Integrated Metabolomic and Proteomic Analyses Reveal Differential Regulation of the Citrate Cycle and Pentose Phosphate Pathway by Cannabis sativa Extract and the Synthetic Cannabinoid HU210 in GT1-7 Neuronal Cells: Sequential Extraction of Metabolites and Proteins from a Single Cell Pellet.

Natori Y.; Mizuno D.; Doshi M.; Kawahara M.; Ishii A. · Metabolites · 2026

**Background & Methods** This integrated metabolomic and proteomic study compared Cannabis sativa (CS) extract containing THC and other cannabinoids with the synthetic cannabinoid agonist HU210 in GT1-7 neuronal cells. Metabolites and proteins were sequentially extracted from single-cell pellets and analyzed using gas chromatography-tandem mass spectrometry and nano-liquid chromatography-quadrupole-Orbitrap mass spectrometry, with cytotoxicity assessed via lactate dehydrogenase assay. **Key Findings** • CS extract (2000 µg/mL delivering 166 nM THC, 800 nM tetrahydrocannabinolic acid, and 11.3 nM cannabidiol) and HU210 (20 µM) showed comparable cytotoxicity at 24 hours but produced opposite metabolic responses at the 3-hour non-cytotoxic timepoint: HU210 caused coordinated metabolic mobilization while CS induced broad metabolic suppression. • HU210 significantly altered both metabolome and proteome (8 of 2,720 proteins changed), whereas CS altered primarily the metabolome with minimal protein changes, indicating differential mechanisms of action despite similar downstream cytotoxic effects. • HU210 decreased citrate while increasing 2-ketoglutarate, fumarate, malate, gluconate, and ribose-5-phosphate, accompanied by upregulation of eight key enzymes in the citrate cycle and pentose phosphate pathway; CS showed opposite metabolite directional changes without corresponding enzymatic upregulation. **Dosage & Administration** CS extract: 2000 µg/mL (non-cytotoxic at 3 hours, cytotoxic at 24 hours); HU210: 20 µM (non-cytotoxic at 3 hours, cytotoxic at 24 hours). CS delivered approximately 166 nM THC per exposure. **Safety & Adverse Effects** Cytotoxicity observed at 24 hours for both CS (2000 µg/mL) and HU210 (20 µM). At pre-lethal timepoints (3 hours), neither compound produced reported adverse effects beyond metabolic alterations. **Evidence Quality** This is an in vitro cell culture study with significant limitations: findings are restricted to GT1-7 neuronal cells and may not translate to whole organism or human biology. The review-type designation and 2026 publication date suggest synthesis of existing data rather than novel primary research. Lack of in vivo validation, limited cell type representation, and absence of clinical correlation substantially limit evidence quality and applicability to clinical practice. Results provide mechanistic insights but cannot establish clinical safety or efficacy profiles.

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