Compensatory Growth Is Accompanied by Changes in Insulin-Like Growth Factor 1 but Not Markers of Cellular Aging in a Long-Lived Seabird.

AbstractDeveloping organisms often plastically modify growth in response to environmental circumstances, which may be adaptive but is expected to entail long-term costs. However, the mechanisms that mediate these growth adjustments and any associated costs are less well understood. In vertebrates, one mechanism that may be important in this context is the highly conserved signaling factor insulin-like growth factor 1 (IGF-1), which is frequently positively related to postnatal growth and negatively related to longevity. To test this idea, we exposed captive Franklin's gulls (Leucophaeus pipixcan) to a physiologically relevant nutritional stressor by restricting food availability during postnatal development and examined the effects on growth, IGF-1, and two potential biomarkers of cellular and organismal aging (oxidative stress and telomeres). During food restriction, experimental chicks gained body mass more slowly and had lower IGF-1 levels than controls. Following food restriction, experimental chicks underwent compensatory growth, which was accompanied by an increase in IGF-1 levels. Interestingly, however, there were no significant effects of the experimental treatment or of variation in IGF-1 levels on oxidative stress or telomeres. These findings suggest that IGF-1 is responsive to changes in resource availability but is not associated with increased markers of cellular aging during development in this relatively long-lived species.

Environmental estrogens inhibit insulin-like growth factor (IGF) receptor mRNA expression, IGF binding, and IGF signaling ex vivo in rainbow trout (Oncorhynchus mykiss).

In this study, we used juvenile rainbow trout to examine the direct effects of selected environmental estrogens (EE), specifically, 17 ß-estradiol (E2), ß-sitosterol (ßS), and 4-n-nonylphenol (NP), on target tissue sensitivity to insulin-like growth factor (IGF) as assessed by expression of IGF receptor type 1 (IGFR1) mRNAs and IGF-1 binding capacity, as well as on the cell signaling pathways through which EE exert their effects. E2 and NP inhibited IGFR1A and IGFR1B mRNA expression in a time- and concentration-related manner in gill and muscle; however, ßS had no effect on expression of IGFR1 mRNAs in either tissue. NP reduced 125I-IGF binding in gill and E2 and NP reduced 125I-IGF in white muscle; ßS had no effect on 125I-IGF binding in either gill or white muscle. Treatment of gill filaments with either E2 or NP rapidly deactivated (via reduced proportion of phosphorylation) JAK2, STAT5, Akt, and ERK; ßS had no effect on the activation state of any cell signaling elements tested. The effects of EE on IGFR mRNA expression in gill were estrogen receptor (ER) dependent as the inhibitory effects were rescued by the ER antagonist, ICI 182,780. All EE tested blocked growth hormone (GH)-stimulated IGFR mRNA expression in gill filaments. GH-stimulated activation of JAK2, STAT5, Akt, and ERK were blocked by E2, ßS, and NP. Lastly, E2 and NP stimulated suppressor of cytokine signaling 2 (SOCS-2) mRNA expression, an effect that also was ER dependent. These results indicate that EE directly reduce the sensitivity of peripheral tissues to IGF by reducing mRNA and functional expression of IGFRs. Such inhibitory actions of EE are mediated, at least in part, by ER-dependent mechanisms that deactivate JAK, STAT, Akt, and ERK and enhance expression of SOCS-2. These findings together with our previous results show that EE retard growth of post-embryonic rainbow trout through widespread direct effects on the GH-IGF system, specifically, by reducing tissue sensitivity to GH, inhibiting IGF production, reducing tissue sensitivity to IGF, and by deactivating post-receptor IGF cell signaling pathways.

Environmental estrogens inhibit the expression of insulin-like growth factor mRNAs in rainbow trout in vitro by altering activation of the JAK-STAT, AKT-PI3K, and ERK signaling pathways.

