Cristina Otero-Rodiño

Central administration of oleate or octanoate activates hypothalamic fatty acid sensing and inhibits food intake in rainbow trout

If levels of fatty acids like oleate and octanoate are directly sensed through different fatty acid (FA) sensing systems in hypothalamus of rainbow trout, intracerebroventricular (ICV) administration of FA should elicit effects similar to those previously observed after intraperitoneal (IP) treatment. Accordingly, we observed after ICV treatment with oleate or octanoate decreased food intake accompanied in hypothalamus by reduced potential of lipogenesis and FA oxidation, and decreased potential of ATP-dependent inward rectifier potassium channel (K(+)ATP). Those changes support direct FA sensing through mechanisms related to FA metabolism and mitochondrial activity. The FA sensing through binding to FAT/CD36 and subsequent expression of transcription factors appears to be also direct but an interaction with peripheral hormones cannot be rejected. Moreover, decreased expression of NPY and increased expression of POMC were observed in parallel with the activation of FA sensing systems and decreased food intake. These results allow us to suggest the involvement of at least these peptides in controlling the decreased food intake noted after oleate and octanoate treatment in rainbow trout.

Ghrelin modulates hypothalamic fatty acid-sensing and control of food intake in rainbow trout.

There is no information available on fish as far as the possible effects of ghrelin on hypothalamic fatty acid metabolism and the response of fatty acid-sensing systems, which are involved in the control of food intake. Therefore, we assessed in rainbow trout the response of food intake, hypothalamic fatty acid-sensing mechanisms and expression of neuropeptides involved in the control of food intake to the central treatment of ghrelin in the presence or absence of a long-chain fatty acid such as oleate. We observed that the orexigenic actions of ghrelin in rainbow trout are associated with changes in fatty acid metabolism in the hypothalamus and an inhibition of fatty acid-sensing mechanisms, which ultimately lead to changes in the expression of anorexigenic and orexigenic peptides resulting in increased orexigenic potential and food intake. Moreover, the response to increased levels of oleate of hypothalamic fatty acid-sensing systems (activation), expression of neuropeptides (enhanced anorexigenic potential) and food intake (decrease) were counteracted by the simultaneous treatment with ghrelin. These changes provide evidence for the first time in fish of a possible modulatory role of ghrelin on the metabolic regulation by fatty acid of food intake occurring in the hypothalamus.

Evidence for the Presence of Glucosensor Mechanisms Not Dependent on Glucokinase in Hypothalamus and Hindbrain of Rainbow Trout (Oncorhynchus mykiss).

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6h after intraperitoneal injection of 5 mL.Kg-1 of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin.Kg-1 body mass), or D-glucose (hyperglycaemic treatment, 500 mg.Kg-1 body mass). Half of tanks were kept at a 10 Kg fish mass.m-3 and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass.m-3) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.

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.

Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss)

We hypothesize that ceramides are involved in the regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with C6:0 ceramide on food intake. In a second experiment, we assessed the effects in brain areas of ceramide treatment on neuropeptide expression, fatty acid-sensing systems, and cellular signaling pathways. Ceramide treatment induced a decrease in food intake, a response opposed to the orexigenic effect described in mammals, which can be related to enhanced mRNA abundance of cocaine and amphetamine-related transcript and proopiomelanocortin and decreased mRNA abundance of Agouti-related protein and neuropeptide Y. Fatty acid-sensing systems appear to be inactivated by ceramide treatment. The mRNA abundance of integrative sensors AMPK and sirtuin 1, and the phosphorylation status of cellular signaling pathways dependent on protein kinase B, AMPK, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FoxO1) are generally activated by ceramide treatment. However, there are differences between hypothalamus and hindbrain in the phosphorylation status of AMPK (decreased in hypothalamus and increased in hindbrain), mTOR (decreased in hypothalamus and increased in hindbrain), and FoxO1 (increased in hypothalamus and decreased in hindbrain) to ceramide treatment. The results suggest that ceramides are involved in the regulation of food intake in rainbow trout through mechanisms comparable to those characterized previously in mammals in some cases.

Daily rhythms in activity and mRNA abundance of enzymes involved in glucose and lipid metabolism in liver of rainbow trout, Oncorhynchus mykiss. Influence of light and food availability

The present research aimed to investigate in a model of teleost fish (rainbow trout) the existence of daily changes in activity and mRNA abundance of several proteins involved in major pathways of carbohydrate and lipid metabolism in liver, and to test whether or not both the light–dark cycle and food availability might influence such rhythms. For this purpose, four cohorts of animals previously adapted to normal housing conditions (12L:12D; Lights on at ZT0; feeding time at ZT2) were subjected to: normal conditions (LD); 48-h constant darkness (DD); 96-h food deprivation (LD + Fasting); or constant darkness and food deprivation (DD + Fasting) respectively. After such time periods, fish were sacrificed and sampled every 4-h on the following 24-h period (ZT/CT0, 4, 8, 12, 16, 20 and 0′). Our results reveal that cortisol and all the analysed genes (gk, pepck, g6pase, pk, glut2, hoad and fas) exhibited well defined daily rhythms, which persisted even in the absence of light and/or food indicating the endogenous nature of such rhythms. Even when the variations of enzyme activities were not significant, their rhythms mostly paralleled those of the respective gene expression. The rhythms of mRNA abundance were apparently dependent on the presence of food, but the light/dark cycle also influenced such rhythms. Since cortisol does not appear to be mainly involved in generating such daily rhythms in liver, alternative mechanisms might be involved, such as a direct interaction between metabolism and the circadian system.

