# Central Activation of Chaperone-Mediated Autophagy reduces Appetite by Fine-tuning Hypothalamic Amino Acid Pools: New Insights from Fish

Steffi Reji, Emilio J. Vélez, Ayelén M Blanco, Cecile Heraud, Vincent Véron, Karine Dias, Alexandre Stella, Odile Burlet-Schiltz, Simon Schnebert, Jérôme Roy, et al.

► To cite this version:

Steffi Reji, Emilio J. Vélez, Ayelén M Blanco, Cecile Heraud, Vincent Véron, et al. Central Activation of Chaperone-Mediated Autophagy reduces Appetite by Fine-tuning Hypothalamic Amino Acid Pools: New Insights from Fish. American Journal of Physiology - Cell Physiology, 2026, 330 (3), pp.C590-C604. 10.1152/ajpcell.00765.2025. hal-05506447

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RESEARCH ARTICLE

# Central activation of chaperone-mediated autophagy reduces appetite by fine-tuning hypothalamic amino acid pools: new insights from fish

Steffi Reji,¹ Ⓓ Emilio J. Vélez,¹,² Ⓓ Ayelén M. Blanco,³ Ⓓ Cecile Heraud,¹ Vincent Véron,¹ Karine Dias,¹ Ⓓ Alexandre Stella,⁴,⁵ Odile Burlet-Schiltz,⁴,⁵ Simon Schnebert,¹ Ⓓ Jérôme Roy,¹ Ⓓ Florian Beaumatin,¹ Beth Cleveland,⁶ José L. Soengas,³ and Iban Seiliez¹

¹UMR 1419 Nutrition Métabolisme et Aquaculture, E2S UPPA, INRAE, Université de Pau et des Pays de l'Adour, Saint-Pée-sur-Nivelle, France; ²Department of Cell Biology, Physiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain; ³Centro de Investigación Mariña, Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, Vigo, Spain; ⁴Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, Université de Toulouse, UPS, Toulouse, France; ⁵Infrastructure Nationale de Protéomique (ProFI), FR 2048, Toulouse, France; and ⁶National Center for Cool and Cold Water Aquaculture, ARS/USDA, Kearneysville, West Virginia, United States

## Abstract

Chaperone-mediated autophagy (CMA) is a key lysosomal proteolytic pathway essential for cellular homeostasis and metabolism, with dysfunction linked to various human diseases. Although extensively studied in humans and mice, CMA was only recently identified in fish, paving the way for novel and evolutionary research perspectives. Here, we demonstrate a role for CMA in regulating feed intake (FI) in rainbow trout (*Oncorhynchus mykiss*), a major aquaculture species and a widely used model in numerous research fields, including physiology, evolutionary genetics, toxicology, immunology, and nutrition. Specifically, we observed that feed deprivation induces an increase in the CMA activation score—a reliable proxy for CMA activity—in the hypothalamus, a central brain region involved in the regulation of feeding behavior. To probe its functional relevance, we intracerebroventricularly (ICV) injected the CMA activator CA77.1 and found a significant reduction in FI levels, suggesting a regulatory role for CMA in appetite. Further analysis suggested that CMA may regulate FI partly through changes in hypothalamic free amino acid availability, with ribosomal protein degradation potentially contributing to this mechanism. Through this mechanism, CMA may play a critical role in the precise regulation of satiety and also represent a promising target for therapeutic strategies aimed at treating metabolic disorders, as well as for nutritional interventions to improve feed efficiency and promote more sustainable growth practices in aquaculture.

**NEW & NOTEWORTHY** This study shows in vivo, in a nonmammalian vertebrate, that chaperone-mediated autophagy (CMA) regulates appetite. It demonstrates that hypothalamic CMA activity increases during feed deprivation and that pharmacological activation of CMA suppresses feed intake by locally modulating amino acid availability. These findings reveal a previously uncharacterized link between CMA, hypothalamic amino acid pools, and appetite regulation, and suggest a novel intracellular mechanism linking proteostasis to nutrient sensing and feeding behavior in vertebrates.

appetite; autophagy; chaperone-mediated autophagy; fish; hypothalamus

## INTRODUCTION

Appetite regulation is a conserved physiological process that maintains energy balance through hypothalamic integration of peripheral hormonal and metabolic signals such as leptin, insulin, ghrelin, and nutrients (1, 2). Although major advances have clarified the neuroendocrine and molecular pathways governing the feeding behavior (3, 4), the intracellular mechanisms by which hypothalamic neurons adapt to metabolic stress and nutrient fluctuations remain incompletely understood (5, 6).

Among these intracellular processes, autophagy—a lysosome-mediated degradation pathway—has emerged as a

critical mechanism by which hypothalamic neurons adapt to changes in nutrient status. Of the three main types of autophagy—macroautophagy (MA), microautophagy, and chaperone-mediated autophagy (CMA)—MA is the most extensively studied in the central regulation of feeding. In a foundational study, Kaushik et al. (7) demonstrated that MA is activated in agouti-related peptide (AgRP) neurons during fasting, promoting the mobilization of lipid stores and enhancing the production of orexigenic AgRP neuropeptides. The authors emphasized that MA-mediated lipid mobilization within these neurons is essential for maintaining energy balance, noting that MA inhibition not only

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<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>

suppresses AgRP expression but also elevates α-melanocyte-
stimulating hormone (α-MSH) levels and reduces adiposity,
highlighting the metabolic consequences of impaired auto-
phagic flux in feeding circuits (7). In subsequent work, the
same group reported that MA disruption in pro-opiomelano-
cortin (POMC) neurons results in decreased α-MSH levels
and increased adiposity, indicating that MA exerts distinct,
cell-type-specific roles within hypothalamic networks (8).
Building on these findings, Oh et al. (9) later demonstrated
that hypothalamic AMPK regulates feeding behavior by
modulating MA-driven changes in neuropeptide expression.
Collectively, these studies establish MA as a key intracellular
mechanism through which hypothalamic neurons sense and
adapt to nutrient availability, coordinating appropriate
behavioral and physiological responses.

In contrast, far less is known about the role of CMA in the hypothalamus. CMA is a selective autophagic pathway that targets cytosolic proteins containing KFERQ-like motifs for lysosomal degradation, via binding to HSC70 and translocation through the lysosome-associated membrane protein type 2A (LAMP2A), the rate-limiting factor in CMA (10). Unlike MA, CMA allows direct and specific degradation of individual proteins without vesicle formation. This pathway plays key roles in metabolic regulation, protein quality control, and cellular stress responses (10–12).

Despite its recognized systemic importance, the role of CMA in hypothalamic nutrient sensing remains poorly understood. Portovedo et al. (13) reported alterations in the expression of LAMP2A and HSC70 in the hypothalamus of mice fed a high-fat diet, as well as in hypothalamic cell lines treated with palmitic acid, suggesting a possible involvement of CMA in central metabolic regulation. Supporting this, Espinosa et al. (14) showed that exposure of POMC-like neurons to saturated fatty acids, including palmitic and stearic acids, suppresses CMA activity by reducing LAMP2A protein levels, thereby impairing insulin signaling and potentially disrupting energy homeostasis. However, despite these insights, direct in vivo evidence linking hypothalamic CMA to the regulation of feeding behavior remains scarce, underscoring a critical gap in our understanding of its physiological function.

