lamprey liver and horse spleen. On the other hand, the rate of superoxide-independent iron release was of comparable magnitude for all of the species examined. The rate of superoxide-dependent iron release was related neither to the iron: protein ratios nor to the subunit size of the ferritins. However, it is significant that the ferritins with a high rate of superoxide-dependent iron release came from tissues known to be susceptible to iron damage. It is thus proposed that the resistance of lamprey liver ferritin to the mobilization of iron by superoxide ions accounts in part for the tolerance of the lamprey liver to high iron loads.

Lipid analysis of lavage samples from the equine guttural pouch (auditory tube diverticulum).

The guttural pouch is a large, air-filled diverticulum of the auditory tube, present in the horse and other species. Lipid analysis of saline lavage from the equine guttural pouch has demonstrated the presence of phospholipids and neutral lipids in amounts that are variable but consistently greater than in any other species described. A stain specific for choline-containing phospholipids has demonstrated the presence of phospholipid-containing vesicles only within the cells of subepithelial, seromucoidlike glands, suggesting that these cells incorporate phospholipids in their secretions. The functional significance of surface-active agents in the guttural pouch may be different from that proposed for other species because of the unique anatomical design and the different proposed functions of the guttural pouch.

Impact of complete isletectomy on plasma glucose in the southern hemisphere lamprey Geotria australis.

The adult southern hemisphere lamprey Geotria australis is the only known vertebrate in which it is possible to remove all of the pancreatic islet tissue without damaging other organs. In the 24 hr after isletectomy, the plasma glucose of adult G. australis rose sharply from 5.0 to 11.6 mmol.liter-1 and remained at a similar elevated level throughout the subsequent 5 days of the experiment. The marked hyperglycemia that follows complete isletectomy parallels the results obtained after removal of the majority of the islet tissue from northern hemisphere lampreys and after pancreatectomy in mammals, but contrasts with observations recorded for some other groups of vertebrates.

Digestive enzyme activities and their distribution in the alimentary canal of larvae of the three extant lamprey families.

The activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), lipase (EC 3.1.1.3) and amylase (EC 3.2.1.1) were measured in different regions of the alimentary tract of ammocoetes from each of the three extant lamprey families. In the southern hemisphere speciesGeotria australis (Geotriidae), and even more particularlyMordacia mordax (Mordaciidae), enzymatic activity was almost entirely confined to prominent diverticular extensions which arise at the oesophageal-intestinal junction. However, in the holarcticLampetra richardsoni (Petromyzontidae), which does not possess a diverticulum, the enzymatic activity was highest in the upper anterior intestine. It is not clear whether the presence of significantly higher amylolytic and lower lipolytic activities in the diverticulum ofG. australis than in the exocrine tissue of the other two species reflects interspecific differences in the composition of their diets. The capacity of exocrine tissue extracts for chymotryptic and tryptic digestion was assayed before and afterin vitro exposure to trypsin and enteropeptidase, their respective catalytic activators. Prior to exposure to these exogenous activators, both proteolytic enzymes were fully active inL. richardsoni, partially active inG. australis and totally inactive inM. mordax. Maximal chymotryptic activity was greater inM. mordax than inL. richardsoni andG. australis. In contrast, maximal tryptic activity was greater inL. richardsoni than inG. australis and was very low inM. mordax. Since trypsin is the only known activator of chymotrypsinogen, the negligible activity of trypsin inM. mordax would appear anomalous unless a trypsin inhibitor is present in the protopancreas of this species. Differences in the distribution of enzymatic activity within the alimentary tract of the three species is discussed in relation to proposed phylogenetic relationships amongst the extant lamprey families.

Enhancement of disaturated phosphatidylcholine synthesis by epidermal growth factor in cultured fetal lung cells involves a fibroblast-epithelial cell interaction.

