Strain‐Level Screening of Human Gut Microbes Identifies Three <i>Lactobacillus</i> Strains as Next‐Generation Antidiabetic Probiotics

ABSTRACT Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia. Probiotic supplementation improves glucolipid metabolism by modulating the gut microbiota. However, current research lacks a systematic understanding of the overall hypoglycemic ability of human gut microbes. First, a high‐throughput screening platform based on HepG2 cells was developed to screen candidate strains with hypoglycemic potential. Subsequently, we utilized a T2DM mouse model to investigate the antidiabetic effects and mechanisms of three selected Lactobacillus strains through biochemical analyses, histopathological analyses, RNA‐seq analyses, and metagenomics analyses. We categorized and ranked the hypoglycemic ability of 4811 strains belonging to 8 phyla, 17 orders, 68 genera, and 241 species. The hypoglycemic ability of all tested strains showed a significant normal distribution, and several species with significant efficacy were screened, primarily from Bifidobacterium and Lactobacillus . Subsequently, we found that three Lactobacillus strains significantly improve glycolipid metabolism disorders in T2DM mice, and the mechanisms include inhibiting hepatic gluconeogenesis via the AKT–FOXO1–PEPCK/G6pase pathway and activating GPR41 and GPR43 to bind short‐chain fatty acids, promoting glucagon‐like peptide‐1 (GLP‐1) secretion. These strains also significantly regulate the gut microbiota structure and composition, contributing to their hypoglycemic effects. This study comprehensively examined the hypoglycemic efficacy of 4811 strains for the first time, resulting in a functional map of human gut microbiota for hypoglycemia. Additionally, we investigated the hypoglycemic efficacy and the mechanisms of three Lactobacillus strains. This study provides a rich bacterial pool for the targeted development of probiotics as potential treatments for diabetes and related metabolic diseases.