Here, we hypothesized that hypothalamic CMA is
responsive to changes in nutritional status and may influ-
ence feeding behavior. To address this, we investigated
CMA's functional role in the control of feed intake (FI)
using the rainbow trout (RT, *Oncorhynchus mykiss*), a
widely used model in vertebrate physiology and evolution-
ary biology (15, 16). In this species, evidence indicates the
presence of hypothalamic mechanisms regulating FI
through the integration of metabolic and neuroendocrine
signals (17). These mechanisms include conserved major
nutrient-sensing pathways or components, such as mTOR,
GCN2, AMPK, and fatty acid and amino acid transporters
(17), which, together with conserved core CMA machinery
such as LAMP2A, HSC70, and related cofactors (18, 19),
make RT a relevant model for investigating hypothalamic
CMA function in vertebrates. Its evolutionary position as
a teleost fish also provides a comparative perspective
on fundamental mechanisms of hypothalamic nutrient
sensing and feeding regulation, while acknowledging

species-specific differences with mammals. Importantly,
CMA activity was only recently evidenced in fish (20),
opening new opportunities to explore its conserved func-
tions in vivo. Here, we report that the CMA activation
score, a transcriptional metric derived from the expression
of key CMA components including LAMP2A and associ-
ated factors—initially defined in mice (21) and subse-
quently adapted and validated in RT (19)—is upregulated
in the hypothalamus during food deprivation. This score
has been extensively validated and reliably reflects CMA
activity (19, 21–26), although, as with any transcription-
based measure, changes may not always capture instanta-
neous flux through the CMA pathway. To probe the func-
tional relevance of hypothalamic CMA activation, we then
intracerebroventricularly (ICV) injected a CMA activator
(CA77.1, hereafter CA) and found a significant reduction in
FI levels. In short, CA is a derivative of the retinoic acid-
based CMA activator AR7 (27) that was optimized for in
vivo use (21). This pharmacological agent activates CMA
by inducing the expression of LAMP2A, thereby promoting
the selective degradation of KFERQ-motif-containing pro-
teins without affecting MA. In vivo, CA demonstrates brain
penetrance with favorable pharmacokinetics, effectively
increasing CMA activity in the brains of mice without
detectable toxicity in blood or major organs after chronic
treatment (21). Although CA has been primarily used sys-
temically, we delivered it through ICV in our study to
achieve targeted hypothalamic effects while avoiding
peripheral confounding. As with any pharmacological
approach, potential off-target effects cannot be fully
excluded; however, its validated mechanism and selectiv-
ity for LAMP2A make it a robust tool to probe CMA func-
tion in vivo. Interestingly, our findings show that CMA
activation is associated with increased hypothalamic-free
amino acids and reduced ribosomal protein abundance,
pointing to a potential role of ribosomal protein degrada-
tion in shaping amino acid availability – as previously
observed (28, 29) – and ultimately influencing satiety.
Together, these results demonstrate in vivo, in a nonmam-
malian vertebrate that central activation of CMA is suffi-
cient to modulate appetite, and reveal a mechanistic link
among CMA-driven protein turnover, amino acid homeo-
stasis, and feeding regulation.

MATERIALS AND METHODS

Animals and Experimental Procedures

Two experimental trials were conducted: 1) a feed deprivation—refeeding trial and 2) an ICV injection trial with a specific CMA activator. The objective of the feed deprivation-refeeding trial was to determine whether CMA activity is regulated by feeding status across different brain regions in RT, whereas the ICV injection trial aimed to investigate the specific role of hypothalamic CMA in the regulation of FI. As these two experiments were designed to address different questions—namely, whether CMA responds to nutritional status in various brain regions and whether hypothalamic CMA regulates FI—their fasting and refeeding protocols were adapted accordingly.
<figure><img src="image_5.png" /></figure>

**Feeding trial.**

Female RT was reared at the French National Research Institute for Agriculture, Food and Environment (INRAE) experimental facility (IE NIAIADE, see https://doi.org/10.15454/GPYD-AM38), which is authorized for animal experimentation by the French Veterinary Service (A64-495-1 and A40-228-1). All technical staff were trained and certified to conduct animal experiments. The study was performed in compliance with Directive 2010/63/EU on the protection of animals used for scientific purposes, and the National Guidelines for Animal Care of the French Ministry of Research (Decree No. 2013-118, Feb 01, 2013).

Juvenile immature RT (~200 g) was maintained at the INRAE experimental facility (Donzacq, France) under natural photoperiod and a constant water temperature (17 ± 1°C). Fish were randomly distributed into 20 tanks (170 L, 40 fish/tank) and fed twice daily to apparent satiation with a commercial diet (Omega HE SF T3P, Skretting, Fontaine-les-Vervins, France). Experimental groups (4 tanks/treatment) were then subjected to different nutritional conditions: basal (deprived of feed for 1 day), F4 (deprived of feed for 4 days), F4R (deprived of feed for 4 days, then refed 1 h before sampling), F8 (deprived of feed for 8 days), and F8R (deprived of feed for 8 days then refed 1 h before sampling). Fish were weighed, anesthetized, and brain tissues (hypothalamus, telencephalon, and cerebellum) were dissected (Fig. 1A). Samples (n = 2/tank; n = 8/treatment, randomly selected based on earlier studies) were rapidly frozen in liquid nitrogen and stored at -80°C until analysis.

**ICV injection experiment.**

This experiment was carried out at ECIMAT-UVIGO experimental facilities, certified for animal experimentation by the legal authorities. All procedures complied with the Guidelines of the Council of the European Union (2010/63) and the Spanish Government (RD 55/2013, and RD 118/2021) for the use of animals in research.

Juvenile immature RT (initial weight ~100 g) were obtained from a local fish farm (Piscifactoría de la Calle, A Estrada, Spain) and maintained in 100 L tanks at a constant water temperature of 15 ± 0.5°C, under 12 L:12D photoperiod. Water parameters (dissolved oxygen, ammonia, nitrite, and nitrate) were regularly monitored. After acclimatization, fish were randomly distributed into eight fiberglass tanks and assigned to two treatment groups: 1) vehicle [dimethyl sulfoxide (DMSO)] and 2) the CA (CA77.1, MedChemExpress, HY-134923), previously developed and characterized by Bourdenx et al. (21). All details regarding the synthesis, purity, and pharmacokinetic properties of this compound are provided in Ref. 21. No off-target effects of the CA have been reported. Each treatment was replicated in four tanks (n = 4/treatment, 10 fish/tank), such that the tank served as the experimental unit. Importantly, fish from the DMSO and CA groups underwent identical handling, anesthesia, and injection procedures throughout the experiment. The only difference between these groups was the presence or absence of CA in the injected solution. Thus, the DMSO group served as a full sham-handled control, allowing us to isolate the pharmacological effects of CA from any potential confounding effects of manipulation.

