Fasting, Exercise, and Diet to Activate Autophagy in Depression

Background and Rationale Depressive disorders are among the leading causes of disability worldwide and represent a major public health burden. Despite the availability of pharmacological and psychotherapeutic treatments, a substantial proportion of patients do not achieve full remission or experience relapse. Current antidepressant strategies primarily target monoaminergic systems and are often insufficient in addressing the biological heterogeneity of depression.

Emerging evidence suggests that metabolic regulation and cellular stress response pathways may play an important role in the pathophysiology of depression. In particular, associations between metabolic disorders (such as obesity and insulin resistance) and depressive symptoms indicate shared biological mechanisms. This has led to increasing interest in lifestyle-based interventions, including physical activity, dietary modification, and caloric restriction, as potential modulators of both metabolic and neuropsychiatric outcomes.

A central candidate mechanism linking metabolism and brain function is autophagy, a conserved cellular process responsible for the degradation and recycling of damaged proteins and organelles. Autophagy is tightly regulated by nutrient availability and energy status, primarily via the AMPK-mTOR signaling axis. It is activated under energy deprivation and suppressed under nutrient excess. Proper autophagic flux is essential for neuronal homeostasis, immune regulation, and cellular stress adaptation.

Preclinical and emerging clinical evidence suggests that impaired autophagy may be involved in psychiatric disorders, including depression. Furthermore, interventions such as physical exercise, caloric restriction, and certain pharmacological agents have been shown to modulate autophagy-related pathways. However, the direct measurement of autophagic flux in humans under physiological conditions remains methodologically challenging, and its relationship to exercise-induced metabolic and neurobiological changes is not fully understood.

This study aims to address this gap by investigating autophagy-related biological responses to acute physical exercise in humans using a multi-omics approach.

Objectives Primary Objective To investigate whether acute physical exercise induces measurable changes in autophagy-related pathways and associated metabolic, proteomic, transcriptomic, and hormonal markers in humans.

Secondary Objectives To characterize exercise-induced changes in systemic metabolism, inflammatory markers, and stress hormones.

To explore associations between fitness level, body mass index (BMI), and molecular responses to exercise.

To identify potential biomarkers of autophagy activation in peripheral blood and saliva.

To generate mechanistic hypotheses linking metabolic regulation, autophagy, and pathways relevant to mood disorders.

Study Design This is a single-center, exploratory human research study conducted at the University Hospital Zurich in collaboration with exercise physiology facilities.

The study uses a cross-sectional experimental design involving standardized acute exercise stimulation (cardiopulmonary exercise testing, CPET) combined with repeated biological sampling and multi-omics profiling.

Participants will be stratified into four groups based on sex and BMI:

Normal-weight women Normal-weight men Overweight women Overweight men All participants will perform a standardized incremental cycling exercise test under controlled laboratory conditions.

Study Population Approximately 120 healthy adults aged 18-40 years will be included. Participants will be selected based on predefined inclusion and exclusion criteria to ensure medical safety and reduce confounding variables such as chronic disease, medication use, psychiatric disorders, and hormonal influences (e.g., hormonal contraception or pregnancy in women).

Women will be tested during early follicular phase (cycle days 1-5) to minimize hormonal variability.

Study Procedures

Each participant will undergo:

1. Screening and Baseline Assessment Informed consent Medical history and physical screening Assessment of inclusion/exclusion criteria Questionnaires assessing mood, anxiety, and physical activity Serological screening for HIV and hepatitis B/C 2. Physiological Measurements (Pre-exercise) Body composition analysis (DXA) Lung function testing Baseline blood sampling Saliva sampling for cortisol 3. Exercise Intervention (CPET) Participants will perform a standardized graded cycling exercise test on an electromagnetically braked ergometer.

The protocol includes:

15-minute warm-up phase at submaximal intensity Incremental ramp protocol until voluntary exhaustion

Continuous monitoring of:

Oxygen uptake (VO₂) Carbon dioxide production (VCO₂) Heart rate and ECG Blood pressure Respiratory exchange ratio

Key physiological thresholds will be determined:

Aerobic threshold Anaerobic threshold Respiratory compensation point 4. Biological Sampling

Repeated biological sampling will be performed at defined time points:

Rest (baseline) End of warm-up (aerobic phase) Peak exercise (maximal exertion) 10 minutes recovery 30 minutes recovery

Samples include:

Venous blood (PBMC isolation and plasma) Capillary blood microsamples (fingertip sampling devices) Saliva (cortisol analysis) Urine (pregnancy test in women) Total blood volume per participant will be approximately 320 mL across all time points.

Laboratory Analyses

Collected samples will be used for multi-layered molecular profiling:

1. Autophagy-Related Analyses LC3B-II-based flux assays in PBMCs Ex vivo stimulation assays with lysosomal inhibition (chloroquine-based approach) Quantification of autophagy-related proteins (e.g., ATG family, ULK1 pathway components) Gene expression profiling of autophagy signaling pathways 2. Metabolomics and Lipidomics Targeted and untargeted metabolomic profiling Energy substrates and oxidative stress markers Polyamine metabolism (e.g., spermidine-related pathways) Steroid hormone profiling via mass spectrometry 3. Proteomics and Transcriptomics Plasma and PBMC proteomic profiling (untargeted and targeted) Phosphoproteomic analysis Single-cell or bulk RNA sequencing of immune cells 4. Inflammatory and Immune Markers Cytokine quantification (e.g., IL-1β, IL-6, IL-10, TNF-α) Markers of immune activation and systemic inflammation 5. Hormonal and Stress Response Measures Cortisol (saliva and plasma) Sex steroid hormones (e.g., estradiol, testosterone, progesterone) Hypothalamic-pituitary-adrenal (HPA) axis-related markers 6. Genomic and Epigenetic Analyses DNA damage mapping (e.g., oxidative lesions, strand breaks) DNA methylation profiling (EPIC array) Gene regulation changes in response to exercise-induced stress

• PBMC Transcriptome [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• PBMC Proteome [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• PBMC Phosphoproteome [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Plasma Proteome [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Targeted Plasma Metabolomics [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Untargeted Metabolomic Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Polyamine Concentrations [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Steroid Hormone Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Inflammatory Marker Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Genome-wide DNA Oxidation Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• Genome-wide DNA Strand Break Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]
• DNA Methylation Profile [Time frame: Baseline (rest), end of aerobic warm-up phase, peak exercise, 10 minutes post-exercise, and 30 minutes post-exercise.]