Changes in the levels and phosphorylation status of Akt, AMPK, CREB, and FoxO1 in hypothalamus of rainbow trout under conditions of enhanced glucosensing activity

ABSTRACT There is no available information about mechanisms linking glucosensing activation in fish and changes in the expression of brain neuropeptides controlling food intake. Therefore, we assessed in rainbow trout hypothalamus the effects of raised levels of glucose on the levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking these processes. We also aimed to assess the changes in the levels and phosphorylation status of two proteins possibly involved in the modulation of these transcription factors: Akt and AMPK. Therefore, in pooled preparations of hypothalamus incubated for 3 and 6 h in the presence of 2, 4 or 8 mmol l−1 d-glucose, we evaluated the response of parameters related to glucosensing mechanisms, neuropeptide expression and levels and phosphorylation status of the proteins of interest. The activation of hypothalamic glucosensing systems and the concomitant enhanced anorectic potential occurred in parallel with activation of Akt and inhibition of AMPK. The changes in these proteins relate to neuropeptide expression through changes in the level and phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses to increased glucose. Summary: The activation of glucosensing systems in rainbow trout hypothalamus leading to changes in the regulation of food intake occurs in parallel with changes in the abundance and phosphorylation status of Akt, AMPK, CREB and FoxO1.

Intracerebroventricular ghrelin treatment affects lipid metabolism in liver of rainbow trout (Oncorhynchus mykiss).

We aimed to elucidate in rainbow trout (Oncorhynchus mykiss) the effects of central ghrelin (GHRL) treatment on the regulation of liver lipid metabolism, and the possible modulatory effect of central GHRL treatment on the simultaneous effects of raised levels of oleate. Thus, we injected intracerebroventricularly (ICV) rainbow trout GHRL in the presence or absence of oleate and evaluated in liver variables related to lipid metabolism. Oleate treatment elicited in liver of rainbow trout decreased lipogenesis and increased oxidative capacity in agreement with previous studies. Moreover, as demonstrated for the first time in fish in the present study, GHRL also acts centrally modulating lipid metabolism in liver, resulting in increased potential for lipogenesis and decreased potential for fatty acid oxidation, i.e. the converse effects to those elicited by central oleate treatment. The simultaneous treatment of GHRL and oleate confirmed these counteractive effects. Thus, the nutrient sensing mechanisms present in hypothalamus, particularly those involved in sensing of fatty acid, are involved in the control of liver energy metabolism in fish, and this control is modulated by the central action of GHRL. These results give support to the notion of hypothalamus as an integrative place for the regulation of peripheral energy metabolism in fish.

Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus–pituitary–interrenal (HPI) axis

We previously demonstrated in rainbow trout that the decrease in circulating levels of fatty acid (FA) induced by treating fish with SDZ WAG 994 (SDZ) induced a counter-regulatory response in which the activation of the hypothalamus–pituitary–interrenal (HPI, equivalent to mammalian hypothalamus–pituitary–adrenal) axis was likely involved. This activation, probably not related to the control of food intake through FA sensor systems but to the modulation of lipolysis in peripheral tissues, liver and Brockmann bodies (BB, the main site of pancreatic endocrine cells in fish), would target the restoration of FA levels in plasma. To assess this hypothesis, we lowered circulating FA levels by treating fish with SDZ alone, or SDZ in the presence of metyrapone (an inhibitor of cortisol synthesis). In liver, the changes observed were not compatible with a direct FA-sensing response but with a stress response, which allows us to suggest that the detection of a FA decrease in the hypothalamus elicits a counter-regulatory response in liver, resulting in an activation of lipolysis to restore FA levels in plasma. The activation of these metabolic changes in liver could be attributable to the activation of the HPI axis and/or to the action of sympathetic pathways. In contrast, in BB, changes in circulating FA levels induce changes in several parameters compatible with the function of FA-sensing systems informing about the decrease in circulating FA levels.

Glucosensing in liver and Brockmann bodies of rainbow trout through glucokinase-independent mechanisms☆

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) are present in the liver and Brockmann bodies (BB) of rainbow trout, and are affected by stress. We evaluated in these tissues changes in parameters related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and SGLT-1 6h after intraperitoneal injection of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment), or d-glucose (hyperglycaemic treatment). Half of tanks were kept at normal stocking density (NSD; 10kgfishmass·m(-3)) whereas the remaining tanks were kept at high stocking density (HSD; 70kgfishmass·m(-3)). The results provide for the first time in fish evidence for the presence of putative glucosensor systems based on mitochondrial activity and sweet taste receptor in liver whereas in BB systems based on LXR, mitochondrial activity, sweet taste receptor, and SGLT-1 could be operative. We also obtained for the first time in fish evidence for the functioning of integrative metabolic sensors in response to changes in nutrient levels since changes in the mRNA abundance of sirtuin 1 (SIRT-1) were observed in response to increased glucose levels. The stress conditions elicited by HSD altered the response of the glucosensor systems based on mitochondrial activity, sweet taste receptor, and SGLT-1 in the liver, and LXR and SGLT-1 in the BB.