Fish were hand-fed to visual satiation once daily, and welfare was monitored each day. One hour after feeding, fish were lightly anaesthetized with 2-phenoxyethanol (Sigma, 0.02% vol/vol), weighed, and received ICV injection of 1 µL·100 g⁻¹ body wt of either DMSO alone (control) or containing CA (1 µg/kg body wt). CA stock solution was acquired at 10 mM (2.98 mg/mL) in DMSO, and the working solution for injection was adjusted to 0.1 µg/µL. For each 1 µL injection/100 g body wt, 0.035 µL of CA stock was diluted in 0.965 µL of saline solution. The vehicle solution contained the same final DMSO concentration as the CA solution, corresponding to 3.4% (vol/vol) DMSO per injection. Preliminary experiments were performed using DMSO alone or DMSO containing different concentrations of CA to assess potential behavioral effects, and no adverse responses or abnormal behaviors were observed at the doses selected for the present study.

ICV administration was performed as previously described (30). In brief, fish were placed on a plexiglass board with Velcro straps adjusted to hold them in place. A 10 µL Hamilton syringe was aligned with the sixth preorbital bone at the rear of the eye socket, and inserted at an angle of ~45°, advancing ~3–4 mm through the frontal bone to reach the third ventricle. Accurate placement was previously validated, by injecting methylene blue and confirming the diffusion of the dye into the third ventricle (31). Across all earlier validation experiments, the success rate of injections using methylene blue exceeded 95%. The absence of injury to the ventricular lining was confirmed by microscopic evaluation of the tissue following histological processing of the brain. After administration, fish were placed back into their respective tanks for recovery, and they were deprived of feed for 48 h. This fasting period was chosen to avoid any transient anorectic effects associated with anesthesia or handling and to ensure that subsequent measurements of FI reflected the specific physiological impact of hypothalamic CMA modulation. This 48-h delay is consistent with previous ICV studies in teleosts and has been shown not to affect feeding motivation once fish resume feeding.

After 48 h, fish were refed to satiation, and the feed ingested by fish in each tank was registered. For this, fish were offered a preweighed amount of feed, allowed to eat, and then the uneaten feed was recovered, dried, and weighed as described previously (32). All behavioral observations and FI assessments were conducted by an observer blinded to treatments, and the injector and observer were different individuals to prevent any measurement bias.

In a subsequent experiment, designed for tissue sampling, fish were redistributed into two 100 L tanks (44 fish/tank), corresponding to each treatment (CA vs. DMSO control). Hypothalamic samples were collected at 6, 24, and 48 h after ICV injection (n = 11 fish/treatment/timepoint, randomly selected based on earlier studies).

**RNA Extraction and Quantitative RT-PCR Analysis**

Total RNA was extracted from individual tissue samples using TRIzol reagent (Invitrogen, Carlsbad, California). Between 10 mg and 20mg of tissue/mL of reagent was processed and homogenized using a Precellys tissue
<figure><img src="image_6.png" /></figure>

homogenizer (Bertin Technologies, Montigny le Bretonneux, France). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the SuperScript III RNase H-Reverse Transcriptase kit (Invitrogen) and random primers (Promega,

Charbonnières-les-bains, France). Quantitative real-time RT-PCR was conducted on a Roche LightCycler 480 system (Roche Diagnostics, Neuilly-sur-Seine, France), following the procedure adapted from Plagnes-Juan et al. (33). In brief,

A

<figure><img src="image_7.png" /></figure>

B

<table><thead><tr><th rowspan="2">Effectors</th><th colspan="2">Modulators</th></tr><tr><th>Lysosomal</th><th>Extralysosomal</th></tr></thead><tbody><tr><td>LAMP2A</td><td>GFAP</td><td>NFAT</td></tr><tr><td>HSC70</td><td>RAC1</td><td>NRF2</td></tr><tr><td>HSP90</td><td>PHLPP1</td><td>Rab11</td></tr><tr><td>HSP40</td><td>Ef1a</td><td>NCOR1</td></tr><tr><td></td><td>RICTOR</td><td>RARα</td></tr><tr><td></td><td>AKT</td><td></td></tr><tr><td></td><td>CTSA</td><td></td></tr></tbody></table>

C

<figure><img src="image_8.png" /></figure>

E

<figure><img src="image_9.png" /></figure>

G

<figure><img src="image_10.png" /></figure>

D

<figure><img src="image_11.png" /></figure>

F

<figure><img src="image_12.png" /></figure>

H

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<figure><img src="image_14.png" /></figure>

reactions were carried out in a final volume of 6 µL contain-
ing 2 µL of diluted cDNA (1:76), 0.24 µL of each primer
(100 mmol·L⁻¹), 3 µL of LightCycler 480 SYBR Green I
Master mix, and 0.52 µL of Dnase/Rnase-free water (5 Prime
GmbH, Hamburg, Germany). The amplification protocol
consisted of an initial denaturation and hot-start Taq poly-
merase activation at 95°C for 3 min, followed by 40 cycles of
amplification (20 s at 95°C and 30 s at 60°C). Melting curve
analysis (0.5°C/10 s, from 55°C to 94°C) was systematically
performed at the end of the run to confirm amplification
specificity. Primer sequences are provided in Supplemental
Tables S1 and S2. All reactions were performed in duplicate
and included negative controls (reverse transcriptase- and
cDNA template-free controls). PCR efficiency was assessed
for each primer pair by generating standard curves from
serial cDNA dilutions. Efficiencies ranged between 1.8 and
2.0 in all cases. Gene expression levels were calculated using
the ΔCT method (34). To allow accurate normalization across
samples, Luciferase control RNA (Promega) was added as an
exogenous spike-in control at a concentration of 10 pg/1.9 mg
of tissue before RNA extraction. The use of an external lucifer-
ase RNA calibrator enables correction for variations in RNA
extraction, reverse transcription, and amplification efficien-
cies, and is particularly suited for conditions in which endoge-
nous reference genes may vary (35).

CMA Score

CMA score was adapted to RT and validated previously (19). In brief, each gene within the CMA network (Fig. 1B) was assigned a specific weight. For genes with multiple paralogs in RT, each paralog received a weight of 1/*n*, where *n* is the number of copies. LAMP2A, being the rate-limiting component of CMA, was assigned a total weight of 2, distributed across its paralogs based on their relative contributions to CMA activity. In addition, a modulation score of +1 or -1 was applied depending on whether the gene positively or negatively influences CMA. The final CMA score was obtained by summing all the weighted and modulated values.

Label-Free Proteomics Analysis

Sample preparation.

Proteins were extracted from RT hypothalamus using the
Thermo Scientific T-PER Tissue Protein Extraction Reagent
(Ref. 78510), 48 h after ICV injection with DMSO or CA.
Protein concentrations were determined using the bicin-
chonic acid (BCA) assay (Interchim; ref UP95424A), and
then processed by the ProteoToul Services - Proteomics
Facility of Toulouse (see https://proteotoul.ipbs.fr/
proteotoul-services/). For each sample, 50 µg of dried pro-
tein extracts were solubilized with 25 µL of 5% SDS. Proteins
were submitted to reduction and alkylation of cysteine

residues by addition of TCEP and chloroacetamide to a final
concentration of 10 mM and 40 mM, respectively. Protein
samples were then processed for trypsin digestion on S-trap
Micro devices (Protifi) according to manufacturer's proto-
col, with the following modifications: precipitation was per-
formed using 216 µL S-Trap buffer, 4 µg trypsin was added
per sample for digestion in 25 µL ammonium bicarbonate
50 mM.

