Phenethylamine in chlorella alleviates high-fat diet-induced mouse liver damage by regulating generation of methylglyoxal

The study investigates whether trace amounts of phenethylamine (PHA), identified in hot water extract of Chlorella pyrenoidosa (WEC), can alleviate high-fat diet (HFD)-induced oxidative stress and liver damage in mammals. Prior work showed PHA at very low doses extended lifespan in Drosophila Sod1 mutants, suggesting antioxidant-related benefits. HFD promotes hepatic lipid deposition, lipid peroxidation, reduced antioxidant enzyme activities, and progression to NAFLD/NASH. Methylglyoxal, produced from glycolytic intermediates (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) normally metabolized by GAPDH, increases intracellular oxidative stress and forms AGEs, contributing to liver pathology. The hypothesis is that trace PHA can mitigate HFD-induced oxidative liver damage by reducing methylglyoxal via increasing hepatic GAPDH, thereby decreasing lipid peroxidation and liver injury.

Background literature highlights: (1) Chlorella pyrenoidosa and its extracts show anti-dyslipidemic and immunomodulatory effects in animals and humans; (2) PHA, a monoamine present in various foods and endogenously derived from phenylalanine, has been used with MAO-B inhibitors for depression, but high doses can cause adverse neurological effects in rodents; (3) HFD-induced NAFLD is associated with increased lipid deposition, lipid peroxidation, and diminished antioxidant defenses; (4) Methylglyoxal elevates oxidative stress and forms AGEs, exacerbating steatosis to NAFLD/NASH; (5) Food compounds can inhibit AGE formation, yet effectively suppressing methylglyoxal generation in vivo remains challenging. These works set the context for testing whether very low-dose PHA modulates GAPDH–methylglyoxal axis to protect the fatty liver.

Design: Male C57BL/6J mice (7 weeks old, 21–23 g) were acclimated then randomized (n=6/group) into four groups for 12 weeks: ND (normal diet), HFD (high-fat diet), WEC (HFD plus water extract of Chlorella), and PHA (HFD plus phenethylamine). Dosing: WEC 100 mg/kg bodyweight in drinking water; PHA 10 µg/kg bodyweight in drinking water, chosen based on prior Drosophila dosing and mouse intake (~12 µg/kg estimated). Diets: ND vs HFD (High-fat diet 32; 32% crude fat; ~60% calories from fat). Intake of food and water monitored; WEC/PHA additions did not alter water intake. Duration: 12 weeks; euthanasia in morning without fasting. Sample collection: Blood from inferior vena cava (heparinized), plasma separated; liver excised and perfused with cold PBS; samples stored at −30 °C. Outcomes and assays: - Body and liver metrics: bodyweight progression, liver weight, liver-to-bodyweight ratio; hepatic triglycerides (TG) by isopropanol extraction and TG kit. - Plasma biochemistry: AST, ALT, LDL-C, HDL-C, total cholesterol (TC), TG (outsourced clinical assays). - Hepatic lipid peroxidation: TBARS assay using TBA with DTPA/BHA; calibration with TEP-derived malondialdehyde standards. - Antioxidant capacity: SOD-like activity (WST-1-based assay) and GPX-like activity (colorimetric kit) from liver extracts; low-molecular-weight ethanolic fraction tested for SOD-like activity (negligible). - Methylglyoxal: LC-MS/MS after derivatization with 2,3-diaminonaphthalene (DAN); ethyl acetate extraction, ODS-3 column, MRM with acidified aqueous/acetonitrile gradient. - Western blotting: Proteins extracted from liver; due to HFD-lowered GAPDH and β-actin, pre-stained markers (PSM) near target sizes were used for normalization (35 kDa for GAPDH/β-actin, 30 kDa for SOD-1/GPX-1). Primary antibodies against SOD-1, GPX-1, GAPDH; HRP-conjugated secondary antibodies; chemiluminescence detection; band intensities normalized to PSM and common HFD control across gels. - Cysteine (reduced and total): LC-MS/MS with derivatization using 4-vinylpyridine (4-VP) and AccQ; stable isotope internal standard (L-cysteine-13C3,15N); results indicated most hepatic cysteine was in reduced form (no change after DTT). - Statistics: Data as mean ± SD; one-way ANOVA with Dunnett’s test vs HFD; significance p<0.05; trend 0.05<p<0.1. Ethics: Procedures approved by Animal Care Committee (No. 20192).