Epidermal growth factor (EGF) increases the rate of choline incorporation into disaturated phosphatidylcholine in cultured fetal rat type II cells via an indirect mechanism. Whereas-EGF has no effect on the rate of disaturated phosphatidylcholine synthesis when added directly to type II pneumocytes, the growth factor is effective if it is present during preliminary conditioning of the media by lung fibroblasts. This effect is concentration dependent with a maximal effect at 20 ng/ml. When lung fibroblasts are incubated with both glucocorticoids and EGF, there is no significant effect of the growth factor over and above that seen with the steroid alone. This suggests that the two agents might act via a similar mechanism. This is supported by the observation that each inducer leads to the production by lung fibroblasts of a stimulatory factor that has a similar, if not identical, chromatographic elution profile. We conclude that EGF may contribute significantly to the normal onset of lung maturation by elaborating a fibroblast-derived factor that stimulates phosphatidylcholine synthesis in type II pneumocytes.

An increase in the concentration of hepatic iron during the metamorphosis of the lamprey Geotria australis is accompanied by increased superoxide dismutase activity.

The concentration of total non-haem iron and its ferritin iron component, and the activity of the enzyme superoxide dismutase (SOD), were measured in the livers of ammocoetes, metamorphosing animals (stages 1-7) and recently metamorphosed downstream migrants of the lamprey Geotria australis. Total non-haem iron in the liver rose significantly from 0.15-0.55 µg.mg wet weight(-1) in ammocoetes and metamorphosing stages 1-3 to 2.2-2.9 µg.mg(-1) in stages 5-7 and to 8.8 µg.mg(-1) in downstream migrants. The comparable values for ferritin iron were 0.06-0.26, 1.4-2.0, and 5.3 µg.mg(-1). Superoxide dismutase activity fell sharply from 0.39 µg.mg(-1) in large ammocoetes to between 0.07 µg.mg(-1) in stage 1 and 0.15 µg.mg(-1) in stage 6, before rising significantly to 0.26 µg.mg(-1) in stage 7 and 0.35 µg.mg(-1) in downstream migrants. The sharp fall in SOD activity at the beginning of metamorphosis is assumed to be related to the marked decline in plasma iron which occurs at the onset of this non-trophic phase in the life cycle. It is proposed that the subsequent increase in SOD activity in the liver of G. australis with increasing iron represents a mechanism aimed at reducing the potentially toxic effects of iron accumulation. This view is consistent with the significant and positive correlation found between both total non-haem and ferritin iron and SOD activity in the liver of non-trophic animals.

Insulin antagonism of dexamethasone induction of tyrosine aminotransferase in cultured fetal hepatocytes. A correlation between enzyme activity, synthesis, level of messenger RNA and transcription.

Previous studies have shown that insulin depresses the induction of tyrosine aminotransferase by glucocorticoids in cultured fetal rat hepatocytes. However, the site at which this inhibitory effect is exerted was not elucidated, since only enzyme activity was determined in such studies. Therefore, the effect of insulin on tyrosine aminotransferase synthesis, the level of its mRNA as well as the rate of transcription of the gene in isolated nuclei have been determined. The results obtained indicate that in cultures exposed to dexamethasone, Bt2cAMP, insulin and combinations of these additives, there is an excellent correlation between the enzyme activity, enzyme synthesis and the level of mRNA. Run-on transcription experiments indicate that the reduction in the level of mRNA by insulin in dexamethasone-supplemented cultures is the result of a diminished rate of gene transcription.

Exceptional iron concentrations in larval lampreys (Geotria australis) and the activities of superoxide radical detoxifying enzymes.

