Farnesoid X Receptor Activation in Brain Alters Brown Adipose Tissue Function via the Sympathetic System.

The nuclear bile acid (BA) receptor farnesoid X receptor (FXR) is a major regulator of metabolic/energy homeostasis in peripheral organs. Indeed, enterohepatic-expressed FXR controls metabolic processes (BA, glucose and lipid metabolism, fat mass, body weight). The central nervous system (CNS) regulates energy homeostasis in close interaction with peripheral organs. While FXR has been reported to be expressed in the brain, its function has not been studied so far. We studied the role of FXR in brain control of energy homeostasis by treating wild-type and FXR-deficient mice by intracerebroventricular (ICV) injection with the reference FXR agonist GW4064. Here we show that pharmacological activation of brain FXR modifies energy homeostasis by affecting brown adipose tissue (BAT) function. Brain FXR activation decreases the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and consequently the sympathetic tone. FXR activation acts by inhibiting hypothalamic PKA-CREB induction of TH expression. These findings identify a function of brain FXR in the control of energy homeostasis and shed new light on the complex control of energy homeostasis by BA through FXR.

Food-Derived Hemorphins Cross Intestinal and Blood-Brain Barriers In Vitro.

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood-brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.

Protein Digestion-Derived Peptides and the Peripheral Regulation of Food Intake.

The gut plays a central role in energy homeostasis. Food intake regulation strongly relies on the gut-brain axis, and numerous studies have pointed out the significant role played by gut hormones released from enteroendocrine cells. It is well known that digestive products of dietary protein possess a high satiating effect compared to carbohydrates and fat. Nevertheless, the processes occurring in the gut during protein digestion involved in the short-term regulation of food intake are still not totally unraveled. This review provides a concise overview of the current data concerning the implication of food-derived peptides in the peripheral regulation of food intake with a focus on the gut hormones cholecystokinin and glucagon-like peptide 1 regulation and the relationship with some aspects of glucose homeostasis.

Using Caco-2 cells as novel identification tool for food-derived DPP-IV inhibitors.

Dietary proteins have recently been investigated as a new source of DPP-IV inhibitory peptides with limited side effects and promising applications. Numerous studies have highlighted and identified peptide sequences able to inhibit DPP-IV activity in vitro, mostly from milk proteins. However, the correlation to in vivo studies remains scarce because standard in vitro assays with purified enzyme do not accurately simulate key factors impacting peptide bioactivity such as intestinal and brush border enzymes or cellular permeability. Therefore, a DPP-IV activity inhibition assay is here proposed using non differentiated confluent Caco-2 cells to rapidly assess food-derived peptide inhibitory potential in approaching intestinal conditions. DPP-IV gene expression was first checked and specific DPP-IV substrate was used to implement the assay. Using a specific DPP-IV inhibitor confirmed that non differentiated Caco-2 cells express measurable DPPIV activity. This in situ assay was then applied to digests which already demonstrated a DPP-IV inhibitory potential with a standard assay using purified enzyme. Bovine hemoglobin and cuttlefish hydrolysate digests from simulated gastrointestinal digestion exerted a dose response inhibition on DPP-IV activity but displayed different inhibitory potentials.

The serial reaction time task in the rat: effects of D1 and D2 dopamine-receptor antagonists.

Sequential behaviour, probably reflecting procedural learning, has intensively been investigated in humans and monkeys using so-called serial reaction time tasks (SRTT), where serial stimuli are either presented in a random or sequential fashion. Learning of sequences is typically inferred from faster reaction times to such sequences as compared to random blocks of stimuli. Work with such tasks has shown that sequential behaviour seems to be mediated by specific brain systems, including the basal ganglia and the neurotransmitter dopamine. We have recently developed a rat version of the human serial reaction time task, in which rats have to respond to visual stimuli in one of four spatial locations by nose-poking in order to obtain food reward under a fixed ratio schedule (FR13). Here, we used a test version where random and sequential condition phases (10 min each) were alternated within-sessions. In support of our previous work, we found that well-trained (i.e. skilled) rats display superior performance under sequential than random conditions, namely, faster reaction times and higher response accuracies. Furthermore, we investigated the effects of selective dopamine-receptor blockade, by systemically administering SKF 83566, a D1 antagonist (.05-.15 mg/kg), or raclopride, a D2 antagonist (.05-.20 mg/kg), in two separate experiments. Both antagonists impaired responding to the conditioned visual stimuli in a dose-related way, i.e. they decreased, or even blocked, nose-poke rates. In those rats, which kept responding, the speeding of reaction times during sequential conditions was no longer observed with the D1 antagonist, whereas the enhancements in accuracy were preserved, or even enhanced as compared to vehicle. The D2 antagonist also impaired instrumental behaviour, but did not alter sequence effects on accuracy or reaction times. In contrast to responses to the conditioned stimuli, reaction times to the unconditioned stimuli (food pellets) were not substantially affected by either drug. These results are discussed with respect to methodological factors, and the possible role of dopamine for instrumental behaviour, in general, and sequential behaviour, in specific.