Nano LC-MS/MS analysis of proteins.

Tryptic peptides were resuspended in 50 µL of 2% acetonitrile and 0.05% trifluoroacetic acid and analyzed by nano-liquid chromatography (LC) coupled to tandem MS, using an UltiMate 3000 system (NCS-3500RS Nano/Cap System; Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). One microliter of each sample was loaded onto an analytical C18 column (PEPMAP C18, 2 µm, 75 µm × 500 mm; Thermo Fisher Scientific) heated at 45°C, equilibrated in 95% solvent A (5% acetonitrile and 0.2% formic acid) and 5% solvent B (80% acetonitrile and 0.2% formic acid). Peptides were eluted using a 95 min gradient: 0–25% buffer B for 58 min, 25–40% buffer B for 20 min, 40–90% buffer B for 2 min, and 90% buffer B for 5 min. The mass spectrometer was operated in data independent acquisition mode with the Xcalibur software. Full-scan MS resolution was set at 60,000, and the AGC target was 300% with a maximum injection time of 100 ms. The MS1 mass range was set to 400–1,008. For MS2 spectra, AGC target value spectra were set at 1,000%. The DIA isolation scheme consisted of 2 × 75 staggered windows (8 m/z width, 4 m/z offset) covering the 400–1,000 m/z range. The resolution was set to 15,000, injection time was set in automatic mode, and normalized collision energy was set at 30%. Raw data were processed using DIA-NN (v.1.8) (36) using default parameters and the spectral library generated with the DDA analysis. DIA-NN reports were postprocessed using the Proline software v.2.2: norm. No imputation of missing values was performed.

**Bioinformatics analysis of mass spectrometry raw files.**

Raw MS files were processed with the Mascot software (v.2.7.0) for database search and Proline55 for label-free quantitative analysis (v.2.1.2). Data were searched against RT entries of Uniprot protein database (46,650 sequences; 17,861,404 residues). Carbamidomethylation of cysteines was set as a fixed modification, whereas oxidation of methionine was set as variable modifications. Specificity of trypsin/P digestion was set for cleavage after K or R, and two missed trypsin cleavage sites were allowed. The mass tolerance was set to 10 ppm for the precursor and to 20 mmu in tandem MS mode. Minimum peptide length was set to seven amino acids, and identification results

**Figure 1.** Modulation of CMA activation score by feeding status across brain regions. A: graphical depiction of a RT brain highlighting the hypothalamus, telencephalon, and cerebellum. B: CMA network components used to calculate the CMA activation score, grouped by function (effectors and modulators) and subcellular localization (lysosomal and extralysosomal). Components that activate CMA are shown in green, whereas inhibitors are shown in red. C, E, and G: normalized expression (to basal) of each element of the CMA network (organized in functional groups) in the hypothalamus (C), telencephalon (E), and cerebellum (G) of RT under different feeding conditions: fasted for 1 day (basal), fasted for 4 days (F4), fasted for 4 days and refed (F4R), fasted for 8 days (F8), and fasted for 8 days and refed (F8R). D, F, and H: CMA activation score in the hypothalamus (D), telencephalon (F), and cerebellum (H), of RT under the different feeding conditions presented (C, E, and G). Values are presented as means ± SE of *n* = 7 or 8 replicates. (*P* < 0.05; **P** < 0.01; t test). CMA, chaperone-mediated autophagy; RT, rainbow trout.
<figure><img src="image_15.png" /></figure>

were further validated in Proline by the target decoy approach using a reverse database at both a PSM and protein false-discovery rate of 1%. For label-free relative quantification of the proteins across biological replicates and conditions, cross-assignment of peptide ions peaks was enabled inside group with a match time window of 1 min, after alignment of the runs with a tolerance of ±600 s. Median Ratio Fitting computes a matrix of abundance ratios calculated between any two runs from ion abundances for each protein. For each pairwise ratio, the median of the ion ratios is then calculated and used to represent the protein ratio between these two runs. A least-squares regression is then performed to approximate the relative abundance of the protein in each run in the dataset. This abundance is finally rescaled to the sum of the ion abundances across runs. A Student's *t* test (two-tailed *t* test, equal variances) was then performed on log₂ transformed values to analyze differences in protein abundance in all biologic group comparisons. Given the large number of proteins analyzed (*n* = 12,955), multiple-testing correction (Benjamini-Hochberg FDR) was not applied in order to preserve sensitivity to potentially relevant changes. As highlighted by Pascovici et al. (37), conventional multiple testing corrections can be overly strict in proteomics experiments with low power, potentially missing true positives even when biologically meaningful changes exist. Accordingly, we report unadjusted *P* values (*P* < 0.05) in combination with a biological relevance threshold (log₂ fold-change| > ±1.41) to highlight proteins potentially affected by CA injection. This approach reflects the exploratory nature of the study and provides a comprehensive view of proteomic alterations across the dataset.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD068293.

## Polar Metabolites Extraction and Amino Acid Profiles in Hypothalamus Using HPLC-FL Technique

Proteinogenic amino acid profiles were determined in RT hypothalamus samples by HPLC-FL technique after polar metabolite extraction and AccQTag derivatization. Reagents, standards and MS grade organic solvents were purchased either from Waters (CA), Sigma-Aldrich (MO), Interchim (Montluçon, France) or ADL and Prochilab (Lormont, France). Extraction of each hypothalamus was performed by homogenization with Precellys 24 (Bertin Technologies, Montigny-le-Bretonneux, France) in accordance with the instructions of the manufacturer, in 20 mM phosphate and 1 mM EDTA buffer (pH: 6.5 ± 0.1) with 1/5 (m/v) ratio. After the first centrifugation (14,000 g, 20 min, 4°C), deproteinization of the supernatant was performed with a volume-to-volume 10% metaphosphoric acid solution. Following second centrifugation (14,000 g, 5 min, 4°C), the supernatant was filtered with a 0.22 µm PVDF unit. Polar metabolites were recovered through a third and last centrifugation and stored at -20°C until analysis. Amino acids were derivatized using AccQTag Ultra derivatization kit as described in Morin et al. (38), using 10 µL of polar metabolite extracts. Chromatographic analyses were

done with a 5 µL injection using an Acquity H-Class PLUS system coupled to an ACQUITY UPLC Fluo detector and equipped with an AccQTag Ultra RP column from Waters (CA). The 13-min-gradient program was the same as previously described. The fluorescence of the eluate was monitored for an excitation of 266 nm and an emission of 473 nm. Data acquisition was performed using Empower Pro software 3.0 (Waters, CA). Amino acids were identified by comparing retention times to those of amino acids standards (Sigma-Aldrich, No. AAS18). Quantification was based on integrating peak areas and comparing them to standard calibration curves of each amino acid (R² > 0.995). Calibration curves were linear from 1 to 200 pmol/injection for each standard (Supplemental Fig. S1). Calibration parameters for the quantification of each amino acid analyzed using the UHPLC-FL system are provided in Supplemental Table S3. Normalization was made using the weight of each hypothalamic matrix. Only amino acids that were detected and for which no data were missing were included in the analyses; therefore, no specific handling or imputation procedure for missing or undetected amino acids was required.

