Max Cake

The anadromous Geotria australis, one of only three lamprey species representing the early agnathan (jawless) stage of vertebrate evolution in Australia and New Zealand, is declining in abundance. Its adults were caught soon after they had entered rivers on their non-trophic upstream migration and maintained in laboratory tanks for 13–15 months through to spawning. As adult G. australis are susceptible to haemorrhagic septicaemia, they were treated prophylactically and maintained in 3-m3 aquaria supplied with a flow-through charcoal filtration system and UV steriliser. Air temperature and the light : dark regime were constantly adjusted to parallel those in the environment. Males developed the very large suctorial disc and gular pouch characteristic of maturity and both sexes matured at the same time as in the wild. While males frequently showed aggressive behaviour towards each other, the same male and female mated on several occasions. The male coiled around the female and, with his urogenital papilla close to the female’s cloaca, twisted and vibrated, leading to egg release. These eggs formed coagulated clusters as in the wild, with many progressing through to the eight-cell stage. Remarkably, numerous G. australis were still alive 95–392 days after the end of the short spawning period, and one male after a further 119 days. Postspawning survival would be facilitated inter alia by extensive proteolysis, reflected in a shortening of the body. The data in this paper emphasise that G. australis is a highly atypical lamprey and provides invaluable information for conserving this declining species.

This paper has integrated new and past data to elucidate how lipid, protein and glycogen metabolism contribute to generating the ATP required by the southern hemisphere lamprey Geotria australis during its ~ 13–15 months, non-trophic upstream spawning migration. Energy is required for maintenance, swimming, the development of gonads and secondary sexual characters and spawning and post-spawning activities. Plasma and muscle metabolites were measured in animals subjected to an exercise-recovery regime at the commencement and completion of the spawning run. The present study demonstrated the following. At all stages of the migration, plasma glucose and glycerol concentrations increased during exercise and then declined, whereas plasma FFAs exhibited the reverse trend. During exercise and recovery, alanine declined and ammonia increased in the plasma of early migrants, while the opposite occurred in mature males. Following exercise, muscle alanine rose and then declined in early migrants, but declined and then rose in mature males. The composite data emphasise that, while the same catabolic processes are employed by both sexes early in the migration, when animals are immature, they differ markedly between the sexes as they mature and then spawn, reflecting their different demands. Energy is supplied predominantly via anaerobic metabolism in early migrants, but by anaerobic and aerobic metabolism in prespawning females and by aerobic metabolism in mature males and spent females. Although proteolysis is limited early in the migration, it is employed extensively during maturation and particularly by females, which undergo a substantial reduction in length in the lead-up to spawning.

Glucocorticoid induction of pulmonary surfactant involves a mesenchyme-derived protein first characterized in 1978 by Smith and termed fibroblast-pneumocyte factor (FPF). Despite a number of agents having been postulated as being FPF, its identity has remained obscure. In the past decade, three strong candidates for FPF have arisen. This review examines the evidence that keratinocyte growth factor (KGF), leptin or neuregulin-1β (NRG-1β) act as FPF or components of it. As with FPF production, glucocorticoids enhance the concentration of each of these agents in fibroblast-conditioned media. Moreover, each stimulates the synthesis of surfactant-associated phospholipids and proteins in type II pneumocytes. Further, some have unique activities, for example, KGF also minimizes lung injury through enhanced epithelial cell proliferation and NRG-1β enhances surfactant phospholipid secretion and β-adrenergic receptor activity in type II cells. However, even though these agents have attributes in common with FPF, it is inappropriate to specify any one of these agents as FPF. Rather, it appears that each contributes to separate mesenchymal-epithelial signaling mechanisms involved in different aspects of lung development. Given that the production of pulmonary surfactant is essential for postnatal survival, it is reasonable to suggest that several mechanisms independently regulate surfactant synthesis.

Glucocorticoids induce lung fibroblasts to produce fibroblast-pneumocyte factor, a peptide that stimulates type II cells to synthesize pulmonary surfactant. This effect is known to be more apparent in cells derived from female fetuses, a characteristic that has been attributed to sex-linked differences in the fibroblasts. In the current study, it has been shown that dexamethasone enhances both β-adrenergic receptor (β-AR) activity (1.3- to 1.6-fold increase) and (-)-isoproterenol-induced secretion of surfactant (1.8- to 1.9-fold increase) in type II cells. However, fibroblast-conditioned media (FCM), prepared in the presence of dexamethasone, generates a much greater response to (-)-isoproterenol (3.1- to 3.8-fold increase). Furthermore, each of these effects is more pronounced if both cell types are female-derived. It is hypothesized that the enhanced response to glucocorticoids is the result of a synergistic effect between the steroid and a component of FCM. Neuregulin-1β (NRG1β), which is elevated in FCM generated in the presence of dexamethasone, influences not only the rate of surfactant secretion and the β-AR activity in type II cells, but also enhances in both sexes the cellular response to (-)-isoproterenol. These results suggest that NRG1β might be more effective than glucocorticoids in treating prematurely born male infants, which are known to respond poorly to glucocorticoids. Given that glucocorticoids are known to induce higher levels of β-AR mRNA, the effect of NRG1β, alone and in combination with dexamethasone, on β-AR gene expression was measured using qRTPCR. Whereas NRG1β had no effect alone, in combination with dexamethasone it produced up to a 4.2-fold elevation in the level of β-AR mRNA.

It is well established that glucocorticoids elevate the production of fibroblast-pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin-1β (NRG1β) in conditioned medium is elevated 2-fold (P < 0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3-fold the rate of phospholipid synthesis (P < 0.05), suggests that NRG1β is a component of FPF that promotes lung development.

Surfactant production is known to involve a cellular communication between lung fibroblasts and the type II pneumocytes. Glucocorticoids induce the production of a peptide by lung fibroblasts, fibroblast-pneumocyte factor (FPF), which sequentially acts on type II cells to enhance the synthesis of surfactant phospholipid. Our findings show that fibroblast-conditioned medium (FCM), generated in the presence of dexamethasone, not only enhanced surfactant phospholipid synthesis in type II cells but also contained an elevated concentration of neuregulin-1β (NRG1β). Even though it has been earlier proposed that leptin has many of the characteristics of FPF, recent research has revealed that NRG1β also has many similar attributes. In the current study, exposure of the type II cells to a commercially available form of NRG1β (heregulin-1β) directly stimulated by more than three-fold the rate of phospholipid synthesis (p <0.05). Although similar in magnitude, the effect of heregulin-1β appeared to require a longer time of exposure to that reported for leptin. There was no increase in the gene expression of NRG1β when lung fibroblasts were exposed to dexamethasone, irrespective of the concentration of dexamethasone used, or the time of contact of the cells to the steroid. Thus the glucocorticoid-induced increase in the level of NRG1β in FCM was not the result of enhanced expression of the NRG1β gene. The inability of dexamethasone to induce a significant increase in NRG1β gene expression in lung fibroblasts suggests that the elevated concentration of NRG1β might be the result of enhanced cleavage of the membrane-bound neuregulin precursor. In summary, these findings not only support but significantly extend the concept previously promoted that NRG1β plays an essential role in the differentiation and maturation of the lung in the later stages of gestation. Moreover, together these studies suggest that FPF may be a complex mixture of agents capable of motivating surfactant synthesis.

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.

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A2 (PLA2)-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA2-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA2. Furthermore, specific inhibitors of PLA2 such as pbromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA2-like activity may be an intrinsic attribute of albumin.