Marta Librán-Pérez

Evidence of a metabolic fatty acid-sensing system in the hypothalamus and Brockmann bodies of rainbow trout: implications in food intake regulation

Enhanced lipid levels inhibit food intake in fish but no studies have characterized the possible mechanisms involved. We hypothesize that the presence of fatty acid (FA)-sensing mechanisms could be related to the control of food intake. Accordingly, we evaluated in the hypothalamus, hindbrain, and Brockmann bodies (BB) of rainbow trout changes in parameters related to fatty acid metabolism, transport of FA, nuclear receptors, and transcription factors involved in lipid metabolism, and components of the K(ATP) channel after intraperitoneal administration of different doses of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). The increase in circulating LCFA or MCFA levels elicited an inhibition in food intake and induced in the hypothalamus a response compatible with fatty acid sensing in which fatty acid metabolism, binding to cluster of differentiation 36 (CD36), and mitochondrial activity are apparently involved, which is similar to that suggested in mammals except for the apparent capacity of rainbow trout to detect changes in MCFA levels. Changes in those hypothalamic pathways can be related to the control of food intake, since food intake was inhibited when FA metabolism was perturbed (using fatty acid synthase or acetyl-CoA carboxylase inhibitors) and changes in mRNA levels of specific neuropeptides such as neuropeptide Y and proopiomelancortin were also noticed. This response seems to be exclusive for the hypothalamus, since the other center controlling food intake (hindbrain) was unaffected by treatments. The results obtained in BB suggest that at least two of the components of a putative fatty acid-sensing system (based on fatty acid metabolism and binding to CD36) could be present. Therefore, the present study provides, for the first time in fish, evidence for a specific role for FA (MCFA and LCFA) as metabolic signals in hypothalamus and BB, where the detection of those FA can be associated with the control of food intake and hormone release.

Oleic Acid and Octanoic Acid Sensing Capacity in Rainbow Trout Oncorhynchus mykiss Is Direct in Hypothalamus and Brockmann Bodies

In a previous study, we provided evidence for the presence in hypothalamus and Brockmann bodies (BB) of rainbow trout Oncorhynchus mykiss of sensing systems responding to changes in levels of oleic acid (long-chain fatty acid, LCFA) or octanoic acid (medium-chain fatty acid, MCFA). Since those effects could be attributed to an indirect effect, in the present study, we evaluated in vitro if hypothalamus and BB respond to changes in FA in a way similar to that observed in vivo. In a first set of experiments, we evaluated in hypothalamus and BB exposed to increased oleic acic or octanoic acid concentrations changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, reactive oxygen species (ROS) effectors, components of the KATP channel, and (in hypothalamus) neuropeptides related to food intake. In a second set of experiments, we evaluated in hypothalamus the response of those parameters to oleic acid or octanoic acid in the presence of inhibitors of fatty acid sensing components. The responses observed in vitro in hypothalamus are comparable to those previously observed in vivo and specific inhibitors counteracted in many cases the effects of FA. These results support the capacity of rainbow trout hypothalamus to directly sense changes in MCFA or LCFA levels. In BB increased concentrations of oleic acid or octanoic acid induced changes that in general were comparable to those observed in hypothalamus supporting direct FA sensing in this tissue. However, those changes were not coincident with those observed in vivo allowing us to suggest that the FA sensing capacity of BB previously characterized in vivo is influenced by other neuroendocrine systems.

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.

Feeding rainbow trout with a lipid-enriched diet: effects on fatty acid sensing, regulation of food intake and cellular signaling pathways.