## Statistical Analysis

All statistical analyses were performed using GraphPad Prism v.8.0.1 for Windows (GraphPad Software, Inc., see www.graphpad.com), and a *P* value < 0.05 was set as a level of significance. Before performing parametric or nonparametric tests, data normality and homogeneity of variances were assessed using the Shapiro-Wilk test and Bartlett's test, respectively. The differences between two groups were evaluated through a parametric two-tailed unpaired Student's *t* test, or a nonparametric Mann-Whitney test. When specified, a multiple testing correction (Benjamini-Hochberg FDR) was applied to adjust *P* values for the number of comparisons. Percentages of CA-modulated hypothalamic proteins and of the total RT proteome with or without at least one KFERQ-like motif were compared by χ² test.

# RESULTS

## Hypothalamic CMA Senses and Is Regulated by Nutritional Status

To determine whether CMA is regulated by feeding status across different brain regions in RT (Fig. 1A), we quantified the CMA activation score, originally defined in mice (21) and subsequently adapted and validated in RT (19), under a range of nutritional conditions: 16 h postfeeding (basal), 4 days of feed deprivation (F4), 4 days of feed deprivation followed by refeeding (F4R), 8 days of feed deprivation (F8), and 8 days of feed deprivation followed by refeeding (F8R). The CMA activation score is calculated as a weighted average of the expression levels of all known CMA effectors, along with both positive and negative modulators (Fig. 1B), and has been established as a reliable proxy for CMA activation status (19, 21-26). In the hypothalamus, feed deprivation strongly modulated the expression of most CMA network components (Fig. 1C), resulting in a time-dependent increase in the CMA score (Fig. 1D), consistent with progressive CMA activation, whereas refeeding reversed this effect (Fig. 1,
<figure><img src="image_16.png" /></figure>

C and D). By contrast, the telencephalon showed no signifi-
cant changes in CMA score across conditions (Fig. 1, E and F),
indicating that CMA activity in this region is largely insen-
sitive to nutritional status. Interestingly, the cerebellum
exhibited an opposite response to that observed in the
hypothalamus, with a significant decrease in the CMA acti-
vation score at 8 days of feed deprivation (F8) (Fig. 1, G and
H). This suggests that CMA regulation in the cerebellum
may follow distinct, possibly region-specific, mechanisms
compared with the hypothalamus.

Pharmacological Activation of Hypothalamic CMA Reduces FI

To investigate the potential role of hypothalamic CMA
in regulating FI, we proceeded with a pharmacological
approach using a CMA activator known to increase the
expression of *LAMP2A* (21). CA (1 µg/body wt) or DMSO as
control were administered through ICV injection into the
third ventricle of RT, and the fish were feed-deprived until
tissue sampling were performed. To validate CA adminis-
tration and effect, we first measured the mRNA abundance
of both RT *lamp2a* paralogs located in chromosomes 14
and 31, respectively (*lamp2a* chr14 and *lamp2a* chr31) at 6,
24, and 48 h postinjection. The levels of *lamp2a* chr14
remained unchanged, whereas that of *lamp2a* chr31,
whose contribution to CMA appears to be critical (19, 39),
exhibited a significant increase at 6 h postinjection (Fig. 2,
A and B).

We then evaluated the effect of ICV administration of
CA on FI. To this end, RT injected with DMSO alone (con-
trol) or containing CA were feed-deprived for 48 h and
then hand-fed until visually satiated, with the amount of
total feed consumed subsequently recorded. As shown in
Fig. 2C, ICV administration of CA caused a significant
reduction in FI 48 h postinjection. This finding indicates
that activation of hypothalamic CMA can decrease feeding
behavior in RT, supporting its role in the central regula-
tion of energy balance.

To investigate the molecular basis of this effect, we meas-
ured relative mRNA abundance of key hypothalamic appe-
tite-regulating neuropeptides at 6, 24, and 48 h postinjection
(Fig. 2, D–K). Surprisingly, *agrp1*, a classical orexigenic gene,
was significantly upregulated by CA at 24 h compared with
the DMSO control (Fig. 2D), despite the observed reduction
in FI. In addition, mRNA levels of *agrp2*, *npy1a*, *pomca*,
*pomcb*, *cartpt*, *mc4ra*, and *mc4rb* remained unchanged at all
timepoints (Fig. 2, E–K). Together, these results suggest that
neither the increase in *agrp1* nor the unchanged expression
of the other canonical appetite-regulating genes contributes
substantially to the CA-induced reduction in feeding. These
findings suggest that the decrease in FI is not mediated by
classical hypothalamic neuropeptide pathways, or at least
not through the expression of the corresponding genes,
pointing to a novel role for CMA in the central regulation of
energy balance.

Intracerebroventricular CMA Activation Rep programs
Hypothalamic Amino Acid Metabolism

To further investigate the mechanisms underlying the
CA-induced decrease in FI following ICV injection, we

examined the mRNA abundance of key hypothalamic
genes involved in glucose, lipid, and amino acid metabo-
lism, given that cellular energetics is a central process
regulated by CMA (10). As shown in Fig. 3, the mRNA
expression levels of enzymes associated with glucose and
lipid metabolism did not differ between CA- and DMSO-
injected fish (Fig. 3, A–E). In contrast, genes related to
amino acid metabolism (bckde2) and transport (slc38a2
and both slc7a5 paralogs) were significantly upregulated
as early as 6 h postinjection (Fig. 3, F–I). This selective
response suggests that hypothalamic CMA activation pro-
motes amino acid catabolism and transport, potentially
contributing to suppressed FI by altering central nutrient
sensing.

To further assess this, we quantified hypothalamic-free
amino acid levels at 6 h postinjection. CA administration
significantly increased the concentrations of aspartic acid,
glutamine, cysteine, methionine, threonine, histidine, aspar-
agine, alanine, lysine, valine, leucine, and serine, whereas
proline was significantly downregulated (Fig. 4).

Collectively, these results demonstrate that ICV injection
of CA increases hypothalamic-free amino acid availability,
reinforcing the notion that CMA activation modulates cen-
tral amino acid metabolism, which may in turn influence
feeding behavior.

Proteomic Remodeling by CMA Coordinates Amino Acid Mobilization and Oxidative Phosphorylation in the Hypothalamus

Given that CMA functions as a proteolytic system, we next
examined whether the rise in hypothalamic amino acid lev-
els reflected proteome remodeling. We therefore performed
a quantitative proteomic analysis of hypothalamic samples
from CA- and DMSO-injected RT. In total, 12,955 proteins
were identified, of which 179 were significantly downregu-
lated and 220 were significantly upregulated (Fig. 5A and
Supplemental Tables S4 and S5).