Simulated GI digestion of dietary protein: Release of new bioactive peptides involved in gut hormone secretion.

Dietary proteins have been reported to induce a strong feeling of satiety that has been partially explained by gut hormone level increase. Up to date, various protein hydrolysates have demonstrated in vitro and in vivo their potential to stimulate gut hormone secretion related to food intake decrease and their mechanisms of action have just started to be resolved. In this context, this study aimed at identifying new peptide sequences involved in gut hormone secretion released by protein in vitro gastrointestinal digestion. Targeted gut hormones were Cholecystokinin (CCK) and Glucagon-Like Peptide 1 (GLP-1). The activity of DPP-IV was also considered as it strongly modulates GLP-1 action. In a previous study, simulated digestion of dietary protein has generated hydrolysates with enhancing effect on CCK and GLP-1 secretion in STC-1 cells as well as DPP-IV inhibitory properties. Successive purification steps were performed to isolate peptide fractions involved in these bioactivities whose sequence was determined by LC-MS-MS. Three peptide sequences ANVST, TKAVEH and KAAVT were pointed out for their stimulating effects on GLP-1 secretion. The sequence VAAA was isolated for its DPP-IV inhibitory properties. Two peptide groups were strongly involved in CCK release sharing a certain occurrence of aromatic amino acid residues.

Sequential behavior in the rat: a new model using food-reinforced instrumental behavior.

Sequential behavior, probably reflecting procedural learning, has intensively been investigated in humans. This work has mainly been done using so-called serial reaction time tasks. In such tasks, subjects have to respond rapidly to simple visual stimuli appearing at one of four locations by pressing a corresponding response key. Unknown to the subjects, these stimuli can follow a specific repeating sequence. Learning of such a sequence is typically inferred from faster reaction times to sequence as compared to random blocks of stimuli. In contrast to human subjects, the analysis of sequential behavior has received considerably less attention in rodents, possibly due to the lack of analogous animal models there. In order to establish such a model, a method was developed in rats to investigate serial reactions under conditions of random or sequential stimulus presentation. Operant testing chambers were used which consisted of four nose-poke holes with cue lights. These holes were arranged in a square fashion with a pellet receptacle in the center. The task of the rat was to rapidly respond to an illuminated hole by poking into it in order to obtain food. The stimulus locations varied permanently, and these changes pursued either a random or serial order. In three experiments with differing methodological details, responding under such conditions was analyzed with sequences consisting of 6, 12 or 13 positions. Evidence was obtained that rats can improve their performance under sequence as compared to random conditions, for example, with respect to the percentage of reinforcements obtained, or with respect to reaction times. Furthermore, methodological factors, like response requirements, were addressed which may critically affect experimental outcome. Together, this new kind of instrumental task might be useful to analyze sequential performance in the rat, and the brain mechanisms by which it is mediated.

Differential regulation of ABCA1 and ABCG1 gene expressions in the remodeling mouse hippocampus after entorhinal cortex lesion and liver-X receptor agonist treatment.

Entorhinal cortex lesioning (ECL) causes an extensive deafferentation of the hippocampus that is classically followed by a compensatory reinnervation, where apolipoprotein E, the main extracellular lipid-carrier in the CNS, has been shown to play a crucial role by shuttling cholesterol to reconstructing neurons terminals. Hence, we investigated whether the ATP-binding cassette (ABC) transporters -A1 and -G1, known to regulate cellular cholesterol efflux and lipidation of the apolipoprotein E-containing lipoprotein complex are actively involved in this context of brain׳s plastic response to neurodegeneration and deafferentation. We assessed ABCA1 and ABCG1 mRNA and protein levels throughout the degenerative phase and the reinnervation process and evaluated the associated cholinergic sprouting following ECL in the adult mouse brain. We subsequently tested the effect of the pharmacological activation of the nuclear receptor LXR, prior to versus after ECL, on hippocampal ABCA1 and G1 expression and on reinnervation. ECL induced a time-dependent up-regulation of ABCA1, but not G1, that coincided with a significant increase in acetylcholine esterase (AChE) activity in the ipsilateral hippocampus. Pre-ECL, but not post-ECL i.p. treatment with the LXR agonist TO901317 also led to a significant increase solely in hippocampal ABCA1 expression, paralleled by increases in both AchE and synaptophysin protein levels in the deafferented hippocampus. Thus, ABCA1 and -G1 are differentially regulated in the lesioned brain and upon treatment with an LXR agonist. Further, TO901317-induced up-regulation of ABCA1 appears to be more beneficial in a prevention (pre-lesion) than rescue (post-lesion) treatment; both findings support a central role for ABC transporters in brain plasticity.

