Marta Conde-Sieira

Effect of different glycaemic conditions on gene expression of neuropeptides involved in control of food intake in rainbow trout; interaction with stress

SUMMARY To assess mechanisms relating to food intake and glucosensing in fish, and their interaction with stress, we evaluated changes in the expression of orexigenic (NPY) and anorexigenic (POMC, CART and CRF) peptides in central glucosensing areas (hypothalamus and hindbrain) of rainbow trout subjected to normoglycaemic (control), hypoglycaemic (4 mg insulin kg–1) or hyperglycaemic (500 mg glucose kg–1) conditions for 6 h under normal stocking density (NSD; 10 kg fish mass m–3) or under stress conditions induced by high stocking density (HSD; 70 kg fish mass m–3). Hyperglycaemic NSD conditions resulted in decreased mRNA levels of NPY and increased levels of CART and POMC in the hypothalamus as well as increased mRNA levels of CART and CRF in the hindbrain compared with hypo- and normoglycaemic conditions. HSD conditions in normoglycaemic fish induced marked changes in the expression of all peptides assessed: mRNA levels of NPY and CRF increased and mRNA levels of POMC and CART decreased in the hypothalamus, whereas the expression of all four peptides (NPY, POMC, CART and CRF) decreased in the hindbrain. Furthermore, HSD conditions altered the response to changes in glycaemia of NPY and POMC expression in the hypothalamus and CART expression in the hypothalamus and the hindbrain. The results are discussed in the context of food intake regulation by glucosensor systems and their interaction with stress in fish.

Central leptin treatment modulates brain glucosensing function and peripheral energy metabolism of rainbow trout.

The aim of the present study was to obtain evidence for the possible modulatory effect of leptin on glucosensing capacity in hypothalamus and hindbrain of rainbow trout. In a first experiment, trout were injected ICV with saline alone or containing increased doses of leptin (0.3-30 microg microl(-1)). Leptin induced in general in both hypothalamus and hindbrain dose-dependent changes in parameters related to glucosensing (increased glycogenic and glycolytic potentials together with increased GK activity, and increased mRNA levels of genes involved in glucosensing response) compatible with those occurring under hyperglycemic conditions, a situation that is known to produce anorexia. The anorectic action of leptin in our experimental conditions was observed in a second experiment. The specificity of leptin action was tested in a third experiment in which trout were injected ICV with saline, or leptin alone, or leptin plus agents known to inhibit leptin signaling pathways in mammals. The results obtained suggest that the central action of leptin on glucosensing system can be related to the JAK/STAT and IRS-PI(3)K pathways. Finally, we also provide evidence for a peripheral effect of central leptin treatment (increased liver glycogenolytic potential), which could be associated with increased sympathetic activity.

Stress alters food intake and glucosensing response in hypothalamus, hindbrain, liver, and Brockmann bodies of rainbow trout

In fish food intake is altered under stress conditions, and in a fish teleost model like rainbow trout food intake is associated with the activity of the glucosensor systems. Thus, we aimed to evaluate the possible interaction of stress with the response of glucosensor mechanisms in rainbow trout. Thus, we subjected rainbow trout (via intraperitoneal injections) to normoglycaemic (control), hypoglycaemic (4 mg.kg(-1) bovine insulin) or hyperglycaemic (500 mg.kg(-1) glucose body mass) conditions for 5 days under normal stocking density (NSD, 10 kg fish mass·m(-3)) or stress conditions induced by high stocking density (HSD, 70 kg fish mass·m(-3)). The experimental design was appropriate since hypoglycemia and hyperglycemia were observed in fish under NSD whereas in normoglycaemic fish HSD induced changes in stress-related parameters similar to those reported in fish literature, such as increased levels of cortisol and glucose in plasma and decreased levels of glycogen in liver. Food intake did not respond to changes in plasma glucose levels in fish under HSD conditions, in contrast with the decreased food intake observed when glucose levels increased in fish under NSD conditions. Moreover, the changes with the increase in plasma glucose levels in parameters involved in glucosensing in liver, Brockmann bodies (BB), hypothalamus, and hindbrain of fish in NSD either disappeared (DHAP and GAP levels, and GK, PK, and GPase activities in liver; glucose, DHAP and GAP levels in BB; glucose and DHAP levels, and GK and PK activities in hypothalamus; glycogen and DHAP levels, and GSase activity in hindbrain) or changed (cortisol levels in plasma; glycogen and GAP levels, and GSase and FBPase activities in liver; GK and PK activities in BB; GK and PK activities in hindbrain) in fish under HSD. Those changes suggest for the first time in fish the existence of an interaction between glucosensing capacity and stress. The readjustment in the activity of glucosensor systems is also associated with changes in food intake resulting in an inability of the fish to compensate with changes in food intake those of circulating glucose levels as observed in fish under non-stressed conditions.