- HFD successfully induced obesity, fatty liver, liver damage, elevated LDL-C, increased hepatic lipid peroxidation, and reduced SOD/GPX activities. - PHA (10 µg/kg) effects: • Did not significantly change bodyweight or liver weight; liver-to-bodyweight ratio tended lower vs HFD (p<0.1). • Slight but significant reduction in hepatic TG (p<0.05). • Significantly reduced plasma AST and ALT (p<0.05), indicating less liver injury. • Significantly reduced plasma LDL-C and total cholesterol (p<0.05) without affecting HDL-C; plasma TG unchanged. • Significantly reduced hepatic lipid peroxidation (TBARS) (p<0.05). • Significantly increased hepatic SOD-like and GPX-like activities (p<0.05) without increasing SOD-1 or GPX-1 protein levels. • Significantly increased hepatic GAPDH protein levels (p<0.05). • Significantly decreased hepatic methylglyoxal levels (p<0.05). • Significantly increased hepatic cysteine to near ND levels (p<0.05). - WEC (100 mg/kg) showed similar trends but generally weaker effects: significant decreases in LDL-C and TBARS; increases in SOD-like and GPX-like activities; significant increase in GAPDH; significant decrease in methylglyoxal; significant increase in hepatic cysteine; no significant change in hepatic TG or plasma AST/ALT. - The hepatic methylglyoxal level did not differ between ND and HFD, but was significantly reduced by PHA and WEC versus HFD. - Western blot showed HFD reduces housekeeping proteins (notably GAPDH), necessitating normalization to pre-stained markers; PHA and WEC restored GAPDH.

Trace-dose PHA mitigated HFD-induced liver oxidative damage primarily by modulating endogenous metabolic and antioxidant pathways rather than acting as a direct antioxidant. By increasing hepatic GAPDH, PHA likely reduced the pool of glycolytic triose phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate), thereby decreasing formation of methylglyoxal, a reactive carbonyl that elevates oxidative stress and forms AGEs. Lower methylglyoxal appears to reduce oxidation of accumulated hepatic lipids, decreasing malondialdehyde (TBARS) and protecting the hepatic cysteine pool. Preserved cysteine may help maintain activities of sulfhydryl-dependent antioxidant enzymes (SOD/GPX), explaining the increased SOD-like and GPX-like activities despite unchanged protein levels. Thus, PHA interrupts a proposed cycle in fatty liver where reduced GAPDH increases methylglyoxal, which triggers lipid peroxidation and damages antioxidant systems, culminating in liver injury. WEC recapitulates these effects—likely due in part to its PHA content—though with lower potency at the tested dose. The findings introduce a GAPDH–methylglyoxal axis as a nutritional target for alleviating NAFLD features with exceedingly low doses of a dietary amine.

Trace amounts of phenethylamine (10 µg/kg) and Chlorella water extract (100 mg/kg) attenuate HFD-induced hepatic oxidative stress and liver injury in mice, primarily by increasing hepatic GAPDH, reducing methylglyoxal, decreasing lipid peroxidation, and preserving hepatic cysteine and antioxidant enzyme activities. These benefits occur with minimal impact on hepatic fat accumulation and bodyweight. The data support a novel nutritional approach targeting the GAPDH–methylglyoxal pathway to protect fatty liver. Future research should elucidate the molecular mechanism by which PHA upregulates GAPDH (e.g., roles of hypoxia-inducible factor and related regulators), determine the mechanism underlying LDL-C reduction by trace PHA, assess dose–response and safety, test efficacy in diverse NAFLD/NASH models and in female mice, and evaluate translational relevance in human studies.

- Sample size was modest (n=6/group) and limited to male C57BL/6J mice. - Western blot normalization required pre-stained markers because HFD altered housekeeping proteins (GAPDH, β-actin), which may introduce variability. - Mechanistic causality for GAPDH upregulation by PHA was not established; regulators (e.g., HIF) were not measured. - Methylglyoxal-related AGEs and comprehensive oxidative stress markers beyond TBARS were not quantified. - The study duration and single-dose design limit dose–response and long-term safety assessment; potential off-target effects of PHA were not evaluated. - WEC contains multiple components; while PHA likely contributes, exact attribution of WEC effects remains inferential. - Findings in mice may not directly generalize to humans.