Environmental estrogens (EE) have been found to disrupt a host of developmental, reproductive, metabolic, and osmoregulatory process in a wide-range of animals, particularly those in aquatic ecosystems where such compounds concentrate. Previously, we showed that EE inhibited post-embryonic organismal growth of rainbow trout in vivo, but the precise mechanism(s) through which EE exert their growth inhibiting effects remain unknown. In this study, we used rainbow trout (Oncorhynchus mykiss) as a model to investigate the direct effects of 17ß-estradiol (E2), ß-sitosterol (ßS), and 4-n-nonylphenol (NP) on the synthesis of insulin-like growth factors (IGFs) and to elucidate the mechanism(s) by which EEs exert such effects. E2, ßS, and NP significantly inhibited the expression of both IGF-1 and IGF-2 mRNAs in liver and gill in a time- and concentration-related manner. Although the response evoked by each EEs on the expression of IGF mRNAs was similar, the potency and efficacy varied with EE; the rank order potency/efficacy was as follows: E2 > NP > ßS. The effects of EEs on the expression of IGF mRNAs was blocked by the estrogen receptor (ER) antagonist, ICI 182780. The mechanism(s) through which EEs inhibit IGF mRNA expression were investigated in isolated liver cells in vitro. EE treatment deactivated JAK, STAT, ERK, and AKT. Moreover, blockade of growth hormone (GH)-stimulated IGF expression by EE was accompanied by deactivation of JAK, STAT, ERK, and AKT. EEs also increased the expression of suppressor of cytokine signaling 2 (SOCS-2), a known inhibitor of JAK-2--an action that also was blocked by ICI 182780. These results indicate that EEs directly inhibit the expression of IGF mRNAs by disrupting GH post-receptor signaling pathways (e.g., JAK, STAT, ERK, and AKT) in an ER-dependent manner.

Changes in processes downstream of the hypothalamus are associated with seasonal follicle development in a songbird, the dark-eyed junco (Junco hyemalis).

Mechanisms related to seasonal reproductive timing in vertebrates have received far more study in males than in females, despite the fact that female timing decisions dictate when rearing of offspring will occur. Production and release of gonadotropin-releasing hormone (GnRH) by the hypothalamus stimulates the pituitary to secrete gonadotropins, initiating the beginning stages of gonadal recrudescence and production of the sex steroids, testosterone and estradiol, which are necessary to prime the liver for secretion of yolk precursors in breeding female birds. While stimulation by the hypothalamus can occur during the pre-breeding period, egg development itself is likely regulated downstream of the hypothalamus. We used GnRH challenges to examine variation in breeding-stage-specific patterns of pituitary and ovarian responsiveness in free-living female dark-eyed juncos (Junco hyemalis) and also examined the ovary and liver for variation in mRNA expression of candidate genes. Baseline LH levels increased during the transition from pre-breeding to egg-development, however no significant difference was observed in post-GnRH injection levels for LH or sex steroids (testosterone and estradiol). Interestingly, a stage by time-point interaction was observed, with post-GnRH LH levels increasing over baseline during the pre-breeding stage, but not during the egg-development stage. We observed a decrease in liver mRNA expression of estradiol receptor-alpha, and glucocorticoid and mineralocorticoid receptors and a decrease in glucocorticoid receptor expression levels in the ovary. A decline in FSH receptor expression across stages was also observed in the ovary. Combined, our data suggest seasonal variation in female's sensitivity to signals of HPG activity and energetic or stress signals. These data provide additional insight into the physiological mechanisms regulating onset of clutch initiation.

Insulin and insulin-like growth factor-1 modulate the lipolytic action of growth hormone by altering signal pathway linkages.

Growth hormone (GH) has many actions in vertebrates, including the regulation of two disparate metabolic processes: growth promotion (anabolic) and the mobilization of stored lipids (catabolic). Our previous studies showed that GH stimulated IGF-1 production in hepatocytes from fed rainbow trout, but in cells from fasted fish GH stimulated lipolysis. In this study, we used rainbow trout (Oncorhynchus mykiss) to elucidate regulation of the mechanisms that enable cells to alter their lipolytic responsiveness to GH. In the first experiment, cells were removed from either fed or fasted fish, conditioned in medium containing serum (10%) from either fed or fasted fish, then challenged with GH. GH stimulated the expression of hormone sensitive lipase (HSL), the primary lipolytic enzyme, in cells from fasted fish conditioned with "fasted serum" but not in cells from fasted fish conditioned in "fed serum." Pretreatment of cells from fed fish with "fasted serum" resulted in GH-stimulated HSL expression, whereas GH-stimulated HSL expression in cells from fasted fish was blocked by conditioning in "fed serum." The nature of the conditioning serum governed the signaling pathways activated by GH irrespective of the nutritional state of the animals from which the cells were removed. When hepatocytes were pretreated with "fed serum," GH activated JAK2, STAT5, Akt, and ERK pathways; when cells were pretreated with "fasted serum," GH activated PKC and ERK. In the second study, we examined the direct effects of insulin (INS) and insulin-like growth factor (IGF-1), two nutritionally-regulated hormones, on GH-stimulated lipolysis and signal transduction in isolated hepatocytes. GH only stimulated HSL mRNA expression in cells from fasted fish. Pretreatment with INS and/or IGF-1 abolished this lipolytic response to GH. INS and/or IGF-1 augmented GH activation of JAK2 and STAT5 in cells from fed and fasted fish. However, INS and/or IGF-1 eliminated the ability of GH to activate PKC and ERK from fasted cells. These results indicate that INS and IGF-1 determine the signaling pathways activated by GH and whether or not a lipolytic response ensues. Such hormone-receptor-signal pathway linkages provide insight into the molecular basis of GH multifunctionality and into how cellular responses to GH can be adjusted to meet physiological (e.g., nutritional), developmental, or other conditions.