Daily Rhythmic Expression Patterns of Clock1a, Bmal1, and Per1 Genes in Retina and Hypothalamus of the Rainbow Trout, Oncorhynchus Mykiss

Living organisms show daily rhythms in physiology, behavior, and gene expression, which are due to the presence of endogenous clocks that synchronize biological processes to the 24-h light/dark cycle. In metazoans, generation of circadian rhythmicity is a consequence of specialized tissues known as “master clocks,” having different locations among species. A few studies have described clock-gene expression in fish neural tissues, but none of them assessed clock-gene expression in different discrete regions. The present study was designed to explore the presence/absence of circadian clock-gene expression in the rainbow trout (Oncorhynchus mykiss) retina and hypothalamus. Juvenile fish were acclimated to a 12:12 light (L)-dark (D) cycle. Then, retina and hypothalamus were collected from animals kept under LD conditions or constant darkness (DD) for 24 h. Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) assays were performed to quantify expression of the core circadian genes Clock1a, Bmal1, and Per1 as representative members of the circadian oscillator. All clock genes analyzed in the retina and hypothalamus showed circadian fluctuations. However, gene expression peaked in the rainbow trout hypothalamus with a 3-h (Clock1a and Bmal1) or 6-h (Per1) delay relative to that observed in the retina, the latter showing highest expression levels at zeitgeber times 9 (ZT9) for Clock1a and Bmal1, and at ZT21 for Per1. When exposed to DD, the rhythmic gene expression pattern was maintained for all genes in the rainbow trout retina, but only for Clock1a and Per1 in the hypothalamus. Bmal1 failed to cycle under DD, suggesting that hypothalamic clock function might depend on either several clock-gene isoforms or regulation from external inputs. Overall, these data indicate that representative molecular members of the core circadian clock are present in both the retina and hypothalamus of rainbow trout. (Author correspondence: jmmiguez@uvigo.es)

ACTH-stimulated cortisol release from head kidney of rainbow trout is modulated by glucose concentration

SUMMARY To assess the hypothesis that cortisol release in rainbow trout is modulated by glucose levels, we first evaluated cortisol release [basal and adrenocorticotropic hormone (ACTH)-regulated] by head kidney tissue superfused with medium reflecting hypoglycaemic, normoglycaemic or hyperglycaemic conditions. Next, cortisol release from head kidney fragments in static incubations was assessed in parallel with changes in parameters related to cortisol synthesis (mRNA abundance of StAR, P450scc, 3βHSD and 11βH) and the GK-mediated glucosensing mechanism (levels of glycogen and glucose, activities of GK, GSase and PK, and mRNA levels of GK, GLUT-2, Kir6.x-like and SUR-like). We then evaluated the effects of two inhibitors of glucose transport, cytochalasin B and phlorizin, on cortisol production and glucosensing mechanisms. The ACTH-induced release of cortisol proved to be modulated by glucose concentration such that increased release occurs under high glucose levels, and decreased ACTH-stimulated cortisol release occurs when glucose transport is inhibited by cytochalasin B. The release of cortisol can be associated with increased synthesis as enhanced mRNA abundance of genes related to cortisol synthesis was also noted in high glucose medium. Specific GK immunoreactivity in the cortisol-producing cells (not in chromaffin cells) further substantiates GK-mediated glucosensing in cortisol production. In contrast, no changes compatible with those of glucose levels and cortisol release/synthesis in the presence of ACTH were noted for any other putative glucosensor mechanisms based on LXR, SGLT-1 or Gnat3. These combined results are the first evidence for a mechanism in fish linking the synthesis and release of a non-pancreatic hormone like cortisol with circulating glucose levels. The relationship was evident for the regulated (ACTH-dependent) pathway and this suggests that under acute stress conditions glucose is important for the regulation of cortisol synthesis and release.