The lin-4 microRNA targets the LIN-14 transcription factor to inhibit netrin-mediated axon attraction.

miR-125 microRNAs, such as lin-4 in Caenorhabditis elegans, were among the first microRNAs discovered, are phylogenetically conserved, and have been implicated in regulating developmental timing. Here, we showed that loss-of-function mutations in lin-4 microRNA increased axon attraction mediated by the netrin homolog UNC-6. The absence of lin-4 microRNA suppressed the axon guidance defects of anterior ventral microtubule (AVM) neurons caused by loss-of-function mutations in slt-1, which encodes a repulsive guidance cue. Selective expression of lin-4 microRNA in AVM neurons of lin-4-null animals indicated that the effect of lin-4 on AVM axon guidance was cell-autonomous. Promoter reporter analysis suggested that lin-4 was likely expressed strongly in AVM neurons during the developmental time frame that the axons are guided to their targets. In contrast, the lin-4 reporter was barely detectable in anterior lateral microtubule (ALM) neurons, axon guidance of which is insensitive to netrin. In AVM neurons, the transcription factor LIN-14, a target of lin-4 microRNA, stimulated UNC-6-mediated ventral guidance of the AVM axon. LIN-14 promoted attraction of the AVM axon through the UNC-6 receptor UNC-40 [the worm homolog of vertebrate Deleted in Colorectal Cancer (DCC)] and its cofactor MADD-2, which signals through both the UNC-34 (Ena) and the CED-10 (Rac1) downstream pathways. LIN-14 stimulated UNC-6-mediated axon attraction in part by increasing UNC-40 abundance. Our study indicated that lin-4 microRNA reduced the activity of LIN-14 to terminate UNC-6-mediated axon guidance of AVM neurons.

Function and comorbidities of apolipoprotein e in Alzheimer's disease.

Alzheimer's disease (AD)-the most common type of dementia among the elderly-represents one of the most challenging and urgent medical mysteries affecting our aging population. Although dominant inherited mutation in genes involved in the amyloid metabolism can elicit familial AD, the overwhelming majority of AD cases, dubbed sporadic AD, do not display this Mendelian inheritance pattern. Apolipoprotein E (APOE), the main lipid carrier protein in the central nervous system, is the only gene that has been robustly and consistently associated with AD risk. The purpose of the current paper is thus to highlight the pleiotropic roles and the structure-function relationship of APOE to stimulate both the functional characterization and the identification of novel lipid homeostasis-related molecular targets involved in AD.

Sequential behavior in the rat: role of skill and attention.

The serial reaction time task (SRTT) is a well-established experimental tool to study cognitive and neural mechanisms of sequential performance in humans. We have recently developed a rodent version of the human serial reaction time task, in which rats have to respond to visual stimuli by nose-poking into one of four spatial locations in order to obtain food reward. In this task, rats display superior performance under sequential as compared to random conditions of stimulus presentation. Specifically, the subjects are able to profit from sequential regularities in terms of faster reaction times and higher response accuracy. Here, we studied the effects of violating a single stimulus in rats, which had been intensively trained under sequential conditions, and we asked whether these subjects, when confronted with sequence violations, still attend to the actual stimulus order (that is, show correct responses), or whether their behavior has become fully automated (leading to specific incorrect responses to violated stimulus positions). In two independent experiments using partly differing instrumental set-ups, we found that the responses to non-cued violations of single stimulus positions were mostly correct, that is, the animals were apparently attending to the stimuli. Nevertheless, these reaction times were slowed, which probably reflects cognitive resources necessary to respond correctly to the unexpected irregularities. When quantifying the minority of responses, which were incorrect, we found that most of them were directed to the position, where the stimulus would have appeared if the sequence had not been violated. These responses were faster than the correct ones (to the violated stimulus), which indicates that sequential responding had become partly automated. Together, our data show that both, attention and skill play a role for sequential performance in our SRT task, and that they can be dissected by quantification of specific response types. In future work, the neural correlates underlying these functional mechanisms will have to be addressed.