Environmental estrogens inhibit mRNA and functional expression of growth hormone receptors as well as growth hormone signaling pathways in vitro in rainbow trout (Oncorhynchus mykiss).

Fish in aquatic habitats are exposed to increasing concentrations and types of environmental contaminants, including environmental estrogens (EE). While there is growing evidence to support the observation that endocrine-disrupting compounds (EDCs) possess growth-inhibiting effects, the mechanisms by which these physiological effects occur are poorly understood. In this study, we examined the direct effects of EE, specifically 17ß-estradiol (E2), ß-sitosterol (ßS), and 4-n-nonylphenol (NP), on GH sensitivity as assessed by mRNA expression and functional expression of growth hormone receptor in hepatocytes, gill filaments, and muscle in rainbow trout (Oncorhynchus mykiss). Additionally, we examined the effects of EE on signaling cascades related to growth hormone signal transduction (i.e., JAK-STAT, MAPK, and PI3K-Akt). Environmental estrogens directly suppressed the expression of GHRs in a tissue- and compound-related manner. The potency and efficacy varied with EE; effects were most pronounced with E2 in liver. EE treatment deactivated the JAK-STAT, MAPK, and PI3K-Akt pathways in liver a time-, EE- and concentration-dependent manner. Generally, E2 and NP were most effective in deactivating pathway elements; maximum suppression for each pathway was rapid, typically occurring at 10-30min. The observed effects occurred via an estrogen-dependent pathway, as indicated by treatment with an ER antagonist, ICI 182,780. These findings suggest that EEs suppress growth by reducing GH sensitivity in terms of reduced GHR synthesis and reduced surface GHR expression and by repressing GH signaling pathways.

Nutritional state modulates growth hormone-stimulated lipolysis.

Growth hormone (GH) regulates several processes in vertebrates, including two metabolically disparate processes: promotion of growth, an anabolic action, and mobilization of stored lipid, a catabolic action. In this study, we used hepatocytes isolated from continuously fed and long-term (4weeks) fasted rainbow trout (Oncorhynchus mykiss) as a model to investigate the mechanistic basis of the anabolic and catabolic actions of GH. Our hypothesis was that nutritional state modulates the lipolytic responsiveness of cells by adjusting the signal transduction pathways to which GH links. GH stimulated lipolysis as measured by increased glycerol release in both a time- and concentration-related manner from cells of fasted fish but not from cells of fed fish. Expression of mRNAs that encode the lipolytic enzyme hormone-sensitive lipase (HSL), HSL1 and HSL2, also was stimulated by GH in cells from fasted fish and not in cells from fed fish. Activation of the signaling pathways that mediate GH action also was studied. In cells from fed fish, GH activated the JAK-STAT, PI3K-Akt, and ERK pathways, whereas in cells from fasted fish, GH activated the PLC/PKC and ERK pathways. In hepatocytes from fasted fish, blockade of PLC/PKC and of the ERK pathway inhibited GH-stimulated lipolysis and GH-stimulated HSL mRNA expression, whereas blockade of JAK-STAT or of the PI3K-Akt pathway had no effect on lipolysis or HSL expression stimulated by GH. These results indicate that during fasting GH activates the PLC/PKC and ERK pathways resulting in lipolysis but during periods of feeding GH activates a different complement of signal elements that do not promote lipolysis. These findings suggest that the responsiveness of cells to GH depends on the signal pathways to which GH links and helps resolve the growth-promoting and lipid catabolic actions of GH.