Nutrient Sensing Systems in Fish: Impact on Food Intake Regulation and Energy Homeostasis

Evidence obtained in recent years in a few species, especially rainbow trout, supports the presence in fish of nutrient sensing mechanisms. Glucosensing capacity is present in central (hypothalamus and hindbrain) and peripheral [liver, Brockmann bodies (BB, main accumulation of pancreatic endocrine cells in several fish species), and intestine] locations whereas fatty acid sensors seem to be present in hypothalamus, liver and BB. Glucose and fatty acid sensing capacities relate to food intake regulation and metabolism in fish. Hypothalamus is as a signaling integratory center in a way that detection of increased levels of nutrients result in food intake inhibition through changes in the expression of anorexigenic and orexigenic neuropeptides. Moreover, central nutrient sensing modulates functions in the periphery since they elicit changes in hepatic metabolism as well as in hormone secretion to counter-regulate changes in nutrient levels detected in the CNS. At peripheral level, the direct nutrient detection in liver has a crucial role in homeostatic control of glucose and fatty acid whereas in BB and intestine nutrient sensing is probably involved in regulation of hormone secretion from endocrine cells.

Hypothalamic fatty acid sensing in Senegalese sole (Solea senegalensis):response to long-chain saturated, monounsaturated, and polyunsaturated (n-3) fatty acids

We assessed the presence of fatty acid (FA)-sensing mechanisms in hypothalamus of Senegalese sole (Solea senegalensis) and investigated their sensitivity to FA chain length and/or level of unsaturation. Stearate (SA, saturated FA), oleate (OA, monounsaturated FA of the same chain length), α-linolenate [ALA, a n-3 polyunsaturated fatty acid (PUFA) of the same chain length], and eicosapentanoate (EPA, a n-3 PUFA of a larger chain length) were injected intraperitoneally. Parameters related to FA sensing and neuropeptide expression in the hypothalamus were assessed after 3 h and changes in accumulated food intake after 4, 24, and 48 h. Three FA sensing systems characterized in rainbow trout were also found in Senegalese sole and were activated by OA in a way similar to that previously characterized in rainbow trout and mammals. These hypothalamic FA sensing systems were also activated by ALA, differing from mammals, where n-3 PUFAs do not seem to activate FA sensors. This might suggest additional roles and highlights the importance of n-3 PUFA in fish diets, especially in marine species. The activation of FA sensing seems to be partially dependent on acyl chain length and degree of saturation, as no major changes were observed after treating fish with SA or EPA. The activation of FA sensing systems by OA and ALA, but not SA or EPA, is further reflected in the expression of hypothalamic neuropeptides involved in the control of food intake. Both OA and ALA enhanced anorexigenic capacity compatible with the activation of FA sensing systems.