This study aimed to elucidate the way in which larvae of the lamprey Geotria australis counteract the potential problems of the very high concentrations of non-haem iron they contain and thereby avoid the deleterious effects associated with iron overload in other vertebrates. Particular attention has been paid to ascertaining whether increasing concentrations of iron are accompanied by (i) change to a less readily available form of iron and (ii) an increase in the activity of those detoxifying enzymes responsible for minimizing the production of harmful hydroxyl radicals via the Haber-Weiss reaction. The mean concentrations of haemosiderin and ferritin in larval G. australis were each far higher in the nephric fold than in either the liver or intestine, but all these concentrations were much greater than those in rat liver. Since haemosiderin releases iron far more slowly than ferritin, the iron it contains is much less readily available to catalyse the Haber-Weiss reaction. It is thus relevant that (i) non-haem iron in the nephric fold occurred to a greater extent as large dense haemosiderin granules than as ferritin molecules and (ii) the proportion of iron in the form of haemosiderin rose with increasing concentration of total non-haem iron. A strong correlation was also recorded between the activity of superoxide dismutase in the nephric fold and the concentrations of total non-haem iron and its haemosiderin and ferritin components. This demonstrates that enzyme detoxification of O2.- rises with increasing amounts of iron. The exceptional iron concentrations in the nephric fold were not reflected by a greater measured activity of superoxide dismutase than that found in other tissues. However, the nephric fold was shown to contain an augmentation factor which is presumed to enhance the activity of this enzyme in vivo. The activity of catalase and glutathione peroxidase, which catalyse the breakdown of H2O2 to O2 and water, were each significantly correlated with the concentration of ferritin.

Induction of tyrosine aminotransferase in utero by anti-insulin agents.

The hepatic enzyme tyrosine aminotransferase, normally expressed in very low amounts until shortly after birth, is prematurely induced in foetal rats made diabetic by the administration of streptozotocin in utero. Similarly, the enzyme is precociously induced in foetuses if the circulating insulin concentration is artificially decreased by the administration of anti-insulin serum. These observations support the proposal that the natural decrease in plasma insulin, known to occur at birth, is a major contributor to the postnatal induction of tyrosine aminotransferase.

Characterization and properties of a progesterone receptor in the uterus of the quokka (Setonix brachyurus).

A progesterone receptor system, with a high specificity for progestins, was detected in the uterine tissue of the marsupial, Setonix brachyurus (quokka), using the synthetic progestin 17 alpha, 21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020). The apparent equilibrium dissociation constant of the ligand binding to the cytosolic component was 2.2 nmol/l, and to the nuclear component 4.8 nmol/l. Significant loss of binding ability of the receptor occurred when cytosol was pretreated with dextran-coated charcoal. All binding studies were performed, therefore, in the presence of endogenous steroid which was demonstrated to affect the dissociation constant but have no effect on the estimation of the concentration of binding sites. Cytosolic binding was increased sixfold by oestradiol-17 beta treatment in vivo, and the translocation of the bound complex into the nucleus was effected by progesterone. It is suggested that the binding component described plays a role in the action of progesterone on the uterine tissue of the quokka.

Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes.

Whereas dexamethasone is unable to induce the premature formation of hepatic tyrosine aminotransferase when administered to foetal rats in utero, the steroid can induce the enzyme in foetal rat liver if the liver is first removed from the environment in utero and grown in culture. Dexamethasone produced a significant induction of the enzyme at a concentration of 0.1 nM in cultured foetal hepatocytes, but for optimal induction the cells were exposed to 10 nM for 15 h. Growing the hepatocytes in the presence of physiological concentrations of insulin had no effect on the enzyme activity in control cells. However, the induction of the enzyme by dexamethasone was markedly diminished in the presence of insulin. This effect of insulin is both time-dependent and dose-dependent with significant inhibition being obtained with 1 nM insulin. Growing foetal hepatocytes in the presence of insulin has no effect on either the cellular level of glucocorticoid receptor or on the stability of dexamethasone-receptor complexes to undergo nuclear translocation suggesting that insulin inhibits some event subsequent to translocation. The results are discussed in relation to the postnatal appearance of tyrosine aminotransferase and suggest that the marked decline in the plasma concentration of insulin, that is known to occur at birth, is a major contributor to the postnatal induction of the enzyme.