To gain insight into the mechanisms underlying these
changes, we first performed a STRING functional protein
association network analysis of proteins downregulated by
CA, among which are likely direct CMA targets. This analysis
revealed a densely interconnected network enriched in pro-
teins involved in ribosomal RNA processing and ribosome
biogenesis (Fig. 5B and Supplemental Table S6). An additional
cluster included proteins associated with the spliceosome
complex. Complementary gene ontology (GO) enrichment
analysis confirmed these findings, highlighting a fall in ribo-
somal components and translational machinery (Fig. 5, C–E),
suggesting that CMA activation by CA may promote the deg-
radation of ribosomes and related proteins.

STRING network analysis of proteins upregulated by
CA, likely reflecting compensatory adaptations, revealed a
major cluster related to proton motive force-driven ATP
synthesis and oxidative phosphorylation, and a secondary
cluster related to ion transport by P-type ATPases (Fig. 5F
and Supplemental Table S6). Consistently, GO enrichment
analysis indicated significant overrepresentation of path-
ways related to mitochondrial energy metabolism and
membrane transport (Fig. 5G). Among the most enriched
functions were proton transmembrane transport (Fig. 5H),
<figure><img src="image_17.png" /></figure>

ATP biosynthetic process and oxidative phosphorylation (Fig. 5I). In addition, several GO terms related to ion and amino acid transport were enriched, including amino acid transport, inorganic cation transmembrane transport, and sodium/potassium ion homeostasis.

Together, these findings point to a potential role of ribosomal protein degradation in shaping hypothalamic amino acid availability, while being coupled to enhanced, likely compensatory, mitochondrial energy metabolism and trans-membrane transport capacity.

<figure><img src="image_18.png" /></figure>

lamp2a chr14

<figure><img src="image_19.png" /></figure>

lamp2a chr31

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

DMSO

<figure><img src="image_22.png" /></figure>

CA

<figure><img src="image_23.png" /></figure>

agrp1

<figure><img src="image_24.png" /></figure>

agrp2

<figure><img src="image_25.png" /></figure>

npy1a

<figure><img src="image_26.png" /></figure>

pomca

<figure><img src="image_27.png" /></figure>

pomcb

<figure><img src="image_28.png" /></figure>

cartpt

<figure><img src="image_29.png" /></figure>

mc4ra

<figure><img src="image_30.png" /></figure>

mc4rb
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

**Figure 3.** Transcriptional response of nutritional metabolism-related genes to ICV injection of the CMA activator. mRNA levels of several enzymes related to glucose (A and B), lipid (C–E), and amino acid (F–I) metabolisms in hypothalamus of RT, 6, 24, and 48 h after ICV injection of 1 µL·100 g⁻¹ body wt of either DMSO alone (10 µL/kg body wt) or containing the CMA activator (CA, 1 µg/kg body wt) (n = 5–7, values are means ± SE-fold induction respect to DMSO group). A t test was performed comparing mRNA levels in DMSO and CA-treated RTs at each sampling time (\*\*P < 0.01; \*\*\*P < 0.001). *acy*, ATP citrate lyase; *bckde2*, branched-chain keto acid dehydrogenase subunit E2; CMA, chaperone-mediated autophagy; DMSO, dimethyl sulfoxide; *fasn*, fatty acid synthase; FI, feed intake; *gcka* and *gckb*, glucokinase a and b; ICV, intracerebroventricular; *me1*, malic enzyme 1; RT, rainbow trout; *slc38a2*, solute carrier family 38 member 2; *Slc7a5* solute carrier family 7 member 5.

## Selective Downregulation of KFERQ-Like Motif Proteins Underlies CMA-Associated Hypothalamic Proteome Remodeling

Finally, to explore functional features associated with hypothalamic proteome remodeling, we analyzed the

presence of CMA-targeting motifs in the differentially regulated proteome using the KFERQ Finder app v.0.8 (40) (Supplemental Table S7). As CMA selectively targets proteins with KFERQ-like motifs for degradation, motif enrichment was expected among downregulated proteins. Consistent with this, a significantly higher proportion of

**Figure 2.** Modulation of FI by ICV injection of a pharmacological activator of CMA. A and B: mRNA abundance of both *lamp2a* in hypothalamus of RT, 6, 24, and 48 h after ICV injection of 1 µL·100 g⁻¹ body wt of either DMSO alone (10 µL/kg body wt) or containing the CMA activator (CA, 1 µg/kg body wt). Values are presented as means ± SE of *n* = 5–7 replicates. \*\*P < 0.01; t test. C: average FI in RT 48 h after ICV treatments with DMSO or CA. FI is shown as the percentage of feed ingested with respect to baseline levels (calculated as the average of FI recorded 7 days before experiment) and was normalized to control group (100%). The results are shown as means ± SE, with the tank as the experimental unit (n = 4 tanks/treatment, 10 fish/tank). \*P < 0.05; t test. D–K: mRNA abundance of neuropeptides-coding genes in hypothalamus from DMSO or CA-injected RTs (n = 5–7, values are means ± SE). A t test was performed comparing mRNA levels between DMSO or CA-treated RTs at each sampling time (\*\*P < 0.05). *agrp1* and *agrp2*, agouti-related proteins 1 and 2; *cartpt*, cocaine- and amphetamine-regulated transcript, peptide; CMA, chaperone-mediated autophagy; DMSO, dimethyl sulfoxide; FI, feed intake; ICV, intracerebroventricular; *mc4ra* and *mc4rb*, melanocortin 4 receptor a and b; *npy1a*, neuropeptide y a; *pomca* and *pomcb*, pro-opiomelanocortin a and b; RT, rainbow trout.
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Figure 4.** ICV CMA activation reprograms hypothalamic amino acid metabolism. Volcano plot of hypothalamic-free amino acid levels in RTs 6 h after ICV injection of 1 µL·100 g⁻¹ body wt of either DMSO alone (10 µL/kg body wt) or containing the CMA activator (CA, 1 µg/kg body wt) (n = 3; values represent mean, normalized to hypothalamic tissue weight). t test was performed to compare amino acid levels between groups. Significantly upregulated amino acids (FDR-adjusted *P* < 0.05; fold change > 2) are shown in green, significantly downregulated amino acids (FDR-adjusted *P* < 0.05; fold change < 0.5) in red, and nonsignificant changes in gray. Amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), valine (Val). CMA, chaperone-mediated autophagy; DMSO, dimethyl sulfoxide; ICV, intracerebroventricular; RT, rainbow trout.</figcaption></figure>

downregulated proteins contained KFERQ-like motifs compared with the RT proteome (P = 0.001, χ² test), and motif enrichment was also significantly greater in downregulated than in upregulated proteins (P = 0.02, χ² test) (Fig. 6A). These findings underscore the plausible direct engagement of CMA in orchestrating the degradation of downregulated proteins and further suggest evolutionary conservation of the KFERQ-like motif across vertebrates.

Among KFERQ-type motifs, a distinction is made between canonical sequences and those generated through post-translational modifications (PTMs), such as phosphorylation or acetylation (40). Canonical motifs accounted for the majority of motif-bearing proteins and showed a marked enrichment among downregulated proteins, indicating that they represent the principal substrates of CMA in the hypothalamus of CA-injected fish (Fig. 6B and Supplemental Table S7). PTM-dependent motifs were also detected, with phosphorylation being more frequent than acetylation, although both occurred at much lower levels compared with canonical motifs, suggesting that PTMs may act as secondary regulators that modulate CMA substrate selection. Taken together, these findings suggest that hypothalamic CMA primarily targets proteins harboring canonical KFERQ motifs, whereas PTM-dependent motifs provide an additional layer of regulation to fine-tune proteome remodeling.

