Evidence for the presence in rainbow trout brain of amino acid-sensing systems involved in the control of food intake

Abstract

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

Feeding Stimulation Ability and Central Effects of Intraperitoneal Treatment of L-Leucine, L-Valine, and L-Proline on Amino Acid Sensing Systems in Rainbow Trout: Implication in Food Intake Control

“…In a previous study (Comesaña et al, 2018) on rainbow trout ( Oncorhynchus mykiss ) as a model teleost fish, we demonstrated that intracerebroventricular (ICV) treatment with leucine decreased food intake as expected whereas valine, contrary to mammals, had a clear orexigenic effect. We related the effects on food intake to the presence of amino acid sensing systems (dependent on metabolism of BCAA, metabolism of glutamine, mTOR, T1R receptors, and GCN2) sensitive to leucine in hypothalamus and telencephalon.…”

“…Enzyme activities were assessed at maximum rates after preliminary tests to determine optimal substrate concentrations. BCAT ( EC 2.6.1.42), glutamine synthetase (GLS, EC 6.3.1.2), and GDH ( EC 1.4.1.4) activities were determined as previously described (Comesaña et al, 2018). …”

“…Analyses were performed on 1 μL cDNA (previously diluted 1:1) using MAXIMA SYBR Green qPCR Mastermix (Life Technologies), in a total PCR reaction volume of 15 μL, containing 50–500 nM of each primer. Most transcripts were measured using previously described primers (Comesaña et al, 2018), with the exception of T1R1 and SESN2. For these transcripts, new primers were designed using Primer3 software 1 from sequences available in GenBank (T1R1, XM_021614421.1; SESN2, XM_021572426.1).…”

“…These amino acids were initially selected considering that, in mammals, leucine is the unique activator of amino acid sensing systems in brain whereas valine acts as a negative control (Heeley and Blouet, 2016). However, the previous realization of important differences between trout and mammals (Comesaña et al, 2018) led us to consider extending the investigation to a non-BCAA in this study. Proline was was selected for several reasons: it typically has a high palatability in carnivore fish species (Li et al, 2009; Morais, 2017), including rainbow trout (Jones, 1989), it has very diverse and important roles in cell metabolism and physiology, and is considered a conditionally essential amino acid in fish given that rates of endogenous synthesis are inadequate during early life stages, and possibly also in adults (Li et al, 2009; Wu et al, 2011).…”

“…Furthermore, the requirement of proline for whole-body protein synthesis is the highest of all amino acids, on a per-gram basis (Wu et al, 2011). Finally, in order to establish whether the action of dietary leucine and valine on central amino acid sensors is possibly direct, we carried out a third experiment with a similar IP administration of amino acids to evaluate the response of amino acid sensing mechanisms in the hypothalamus and telencephalon, and compare it with the previous results after ICV administration (Comesaña et al, 2018). In particular, we assessed parameters related to putative amino acid sensing mechanisms based on: (1) metabolism of BCAA, evaluated through branched-chain amino acid aminotransferase (BCAT) activity, and mRNA abundance of branched-chain α-keto acid dehydrogenase E2 subunit (BCKDE2) and branched chain ketoacid dehydrogenase kinase (BCKDK), (2) metabolism of glutamine, evaluated by glutamine synthase (GLS) and glutamate dehydrogenase (GDH) activities, and mRNA abundance of GLS1 and GLS2, (3) mTOR/AMPKα, evaluated by the abundance of their mRNA as well as by their phosphorylation status, (4) T1R receptors, including the umami and sweet taste receptors (which in fish also bind amino acid ligants; Oike et al, 2007), through mRNA abundance of subunits T1R1, T1R2, and T1R3, and (5) GCN2, evaluated by mRNA abundance of eIF2α, SNAT2, and SESN2.…”