Environmental estrogens inhibit growth of rainbow trout (Oncorhynchus mykiss) by modulating the growth hormone-insulin-like growth factor system.

Although environmental estrogens (EE) have been found to disrupt a wide variety of developmental and reproductive processes in vertebrates, there is a paucity of information concerning their effects on organismal growth, particularly postembryonic growth. In this study, we exposed juvenile rainbow trout (Oncorhynchus mykiss) to 17ß-estradiol (E2) ß-sitosterol (ßS), or 4-n-nonylphenol (NP) to assess the effects of EE on overall organismal growth and on the growth hormone-insulin-like-growth factor (GH-IGF) system. EE treatment significantly reduced food conversion, body condition, and body growth. EE-inhibited growth resulted from alterations in peripheral elements of the GH-IGF system, which includes multiple GH receptors (GHRs), IGFs, and IGF receptors (IGFRs). In general, E2, ßS, and NP reduced the expression of GHRs, IGFs, and IGFRs; however, the effects varied in an EE-, tissue-, element type-specific manner. For example, in liver, E2 was more efficacious than either ßS, and NP in reducing GHR expression, and the effect of E2 was greater on GHR 1 than GHR2 mRNA. By contrast, in gill, all EEs affected GHR expression in a similar manner and there was no difference in the effect on GHR1 and GHR 2 mRNA. With regard to IGF expression, all EEs reduced hepatic IGF1 and IGF2 mRNA levels, whereas as in gill, only E2 and NP significantly reduced IGF1 and IGF2 expression. Lastly, E2 and NP reduced the expression of IGFR1A and IGFR1B mRNA expression similarly in gill and red and white muscle, whereas ßS had no effect on expression of IGFR mRNAs. These findings indicate that EEs disrupt post-embryonic growth by reducing GH sensitivity, IGF production, and IGF sensitivity.

Telomere length is longer following diapause in two solitary bee species.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Evolutionary origin and divergence of the growth hormone receptor family: insight from studies on sea lamprey.

Sea lamprey, one of the oldest extant lineages of vertebrates, Agnatha, was used to clarify the evolutionary origin and divergence of the growth hormone receptor (GHR) family. A single full-length cDNA encoding a protein that shares amino acid identity with GHRs and prolactin receptors (PRLRs) previously characterized from teleost fish was identified. Expression of the GHR/PRLR-like transcript was widespread among tissues, including brain, pituitary, heart, liver, and skeletal muscle, which is consistent with the broad physiological roles of GH-family peptides. Phylogenetic analysis suggests that the lamprey possess an ancestral gene encoding a common GHR/PRLR that diverged to give rise to distinct GHRs and PRLRs later in the course of vertebrate evolution. After the divergence of the Actinopterygian and Sarcopterygian lineages, the GHR gene was duplicated in the Actinopterygian lineage during the fish-specific genome duplication event giving rise to two GHRs in teleosts, type 1 GHR and type 2 GHR. A single GHR gene orthologous to the teleost type 1 GHR persisted in the Sarcopterygian lineage, including the common ancestor of tetrapods. Within the teleosts, several subsequent independent duplication events occurred that led to several GHR subtypes. A revised nomenclature for vertebrate GHRs is proposed that represents the evolutionary history of the receptor family. Structural features of the receptor influence ligand binding, receptor dimerization, linkage to signal effector pathways, and, ultimately, hormone function.

Nutrition-regulated lipolysis in rainbow trout (Oncorhynchus mykiss) is associated with alterations in the ERK, PI3K-Akt, JAK-STAT, and PKC signaling pathways.

