Chlorella pyrenoidosa, a freshwater green alga, and its water extract (WEC) have shown various biological activities, including anti-dyslipidemic and immunomodulatory effects. Previous research identified phenethylamine (PHA) in WEC and demonstrated its lifespan-extending effects in Drosophila at low doses, suggesting amelioration of oxidative stress. PHA is found in various foods, and while high doses have shown adverse effects in mammals, the effects of low-dose PHA remain unexplored. This study aimed to investigate the beneficial effects of trace amounts of PHA in mammals, specifically focusing on its impact on high-fat diet (HFD)-induced liver damage. HFD is known to cause hepatic damage through lipid deposition, lipid peroxidation, and decreased antioxidant enzyme activities, contributing to non-alcoholic fatty liver disease (NAFLD). Methylglyoxal, a reactive compound generated from glucose and fructose metabolism, has been implicated in increasing intracellular oxidative stress and contributing to NAFLD progression. This study hypothesized that trace amounts of PHA could mitigate HFD-induced oxidative stress and liver damage by modulating methylglyoxal levels via its interaction with GAPDH.

Previous studies have shown that *Chlorella pyrenoidosa* and its extracts improve dyslipidemia in rats and hamsters fed a high-fat diet. Clinical trials have also reported immunomodulatory effects of chlorella. The research team previously reported that trace amounts of phenethylamine (PHA) extend the lifespan of *Sod1* mutant *Drosophila melanogaster*, indicating a role in ameliorating oxidative stress. Studies have also shown that PHA supplementation, combined with a monoamine oxidase inhibitor, alleviates depression in humans, while high doses cause adverse effects in mice. Regarding HFD-induced liver damage, research highlights increased lipid deposition, lipid peroxidation, and reduced antioxidant enzyme activity. Methylglyoxal is identified as a key player in this process, inducing oxidative stress and contributing to NAFLD progression. While some food compounds show inhibitory effects on advanced glycation end products (AGEs) formation, directly inhibiting methylglyoxal generation remains challenging.

Male C57BL/6J mice were divided into four groups (n=6): a normal diet (ND) group, a high-fat diet (HFD) group, an HFD group treated with WEC (100 mg/kg bodyweight), and an HFD group treated with PHA (10 µg/kg bodyweight). The mice were fed their respective diets for 12 weeks. Plasma and liver samples were collected for biochemical analyses. Plasma AST, ALT, TG, TC, HDL-C, and LDL-C levels were measured. Hepatic TG levels were determined using an enzymatic assay. Lipid peroxidation was assessed using the thiobarbituric acid reactive substances (TBARS) assay. Hepatic SOD-like and GPX-like activities were measured using commercially available kits. Hepatic methylglyoxal levels were determined using LC-MS/MS after derivatization with DAN. Western blotting was used to analyze the protein levels of SOD-1, GPX-1, and GAPDH. Hepatic cysteine levels (reduced and oxidized forms) were measured using LC-MS/MS after derivatization with 4-VP and AccQ. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test.

HFD feeding induced obesity, fatty liver, liver damage, and increased plasma LDL-C, lipid peroxidation, and decreased antioxidant enzyme activities. PHA significantly reduced hepatic lipid peroxidation and liver damage (as indicated by plasma AST and ALT levels), and plasma LDL-C, without significantly decreasing hepatic lipid accumulation. WEC showed similar effects but with less potency. PHA and WEC significantly reduced hepatic methylglyoxal levels and significantly increased hepatic GAPDH protein levels. While SOD-1 and GPX-1 protein levels were not significantly affected, HFD significantly decreased GAPDH protein levels, which was reversed by PHA and WEC administration. Hepatic cysteine levels, which were lower in the HFD group, were significantly increased by PHA and WEC administration. The results indicate that PHA exerts its beneficial effects not by direct antioxidant activity but rather by modulating the expression of endogenous proteins such as GAPDH.

This study demonstrates that trace amounts of PHA, found in WEC, effectively mitigate HFD-induced liver damage in mice. The mechanism appears to involve the upregulation of GAPDH, which reduces methylglyoxal levels, a reactive aldehyde implicated in lipid oxidation and subsequent liver injury. The increase in hepatic cysteine levels further suggests a protective role against the damaging effects of aldehydes. The findings contradict previous research showing adverse effects of higher PHA doses, highlighting the importance of dose-dependent effects. The low dose used in this study is achievable through consumption of various fermented foods, suggesting a potential dietary approach to improve liver function. Future research should focus on the specific mechanisms by which PHA modulates GAPDH expression, potentially involving hypoxia-inducible factor (HIF). Further investigation into the LDL-C-lowering effect of PHA is also warranted.

This research reveals a novel mechanism by which trace amounts of phenethylamine (PHA) alleviate high-fat diet-induced liver damage in mice. PHA's beneficial effects stem from its ability to increase glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels, thereby decreasing methylglyoxal and reducing lipid peroxidation. This suggests potential dietary strategies for improving liver health. Future studies should elucidate the precise mechanisms involved in PHA's regulation of GAPDH and explore its potential in NAFLD prevention and treatment.

The study utilized a mouse model, and the findings might not directly translate to humans. The study focused on a specific high-fat diet and may not be generalizable to other dietary models. The exact mechanisms by which PHA upregulates GAPDH need further investigation.