Direct Effects of D-Chiro-Inositol on Insulin Signaling and Glucagon Secretion of Pancreatic Alpha Cells

Abstract

The insulin resistance state of pancreatic α-cells seems to be related to glucagon hypersecretion in type 2 diabetes. Treatment that can improve the insulin sensitivity of α-cells could control glucagon levels in patients with diabetes mellitus. The aim of this study was to investigate the preventive role of D-chiro-inositol (DCI), which has insulin receptor-sensitizer effects on insulin signaling pathways and glucagon secretion in pancreatic α-TC1 clone 6 cells. Cells were chronically treated with palmitate to induce insulin resistance in the presence/absence of DCI. DCI treatment improved the insulin signaling pathway and restored insulin-mediated glucagon suppression in α-TC1-6 cells exposed to palmitate. These results indicate that DCI treatment prevents the insulin resistance of α-TC1-6 cells chronically exposed to palmitate. Our data provide evidence that DCI could be useful to improve the insulin sensitivity of pancreatic α-cells in diabetes treatment.

Keywords: D-chiro-inositol; insulin resistance; lipotoxicity; α-cells.

Figure 1

Effect of palmitate and D-chiro-inositol (DCI) exposure on cell viability in α-TC1-6 cells. MTT assay in α-TC1-6 cells exposed to increasing concentrations of palmitate or DCI. (A) α-TC1-6 cells exposed to palmitate (0.25, 0.5, 0.75, and 1 mM) for 48 h. (B) α-TC1-6 cells exposed to DCI (0.1, 0.5, 1, and 2 mM) for 48 h. (C) α-TC1-6 cells exposed to DCI (0.1, 0.5, and 1 mM) in the presence of palmitate (0.5 mM) for 48 h. Data are expressed as scatter plots with bar ± standard error of 570 nM absorbance to percentage of control. Experiments were conducted in four biological replicates. One-way ANOVA followed by Bonferroni test (n = 4): * p < 0.05, *** p < 0.001 with respect to controls. n.s., not significant; PA, palmitate; DCI, D-chiro-inositol.

Figure 2

Effect of DCI on glucagon secretion in α-TC1-6 cells. Glucagon secretion in cells treated with DCI (0.1 or 1 mM) for 48 h in control conditions (without palmitate). (A) Acute glucagon levels secreted by α-cells cultured in the presence or absence of DCI; glucagon levels were detected after 48 h of treatment in 2 h cell-conditioned Krebs–Ringer buffer (KRB) (control: 1505 pg/mg protein ± 12 vs. DCI 0.1 mM: 1504 pg/mg protein ± 12 vs. DCI 1 mM: 1539 pg/mg protein ± 13). (B) Chronic glucagon levels, detected directly in Dulbecco’s modified Eagle’s medium (DMEM) at the end of 48 h of treatment in cells cultured in the presence or absence of DCI in control conditions, without palmitate (control: 1825 pg/mg protein ± 14  vs. DCI 0.1 mM: 1774 pg/mg protein ± 9 vs. DCI 1 mM: 1694 pg/mg protein ± 12). Data represent four independent experiments. One-way ANOVA followed by Bonferroni test (n = 4) was used to evaluate statistical significance in DCI treated cells with respect to controls. n.s., not significant; DCI, D-chiro-inositol.

Figure 3

Effect of palmitate on glucagon secretion in α-TC1-6 cells. Insulin-regulated glucagon secretion in control cells and cells treated with palmitate (0.5 mM for 48 h) and/or insulin 10⁻⁹ M. Data represent four independent experiments. Student’s t-test was used to evaluate statistical significance in insulin-stimulated cells with respect to unstimulated cells in the absence and presence of palmitate (control: 1405 pg/mg protein ± 13  vs. control + Ins 10^–9 M: 829 pg/mg protein ± 11; palmitate: 6036 pg/mg ± 12  vs. palmitate + Ins 10^–9 M: 6273 pg/mg protein ± 12 ): ** p < 0.01. n.s., not significant.

Figure 4

Effect of DCI on glucagon secretion in α-TC1-6 cells exposed to palmitate. Acute insulin inhibition of glucagon secretion in control cells and cells treated with 0.1 or 1 mM DCI in the presence or absence of palmitate (0.5 mM for 48 h) and/or insulin 10⁻⁹ M. Data represent four independent experiments. Student’s t-test was used to evaluate statistical significance in insulin-stimulated cells with respect to unstimulated cells in the absence and presence of palmitate (control: 1471 pg/mg protein ± 11 vs. control + Ins 10^–9 M: 870 pg/mg protein ± 10; palmitate: 5852 pg/mg protein ± 10 vs. palmitate + Ins 10^–9 M: 5976 pg/mg protein ± 9); * p < 0.05, ** p < 0.01. One-way ANOVA followed by Bonferroni test (n = 4) was used to evaluate statistical significance in insulin-stimulated cells exposed to increasing DCI concentrations with respect to insulin-stimulated controls in the presence of palmitate. ^### p < 0.001. n.s. not significant; DCI, D-chiro-inositol.

Figure 5

Effect of DCI on insulin receptor (IR) phosphorylation and the IRS-1/AKT pathway in α-TC1-6 cells exposed to palmitate. Immunoprecipitation and Western blot analysis for total IR (Tyr1150/1151-β subunit) (p-IR-β) and total IR; Western blot analysis for p-IRS-1 (Tyr612), total IRS-1 (IRS-1), p-AKT (Ser 473), total AKT (AKT), and β-actin: αTC1-6 cells were treated with DCI (0.1 or 1 mM for 48 h) in the presence or absence of palmitate (0.5 mM for 48 h) and acutely stimulated with insulin (10⁻⁹ M for 5 min). (A) Representative immunoblot of three independent experiments. (B) Means ± SEM of densitometric analysis. One-way ANOVA followed by Bonferroni test (n = 3) was used to evaluate statistical significance in insulin-stimulated cells treated with increasing concentrations of DCI with respect to insulin-stimulated controls in the absence and presence of palmitate: * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., not significant; DCI, D-chiro-inositol.