ABSTRACT Using rainbow trout fed with low-fat or high-fat diets, we aimed to determine whether the response of food intake, mRNA abundance of hypothalamic neuropeptides involved in the metabolic regulation of food intake and fatty acid sensing systems in the hypothalamus and liver are similar to results previously observed when levels of specific fatty acids were raised by injection. Moreover, we also aimed to determine if the phosphorylation state of intracellular energy sensor 5′-AMP-activated protein kinase (AMPK), and proteins involved in cellular signaling such as protein kinase B (Akt) and target of rapamycin (mTOR) display changes that could be related to fatty acid sensing and the control of food intake. The increased levels of fatty acids in the hypothalamus and liver of rainbow trout fed with a high-fat diet only partially activated fatty acid sensing systems and did not elicit changes in food intake, suggesting that the fatty acid sensing response in fish is more dependent on the presence of specific fatty acids, such as oleate or octanoate, rather than to the global increase in fatty acids. We also obtained, for the first time in fish, evidence for the presence and function of energy sensors such as AMPK and proteins involved in cellular signaling, like mTOR and Akt, in the hypothalamus. These proteins in the hypothalamus and liver were generally activated in fish fed the high-fat versus low-fat diet, suggesting that cellular signaling pathways are activated in response to the increased availability of fatty acids. Summary: Feeding rainbow trout with a lipid-enriched diet affects fatty acid sensing and intracellular signaling pathways in the hypothalamus, suggesting an interaction that controls food intake.

Short-term time course of liver metabolic response to acute handling stress in rainbow trout, Oncorhynchus mykiss.

To elucidate the short-term time-course of liver metabolic response in rainbow trout to acute handling stress we subjected rainbow trout to 5min chasing and obtained samples 0 to 480min post-stress. Levels of cortisol, glucose and lactate were measured in plasma, whereas metabolite levels, enzyme activities, mRNA abundance of parameters related to energy metabolism, and glucocorticoid receptors were assessed in liver. Acute stress affected many parameters related to energy metabolism, with most of them turning back to normal levels after 480min. In general, the present results support the existence of two stages in the short-term time-course of metabolic response to handling stress. A first stage occurring few minutes post-stress (15-45min), was characterized by increased mobilization of liver glycogen resulting in increased production of endogenous glucose, reduced use of exogenous glucose and reduced lipogenic potential. A second stage, occurring 60-120min post-stress onwards was characterized by the recovery of liver glycogen levels, the increased capacity of liver for releasing glucose, and the recovery of lipogenic capacity whereas no changes were noted in gluconeogenic potential, which probably needs longer time periods to become enhanced.

Response of hepatic lipid and glucose metabolism to a mixture or single fatty acids: Possible presence of fatty acid-sensing mechanisms.

To assess the hypothesis that an acute dietary fatty acid (FA) supply may improve glucose tolerance in rainbow trout, we orally administered fish with fish oil (FO; 10mL.kg(-1), one time), which were then subjected to a glucose tolerance test and sampled 6h after injection. Parameters related to glucose and lipid metabolism were then assessed. The results suggest that when both nutrients were administered at the same time, an increased potential for lipogenesis occurred concomitantly with a lower level of glycaemia. In a second experiment we administered intraperitoneally a single FA present in the FO mixture such as oleic acid (60 or 300μg.kg(-1)) whereas octanoic acid (60 or 300μg.kg(-1)) was used as negative control (absent from the FO). However, the effects of both FA were similar in reducing the potential of lipid synthesis and oxidation, and in enhancing the potential of glucose synthesis and glycogenesis. Differences found between FO and single FA administration show that response to FA was dependent on the treatment (mixture vs. single FA) but also comply with the idea that an interaction between FA and glucose rather than FA alone are in the origin of the results reported. The administration of individual FA such as oleic and octanoic acid failed in enhancing lipogenesis and reducing plasma glucose levels and thus in explaining results obtained with FO. However, results provide evidence that FA even provided at a low dose play a key role in the regulation of several putative components of a FA sensing system present in rainbow trout liver.

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.

Counter-regulatory response to a fall in circulating fatty acid levels in rainbow trout. Possible involvement of the hypothalamus-pituitary-interrenal axis.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.

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.