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Background: Ice plant (Mesembryanthemum crystallinum) is a vegetable with various therapeutic uses, one of which is its ability to prevent diabetes. The present study examined the insulin secretion effect related to the mechanism of action of ice plant extract (IPE) and its active compound D-pinitol in a rat insulin-secreting β-cell line, INS-1, as well as in diabetic rats. Methods: The glucose-stimulated insulin secretion (GSIS) test and Western blotting were used to measure GSIS. The glucose-stimulated index (GSI) and expression levels of insulin-related pathway factors, including insulin receptor substrate-2 (IRS-2), phosphoinositide 3-kinase (PI3K), Akt, and pancreatic and duodenal homeobox-1 (PDX-1), were measured in INS-1 cells. Results: The results showed that the GSI values were found to be 8.17 ± 0.22 and 12.21 ± 0.22 for IPE (25 μg/mL) and D-pinitol (100 μM), respectively. GSI values increased statistically significantly. In addition, IPE and D-pinitol upregulated the expression of insulin-related pathway factors. These findings indicate that insulin secretion was significantly stimulated by IPE and D-pinitol in the INS-1 cells, partly by upregulating the expression of IRS-2, PI3K, Akt, and PDX-1. Additionally, IPE administration significantly reduced excessive weight gain and improved glucose tolerance by decreasing the OGTT-AUC. It demonstrated liver-function-improving and lipid-lowering effects by reducing serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), triglyceride levels, and total cholesterol levels. Mechanistically, IPE enhances insulin signaling by increasing insulin receptor substrate 1 (IRS-1) phosphorylation and improving glucose metabolism and insulin sensitivity. Conclusions: These results offer important new information on the potential of D-pinitol and IPE as functional foods for improving insulin secretion and managing metabolic dysregulation associated with diabetes. [ABSTRACT FROM AUTHOR]