Previous studies have shown that food deprivation, which occurs naturally in the life cycle of many species of fish, results in cessation of growth and catabolism of stored energy reserves, including lipids. In this study, we used rainbow trout (Oncorhynchus mykiss) to identify the cellular mechanisms involved with this metabolic shift. Fish were placed on one of five dietary regimes--fed continuously for 2 or 4 weeks, fasted continuously for 2 or 4 weeks, or fasted 2 weeks then refed 2 weeks--and the effects on organismal growth and lipid catabolism and on the activation state of signaling elements (e.g., Akt, ERK, JAK-STAT, PKC) in selected tissues were measured. Fasting for either 2 or 4 weeks significantly retarded growth in terms of body weight, body length, and body condition; refeeding restored growth such that body length and body condition were similar to measures seen in continuously fed fish. Fasting activated lipid catabolism by stimulating the mRNA expression and catalytic activity of hormone-sensitive lipase (HSL). Two HSL-encoding mRNAs have been characterized, and the expression of both forms of mRNA in 2- and 4-week fasted fish were significantly elevated over levels in fed fish in all tissues. In adipose tissue, liver, and white muscle, HSL activity was significantly elevated in 2- and 4-week fasted fish compared to fed animals; whereas in red muscle, HSL activity was significantly elevated compared to fed fish after 4 weeks of fasting. Refeeding reversed both fasting-associated HSL mRNA expression and HSL activity. Fasting resulted in the deactivation of Akt, JAK2, and STAT5 in adipose tissue, liver, and red and white muscle. By contrast, fasting activated ERK and PKC in all tissues measured. Refeeding reversed fasting-associated alterations in the activation state of all signal elements. These findings suggest that deactivation of Akt and JAK-STAT in conjunction with activation of ERK and PKC underlie fasting-associated growth retardation and lipolysis.

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT.

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.

Rainbow trout (Oncorhynchus mykiss) possess two hormone-sensitive lipase-encoding mRNAs that are differentially expressed and independently regulated by nutritional state.

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogenetic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.

Isolation, characterization, and distribution of somatostatin receptor subtype 2 (SSTR 2) mRNA in rainbow trout (Oncorhynchus mykiss), and regulation of its expression by glucose.

In this study, cDNA for a somatostatin receptor variant (somatostatin receptor subtype 2, SSTR 2) was isolated, cloned, and sequenced from rainbow trout. A 1821-nt cDNA was isolated and found to contain a single initiation site 387-nt from the most 5' end, an open reading frame of 1116-nt, and a single putative polyadenylation site 189-nt from the most 3' end. The encoded protein contains 372 amino acids and contains seven membrane-spanning domains. Based on structural analysis, the protein was identified as a subtype 2 SSTR. These data support the emergence of a multigenic SSTR family early in the course of vertebrate evolution, concomitant with or perhaps prior to the divergence of boney fish. The distribution of SSTR 2 mRNA in tissues was determined by quantitative real time-PCR (QRT-PCR). SSTR 2 was most abundant in the brain (where it was detected in the telencephalon, optic tectum, and hypothalamus), skeletal muscle, and liver, but it also was present in the endocrine pancreas (Brockmann body) and various regions of the gastrointestinal tract (esophagus, stomach, intestine). SSTR 2 mRNA was most abundant in the brain, muscle, and liver. In vitro the Brockmann body and liver with increasing concentrations of glucose (1, 4, 10mM) resulted in increased expression of SSTR 2 mRNA. These findings contribute to the understanding of the evolution of the SSTR family and provide insight into the roles of SSTR 2 in fish.

No evidence of physiological declines with age in an extremely long-lived fish.

Although the pace of senescence varies considerably, the physiological systems that contribute to different patterns of senescence are not well understood, especially in long-lived vertebrates. Long-lived bony fish (i.e., Class Osteichthyes) are a particularly useful model for studies of senescence because they can readily be aged and exhibit some of the longest lifespans among vertebrates. In this study we examined the potential relationship between age and multiple physiological systems including: stress levels, immune function, and telomere length in individuals ranging in age from 2 to 99 years old in bigmouth buffalo (Ictiobus cyprinellus), the oldest known freshwater teleost fish. Contrary to expectation, we did not find any evidence for age-related declines in these physiological systems. Instead, older fish appeared to be less stressed and had greater immunity than younger fish, suggesting age-related improvements rather than declines in these systems. There was no significant effect of age on telomeres, but individuals that may be more stressed had shorter telomeres. Taken together, these findings suggest that bigmouth buffalo exhibit negligible senescence in multiple physiological systems despite living for nearly a century.

TA-65 does not increase telomere length during post-natal development in house sparrow chicks (Passer domesticus).

Telomeres, protective caps at the end of chromosomes, are often positively related to lifespan and are thought to be an important mechanism of organismal aging. To better understand the casual relationships between telomere length and longevity, it is essential to be able to experimentally manipulate telomere dynamics (length and loss rate). Previous studies suggest that exposure to TA-65, an extract from the Chinese root Astragalus membranaceus, activates telomerase, lengthens telomeres, increases the growth of keratin-based structures, and boosts the immune system in adults. However, telomere loss is expected to be greatest during early life but whether TA-65 has similar effects during this life stage is currently unknown. Here, we experimentally exposed free-living house sparrow (Passer domesticus) chicks to TA-65 during post-natal development and examined the effects on telomere length and loss, growth of keratin-based structures, and a measure of cellular immunity. Contrary to expectation, the growth of keratin-based structures was reduced in TA-65 chicks and in the second year of the study, chicks exposed to TA-65 experienced more telomere loss than controls. Thus, the effects of TA-65 on telomeres and keratin-based structures differ across life stages and future research will be necessary to determine the mechanisms underlying these age-specific effects.