CRF treatment induces a readjustment in glucosensing capacity in the hypothalamus and hindbrain of rainbow trout

SUMMARY Stress conditions induced in rainbow trout a readjustment in the glucosensing response of the hypothalamus and hindbrain such that those sensors did not respond properly to changes in glucose levels, as demonstrated in previous studies. To evaluate the hypothesis that corticotropin-releasing factor (CRF) could be involved in that response, we have incubated the hypothalamus and hindbrain of rainbow trout at different glucose concentrations in the presence of different concentrations of CRF. Under those conditions, we evaluated whether parameters related to glucosensing [the levels of glucose, glycogen and glucose 6-phosphate, the activities of glucokinase (GK), glycogen synthase (GSase) and pyruvate kinase (PK), and mRNA abundance of transcripts for GK, Glut2, Kir.6-like and sulfonylurea receptor (SUR)-like] are modified in the presence of CRF in a way comparable to that observed under stress conditions. We obtained evidence allowing us to suggest that CRF could be involved in the interaction between stress and glucosensing as CRF treatment of the hypothalamus and hindbrain in vitro induced a readjustment in glucosensing parameters similar to that previously observed under stress conditions in vivo. We had also previously demonstrated that stress elicits alterations in food intake in parallel with the readjustment of glucosensing systems. Here, we provide evidence that the mRNA abundance of several of the neuropeptides involved in the regulation of food intake, such as neuropeptide Y (NPY) or cocaineand amphetamine-regulated transcript (CART), is affected by CRF treatment, in such a way that their expression does not respond to changes in glucose levels in the same way as controls, allowing us to suggest that the food intake response that is integrated by changes in those peptides and known to be reduced by stress could be also mediated by CRF action in glucosensing areas.

Is plasma cortisol response to stress in rainbow trout regulated by catecholamine-induced hyperglycemia?

Based on previous studies we hypothesize that under stress conditions catecholamine-induced hyperglycemia contributes to enhance cortisol production in head kidney of rainbow trout. Therefore, treatment with propranolol (β-adrenoceptor blocker) should reduce the hyperglycemia elicited by stress and, therefore, we expected reduced glucosensing response and cortisol production in head kidney. Propranolol treatment was effective in blocking most of the effects of catecholamines in liver energy metabolism resulting in a lower glycemia in stressed fish. The decreased glycemia of stressed fish treated with propranolol was observed along with reduced transcription of genes involved in the cortisol synthetic pathway, which supports our hypothesis. However, changes in putative glucosensing parameters assessed in head kidney were scarce and in general did not follow changes noted in glucose levels in plasma. Furthermore, circulating cortisol levels did not change in parallel with changes in glycemia. As a whole, the present results suggest that glycemia could participate in the regulation of cortisol synthetic pathways but other factors are also likely involved. Propranolol effects on trout stress response were different depending on time passed after stress onset; the direct or indirect involvement of catecholaminergic response in the regulation of cortisol production and release deserves further investigation.

Melatonin treatment alters glucosensing capacity and mRNA expression levels of peptides related to food intake control in rainbow trout hypothalamus.

As demonstrated in previous studies, the functioning of brain glucosensing systems in rainbow trout is altered under stress conditions in a way that they are unable to respond properly to changes in glucose levels. Melatonin has been postulated as necessary for homeostatic control of energy metabolism in several vertebrate groups, and in fish it has been suggested as an anti-stress molecule. To evaluate the possible effects of melatonin on glucosensing, we have incubated hypothalamus and hindbrains of rainbow trout at different glucose concentrations in the presence of increased doses (0.01, 1, and 100nM) of melatonin assessing whether or not the responses to changes in glucose levels of parameters related to glucosensing (glucose, glycogen and glucose 6-phosphate levels, activities of GK, GSase and PK, and mRNA content of GK, GLUT2, Kir6.x-like, and SUR-like) are modified in the presence of melatonin. While no effects of melatonin were observed in hindbrain, in hypothalamus melatonin treatment up-regulated glucosensing parameters, especially under hypo- and normo-glycaemic conditions. The effects of melatonin in hypothalamus occurred apparently through MT(1) receptors since most effects were counteracted by the presence of luzindole but not by the presence of 4-P-PDOT. Moreover, melatonin treatment induced in hypothalamus increased mRNA expression levels of NPY and decreased mRNA levels of POMC, CART, and CRF. A role of the hormone in daily re-adjustment of hypothalamic glucosensor machinery is discussed.