To further investigate regulatory mechanisms, we analyzed KFERQ motifs within the functionally enriched biological processes of downregulated proteins. A substantial fraction of proteins across multiple GO categories contained KFERQ-like motifs, supporting their potential recognition as

CMA substrates (Fig. 6C and Supplemental Table S7). The distribution of canonical, acetylated, and phosphorylated motifs, however, varied across categories. Notably, proteins involved in mRNA splicing and cytoplasmic translation showed a higher prevalence of acetylated motifs, whereas phosphorylated KFERQ-containing proteins were comparatively represented across pathways. In contrast, more than half of the proteins linked to ncRNA processing carried canonical motifs, followed by those associated with rRNA processing (Fig. 6C). Together, these findings suggest that motif recognition, in combination with PTM-dependent regulation, underlies the selective targeting of proteins for CMA during hypothalamic proteome remodeling.

## DISCUSSION

The regulation of appetite is a fundamental physiological process that allows organisms to maintain energy homeostasis in response to internal nutrient cues and external environmental changes (41, 42). Central to this process is the hypothalamus which integrates hormonal and nutrient signals to orchestrate feeding behavior (41, 43). Although considerable progress has been made in understanding neuropeptide- and hormone-driven control of appetite (44), the intracellular mechanisms by which hypothalamic neurons sense and adapt to nutrient availability remain less well characterized.

Our findings provide new insight into this gap by demonstrating that CMA, a selective lysosome-dependent degradation pathway, contributes to hypothalamic nutrient sensing and the control of feeding behavior. Using RT as a model, we demonstrate that hypothalamic CMA is selectively responsive to nutritional status, being upregulated during feed deprivation and rapidly downregulated upon refeeding. This region-specific regulation highlights the hypothalamus as a central hub where CMA dynamically responds to energy deficits, complementing previous reports showing the sensitivity of hypothalamic neurons to metabolic stress through MA (7–9). Importantly, our work extends these observations to CMA, a selective autophagic pathway whose functional contribution to central energy homeostasis has been largely unexplored. Previous studies in mice have suggested such a role: Portovedo et al. (13) reported that high-fat diet feeding or palmitic acid exposure alters hypothalamic LAMP2A and HSC70 expression, and Espinosa et al. (14) demonstrated that saturated fatty acids suppress CMA in POMC-like neurons, impairing insulin signaling. Although these studies hinted at CMA involvement, direct in vivo evidence linking hypothalamic CMA to feeding behavior was lacking, evidence our study now provides.

To explore the role of hypothalamic CMA in regulating feeding behavior, we used a pharmacological strategy using a CMA activator shown to enhance LAMP2A expression (21). It should be noted that CA, while primarily increasing LAMP2A expression, is a small-molecule compound that could theoretically influence off-target pathways or broader cellular stress responses. Therefore, one cannot exclude that some of the observed effects might reflect unintended or compensatory actions rather than being solely attributable to CMA activation. However, to date, no off-target effects have been reported in studies
<figure><img src="image_43.png" /></figure>

using this compound, supporting its relative specificity for CMA activation. ICV administration of the CMA activator significantly reduced FI, indicating that CMA activity in this region can modulate feeding behavior. These findings

suggest that CMA functions not only as a metabolic sensor but also as an active effector that directs energy balance through the regulation of hypothalamic signaling circuits. Notably, the observed anorexigenic effect was associated

A

<figure><img src="image_44.png" /></figure>

C

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F

<figure><img src="image_46.png" /></figure>

B

<figure><img src="image_47.png" /></figure>

D

<figure><img src="image_48.png" /></figure>

E

<figure><img src="image_49.png" /></figure>

G

<figure><img src="image_50.png" /></figure>

H

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

I

<figure><img src="image_53.png" /></figure>
<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /><figcaption>**Figure 6.** Frequency and types of KFERQ-like motifs in the hypothalamic proteome affected by CMA activation. A: percentage of CA-modulated hypothalamic proteins and of the total RT proteome with or without at least one KFERQ-like motif. A $\chi^2$ test was performed comparing the different groups (*P* < 0.05; **P** < 0.01). B: distribution of KFERQ-like motif classes among CA-modulated hypothalamic proteins and the total RT proteome. Motifs are ranked in the following order: canonical > phosphorylation-generated > acetylation-generated. Proteins containing multiple motif types were assigned to the group corresponding to the highest-ranking motif. C: relative abundance of the indicated KFERQ-like motif classes within functionally enriched biological processes among downregulated proteins. CMA, chaperone-mediated autophagy; RT, rainbow trout.</figcaption></figure>

with selective changes in amino acid metabolism. CMA activation increased the hypothalamic concentrations of several free amino acids, including branched-chain amino acids, and upregulated genes involved in amino acid catabolism and transport. These results align with, but also extend, a growing body of evidence demonstrating that central amino acid availability directly influences feeding behavior. Pioneering work first reported in rats that direct administration of a balanced mixture of amino acids into the hypothalamic parenchyma reduced food intake within 1 h of injection (45). This early observation was subsequently reinforced by several studies showing that administration of physiologically relevant amounts of leucine into the third ventricle or discrete nutrient-sensing regions of fasted rodent brains reduces energy intake

during the refeeding period (46–49). Beyond mammals, evidence from avian and fish models indicates that hypothalamic amino acid sensing is a conserved feature of vertebrate energy balance, albeit with species-specific outcomes. In avian models for example, ICV injection of leucine stimulates FI while upregulating the expression of orexigenic neuropeptides such as *NPY* and *AGRP*, whereas glutamate suppresses feeding and increases expression of anorexigenic markers including *CRH* and *MC4R* (50). Similarly, methionine, lysine, and taurine reduce feeding in chicks, with taurine in addition to enhancing *POMC* and *MC3R* expression. In fish, comparable complexity has been reported: ICV leucine decreases FI in RT and is accompanied by suppression of *npy/agrp* and stimulation of *pomc* mRNA abundance (51), whereas ICV valine exerts

