Hexane Extract of Orthosiphon stamineus Induces Insulin Expression and Prevents Glucotoxicity in INS-1 Cells

Abstract

Background: Hyperglycemia, a characteristic feature of diabetes, induces glucotoxicity in pancreatic β-cells, resulting in further impairment of insulin secretion and worsening glycemic control. Thus, preservation of insulin secretory capacity is essential for the management of type 2 diabetes. In this study, we evaluated the ability of an Orthosiphon stamineus (OS) extract to prevent glucotoxicity in insulin-producing cells.

Methods: We measured insulin mRNA expression and glucose-stimulated insulin secretion (GSIS) in OS-treated INS-1 cells after exposure to a high glucose (HG; 30 mM) concentration.

Results: The hexane extract of OS elevated mRNA expression of insulin as well as pancreatic and duodenal homeobox-1 of INS-1 cells in a dose-dependent manner. The hexane OS extract also increased the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) in a concentration-dependent manner. Additionally, Akt phosphorylation was elevated by treatment with 100 and 200 µmol of the hexane OS extract. Three days of HG exposure suppressed insulin mRNA expression and GSIS; these expressions were restored by treatment with the hexane OS extract. HG elevated peroxide levels in the INS-1 cells. These levels were unaffected by OS treatment under both normal and hyperglycemic conditions.

Conclusion: Our results suggested that the hexane extract of OS elevates insulin mRNA expression and prevents glucotoxicity induced by a 3-day treatment with HG. This was associated with the activation of PI-3K and Akt.

Keywords: Glucose-stimulated insulin secretion, Insulin mRNA; Glucotoxicity; Orthosiphon stamineus.

Fig. 1. The effects of various fractions of Orthosiphon stamineus (OS) extract on insulin mRNA expression in INS-1 cells under normal and hyperglycemic (exposure to high glucose [HG] for 3 days) conditions. The cells were treated with each OS extract (200 µM) for 12 hours. Bars represent the mean±standard error of three separate experiments. BuOH, n-butanol; EtOAc, ethylacetate. ^aP<0.05, ^bP<0.01, and ^cP<0.001 versus the untreated cells under normal conditions, ^dP<0.05, ^eP<0.01, and ^fP<0.001 versus the untreated HG control.

Fig. 2. The effects of various concentrations of Orthosiphon stamineus (OS) extract on the mRNA expression of (A) insulin and (B) pancreatic and duodenal homeobox-1 (PDX-1) in INS-1 cells. Cells were treated with the hexane OS extract at concentrations of 0, 50, 100, and 200 µM for 12 hours. Bars represent the mean±standard error of three separate experiments.

^a,b,cValues that do not share a common superscript are significantly different at P<0.05.

Fig. 3. Effect of the hexane Orthosiphon stamineus (OS) extract on glucose-stimulated insulin secretion (GSIS) in INS-1 cells. OS extract treatment (200 µM for 12 hours) restored GSIS that was completely suppressed by exposure to high glucose (HG) for 3 days. Bars represent the mean±standard error of three separate experiments. ^aP<0.05, ^bP<0.01.

Fig. 4. The effects of various doses of Orthosiphon stamineus (OS) extract on (A) phosphatidylinositol 3-kinase (PI3K) and (B) Akt phosphorylation in INS-1 cells. Cells were treated with the hexane OS extract at concentrations of 0, 50, 100, and 200 µM for 12 hours. Bars represent the mean±standard error of three separate experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

^a,b,cValues that do not share a common superscript are significantly different at P<0.05.

Fig. 5. Effects of the hexane Orthosiphon stamineus (OS) extract on intracellular peroxide levels in INS-1 cells under normal and high glucose (HG) conditions. Cells were treated with 200 µM of the hexane extract for 12 hours. Bars represent the mean±standard error of three separate experiments. ^aP<0.01, ^bP<0.001 versus the untreated cells cultured under normal conditions.