Somatostatin inhibits basal and growth hormone-stimulated hepatic insulin-like growth factor-I production.

Growth of vertebrates is controlled by the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system, and somatostatins (SSs) have been shown to inhibit growth by reducing the release of growth hormone (GH) from the pituitary. In this study, we used rainbow trout to assess the effects of SSs on the production of IGF-I. Somatostatin-14 (SS-14-I) implantation for 15 days significantly reduced steady-state levels of IGF-I mRNA in liver and lowered IGF-I concentration in plasma compared to control animals. The direct effects of SSs were examined on hepatocytes incubated in vitro. SS-14-I inhibited basal and GH-stimulated IGF-I mRNA expression. SS-14-I inhibition of GH-stimulated IGF-I expression was concentration- and time-dependent; the ED(50) was ca. 40 ng/ml and the maximum response was observed after 6h. All SS isofoms tested, including the N-terminally extended form of SS-14-I, SS-28-I, and the [Tyr(7), Gly(10)]-substituted forms of SS, SS-14-II, SS-25-II and SS-28-II, inhibited GH-stimulated IGF-I mRNA expression. The inhibitory effects of SS-14-I on steady-state levels of IGF-I mRNA resulted from reduced IGF-I mRNA transcription and not from altered mRNA stability. SS-14-I also reduced basal and GH-stimulated release of IGF-I into culture medium. These results indicate that SSs regulate growth in an extrapituitary manner by reducing hepatic IGF-I biosynthesis and secretion.

The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and ß in the tobacco hornworm, Manduca sexta.

As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-ß dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. SUMMARY STATEMENT: As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar.

Telomere correlations during early life in a long-lived seabird.

Telomere dynamics in blood cells have been linked to aging in a variety of organisms. However, whether blood telomeres are correlated with telomeres in other parts of the body is not well known, especially during early life when telomere loss is expected to be most rapid. We investigated this question in Franklin's gulls (Leucophaeus pipixcan) by measuring telomere lengths in blood and several other tissues including: heart, liver, and skeletal muscle at the end of embryonic (n=31) and post-natal development (n=20). In late-stage embryos, blood telomeres were significantly positively correlated with heart and skeletal muscle, but not liver telomeres. However, at the end of post-natal development, there were no significant correlations among blood telomeres and telomeres in any other tissues. In late-stage embryos, heart telomeres were significantly longer than blood, liver, and skeletal muscle telomeres, but at the end of post-natal development telomere lengths did not significantly differ among tissues. These results suggest that blood telomere length is not necessarily indicative of other tissues at all stages of development and highlights the importance of understanding any functional consequences of tissue specific telomere dynamics in early life.

Regulation of pancreatic somatostatin gene expression by insulin and glucagon.

Rainbow trout were used as a model system to study the effects of insulin and glucagon on the expression of preprosomatostatins (PPSS). We previously showed that the endocrine pancreas of trout contains three mRNAs that encode for distinct somatostatin-containing peptides: PPSS I, which contains somatostain-14 (SS-14) at its C-terminus, and two separate PPSS IIs, PPSS II' and PPSS II'', each containing [Tyr7, Gly10]-SS-14 at their C-terminus. Rainbow trout injected (100 ng/g body weight) with insulin displayed elevated expression of PPSS II' and PPSS II'' mRNAs. Glucagon-injected (100 ng/g body weight) animals displayed elevated pancreatic expression of all PPSS mRNAs compared to saline-injected control animals. Insulin directly stimulated the expression of pancreatic PPSS II' and PPSS II'' mRNAs in vitro in a dose-dependent manner in the presence of 4mM glucose. Glucagon, in the presence of 10mM glucose, directly stimulated the expression of all PPSS mRNAs in a dose-dependent manner in vitro. These results indicate that the pancreatic expression of PPSS mRNAs is differentially regulated by insulin and glucagon and that the regulatory pattern is dependent on glucose concentration.