**Figure 5.** ICV administration of the CMA activator induces extensive remodeling of the hypothalamic proteome. A: volcano plot of the quantitative proteomics analysis of hypothalamus of RTs 48 h after ICV injection of 1 µL·100 g⁻¹ body wt of either DMSO alone (10 µL/kg body wt) or containing the CMA activator (CA, 1 µg/kg body wt). Top left: number of identified proteins (n) and significantly downregulated hits. Top right: number of significantly upregulated hits. Red and green dots indicate significantly down- and upregulated proteins, respectively (*P* < 0.05 and fold change > 1.4). B: STRING functional protein association network analysis of downregulated proteins with CA. C: gene ontology analysis of downregulated proteins with CA. D and E: heatmaps of proteins significantly downregulated by CA within subsets associated with ribosome biogenesis (D) and the spliceosome complex (E) (n = 4/condition). F: STRING functional protein association network analysis of upregulated proteins with CA. G: gene ontology analysis of upregulated proteins with CA. H and I: heatmaps of proteins significantly upregulated by CA within subsets associated with ion transmembrane transport (H) and oxidative phosphorylation (I) (n = 4/condition). CMA, chaperone-mediated autophagy; ICV, intracerebroventricular; RT, rainbow trout.
<figure><img src="image_56.png" /></figure>

the opposite effect by stimulating feeding (51, 52). Together, these studies highlight that hypothalamic amino acid avail- ability can shape appetite regulation in multiple vertebrate species, though the directionality and underlying mecha- nisms differ according to the amino acid and context. In the present study, we clearly observed upregulation of mRNA abundance of several markers of amino acid metabolism and transport such as *bckde2*, *slc38a2*, and *slc7a5* paralogs. These changes are comparable to those observed in the same spe- cies when ICV treatment with leucine resulted in decreased FI (51). However, in contrast to available studies in the same species where ICV treatment with amino acids resulted in parallel changes in FI and the mRNA levels of the classical hypothalamic neuropeptides (e.g., *npy*, *agrp*, *pomc*, and *cartpt*), we did not observe such parallel responses. The only neuropeptidergic change detected was an unexpected upregulation of the orexigenic gene *agrp1* at 24 h, despite the reduction in FI. This may reflect a compensatory tran- scriptional response to reduced AgRP neuronal activity or peptide release, or differential effects of CMA activation on transcriptional versus translational control. Although the precise mechanism remains to be established, these possi- bilities provide a framework to interpret this apparent para- dox. However, we cannot exclude that this *agrp1* response, together with the lack of changes in other hypothalamic neuropeptides, could also be related to differences in the timing of tissue collection or other experimental factors compared with previous studies. In any case, our data rein- force the notion that hypothalamic amino acid dynamics are closely linked to the control of feeding behavior and point to CMA as a novel player in this regulatory network, with the precise nutrient-sensing mechanisms involved yet to be determined.

To further investigate the origin of the increased hypo-
thalamic-free amino acid levels observed after ICV CMA acti-
vation, we performed an unbiased proteomic analysis. Our
results provide mechanistic insight into how CMA repro-
grams hypothalamic metabolism. Proteins downregulated
by CMA were enriched for components of the ribosome
and translational machinery, whereas upregulated pro-
teins, likely reflecting compensatory adaptations, clus-
tered around oxidative phosphorylation and ion transport
pathways. Given that ribosomes constitute one of the larg-
est cellular protein pools, their selective degradation could
contribute to the observed increases in hypothalamic
amino acid levels during nutrient stress (28, 29), although
direct flux measurements will be required to confirm that
ribophagy or CMA-mediated ribosomal degradation is
indeed the source of these liberated amino acids. These
amino acids can then fuel mitochondrial energy produc-
tion. Altogether, these findings highlight CMA as a critical
mediator of metabolic adaptation, coordinating amino
acid mobilization with enhanced energy metabolism to
maintain homeostasis under nutrient stress, consistent
with mechanisms previously described in other models
(10). Importantly, downregulated proteins were highly
enriched in KFERQ-like motifs, highlighting CMA's selec-
tive targeting and the evolutionary conservation of this
recognition signal. Canonical motifs accounted for the
majority of these proteins, whereas PTM-modified motifs,
such as phosphorylation and acetylation, likely provide an

additional layer of regulatory specificity (10, 40, 53). Functional enrichment analysis further revealed that proteins involved in mRNA translation, splicing, and gene expression were preferentially targeted, suggesting that CMA fine-tunes proteome composition to rapidly adjust cellular metabolism. Although this motif enrichment supports CMA's potential role, the presence of KFERQ-like motifs alone does not constitute direct proof of CMA-mediated degradation, and future studies will be needed to experimentally validate the primary CMA substrates in this species.

Together, our results position CMA as a selective and evo-
lutionarily conserved regulator of hypothalamic proteome
composition and feeding behavior. By coupling targeted pro-
tein degradation with amino acid mobilization and energy
metabolism, CMA emerges as a central hub-linking nutrient
status to hypothalamic control of appetite and energy home-
ostasis. While MA is also involved in feeding regulation
through targeted or bulk degradation of organelles and cyto-
plasmic components, and endosomal microautophagy (eMI)
shares substrate recognition of KFERQ-like motifs with
CMA, their respective contributions to hypothalamic nutri-
ent sensing remain unclear. Future studies should explore
how CMA interacts with these complementary autophagic
pathways and delineate the downstream neuroendocrine
mechanisms linking CMA-mediated amino acid mobiliza-
tion to appetite regulation.

DATA AVAILABILITY

The mass spectrometry proteomics data have been deposited
to the ProteomeXchange Consortium through the PRIDE partner
repository with the dataset identifier PXD068293. Supplemental
Tables S1–S7 and Supplemental Fig. S1 as well as all other data-
sets generated during and/or analyzed during the current study
are openly available, along with their statistic report, in the
Recherche Data Gouv repository, at https://doi.org/10.57745/
DXM3CB.

SUPPLEMENTAL MATERIAL

Supplemental Tables S1–S7 and Supplemental Fig. S1: https://
doi.org/10.57745/DXM3CB.

ACKNOWLEDGMENTS

We thank F. Terrier, A. Lanuque, and B. Disazeaux from the
INRAE experimental facility (Donzacq, France) for the preparation
of diets and care of fish. We also thank C. Fernández from
Universidade de Vigo for helping during ICV experiments.

GRANTS

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska Curie actions, Grant Agreements Nos. 945416 and 101030643 and Aquaexcel3.0 Grant Agreement No. 871108. It was also supported in part by the French Ministry of Research through the "Infrastructures Nationales en Biologie et Santé" program to the Proteomics French Infrastructure, ProFI Grants ANR 10-INBS-08 and ANR-24-INBS-0015 (to O.B.S.). The Spanish Agencia Estatal de Investigación and the European Regional
<figure><img src="image_57.png" /></figure>

Development Fund Grant PID2022-1362880B-C31 and Xunta de Galicia Grant ED431C 2025/38 (to J.L.S.).

## DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

## AUTHOR CONTRIBUTIONS

S.R., E.J.V., A.M.B., J.L.S., and I.S. conceived and designed research; S.R., E.J.V., A.M.B., C.H., V.V., K.D., A.S., O.B.-S., and S.S. performed experiments; S.R., E.J.V., A.M.B., C.H., V.V., K.D., A.S., O.B.-S., and S.S. analyzed data; S.R., E.J.V., A.M.B., C.H., V.V., K.D., A.S., O.B.-S., S.S., J.R., F.B., B.C., J.L.S., and I.S. interpreted results of experiments; S.R. and I.S. prepared figures; S.R. and I.S. drafted manuscript; S.R., E.J.V., A.M.B., S.S., J.R., F.B., B.C., J.L.S., and I.S. edited and revised manuscript; S.R., E.J.V., A.M.B., C.H., V.V., K.D., A.S., O.B.-S., S.S., J.R., F.B., B.C., J.L.S., and I.S. approved final version of manuscript.

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