Glucosamine substituted sulfonylureas: IRS-PI3K-PKC-AKT-GLUT4 insulin signalling pathway intriguing agent

Abstract

Normally, skeletal muscle accounts for 70-80% of insulin-stimulated glucose uptake in the postprandial hyperglycemia state. Consequently, abnormalities in glucose uptake by skeletal muscle or insulin resistance (IR) are deemed as initial metabolic defects in the pathogenesis of type 2 diabetes mellitus (T2DM). Globally, T2DM is growing in exponential proportion. The majority of T2DM patients are treated with sulfonylureas in combination with other drugs to improve insulin sensitivity. Glycosylated sulfonylureas (sulfonylurea-glucosamine analogues) are modified analogues of sulfonylurea that have been previously reported to possess antidiabetic activity. The aim of this study was to evaluate the impact of glycosylated sulfonylureas on the insulin signalling pathway at the molecular level using L6 skeletal muscle cell (in vitro) and extracted soleus muscle (ex vivo) models. To create an in vitro model, insulin resistance was established utilizing a high insulin-glucose approach in differentiated L6 muscle cells from Rattus norvegicus. Additionally, for the ex vivo model, extracted soleus muscles, adult Sprague-Dawley rats were subjected to a solution containing 25 mmol L^-1 glucose and 100 mmol L^-1 insulin for 24 hours to induce insulin resistance. After insulin resistance, compounds under investigation and standard medicines (metformin and glimepiride) were tested. The differential expression of PI3K, IRS-1, PKC, AKT2, and GLUT4 genes involved in the insulin signaling pathway was evaluated using qPCR. The evaluated glycosylated sulfonylurea analogues exhibited a significant increase in the gene expression of insulin-dependent pathways both in vitro and ex vivo, confirming the rejuvenation of the impaired insulin signaling pathway genes. Altogether, glycosylated sulfonylurea analogues described in this study represent potential therapeutic anti-diabetic drugs.

Scheme 1. Chemical structures of glycosylated sulfonylurea analogues. Group A (acetylated); 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(benzenesulfonylurea)-d-glucopyranose analogues. Group B (deacetylated); 2-deoxy-2-(benzene sulfonylurea)-d-glucopyranose analogues.

Scheme 2. Experimental grouping and treatment.

Fig. 1. Cytotoxicity assay (MTT) results using L6 skeletal muscle cells. Cells were incubated with increasing concentrations ranging from 50–250 μM of each treatment.

Fig. 2. Relative quantification of IRS-1 expression in glimepiride, metformin, group A (acetylated) and group B (deacetylated) (at 1 μM and 2 μM) treated diabetic groups. (I): In vitro. (II): Ex vivo. The gene was normalized against a geometric mean of two housekeeping genes (β-actin and γ-actin). All the data are presented as mean ± standard error mean where one-way ANOVA with Duncan's multiple range post hoc test was conducted using StatPlus. The statistically different values (p < 0.05) are denoted with different case letters (b–d) as follows: b compared vs. diabetic control group (a); c compared vs. glimepiride; d compared vs. metformin.

Fig. 3. Relative quantification of PI3K expression in glimepiride, metformin, group A (acetylated) and group B (deacetylated) (at 1 μM and 2 μM) treated diabetic groups. (I): In vitro. (II): Ex vivo. The gene was normalized against a geometric mean of two housekeeping genes (β-actin and γ-actin). All the data are presented as mean ± standard error mean where one-way ANOVA with Duncan's multiple range post hoc test was conducted using StatPlus. The statistically different values (p < 0.05) are denoted with different case letters (b–d) as follows: b compared vs. diabetic control group (a); c compared vs. glimepiride; d compared vs. metformin.

Fig. 4. Relative quantification of PKC expression in glimepiride, metformin, group A (acetylated) and group B (deacetylated) (at 1 μM and 2 μM) treated diabetic groups. (I): In vitro. (II): Ex vivo. The gene was normalized against a geometric mean of two housekeeping genes (β-actin and γ-actin). All the data are presented as mean ± standard error mean where one-way ANOVA with Duncan's multiple range post hoc test was conducted using StatPlus. The statistically different values (p < 0.05) are denoted with different case letters (b–d) as follows: b compared vs. diabetic control group (a); c compared vs. glimepiride; d compared vs. metformin.

Fig. 5. Relative quantification of AKT2 expression in glimepiride, metformin, group A (acetylated) and group B (deacetylated) at (1 μM and 2 μM) treated diabetic groups. (I): In vitro. (II): Ex vivo. The gene was normalized against a geometric mean of two housekeeping genes (β-actin and γ-actin). All the data are presented as mean ± standard error mean where one-way ANOVA with Duncan's multiple range post hoc test was conducted using StatPlus. The statistically different values (p < 0.05) are denoted with different case letters (b–d) as follows: b compared vs. diabetic control group (a); c compared vs. glimepiride; d compared vs. metformin.

Fig. 6. Relative quantification of GLUT4 expression in glimepiride, metformin, group A (acetylated) and group B (deacetylated) (at 1 μM and 2 μM) treated diabetic groups. (I): In vitro. (II): Ex vivo. The gene was normalized against a geometric mean of two housekeeping genes (β-actin and γ-actin). All the data are presented as mean ± standard error mean using StatPlus. The data were statistically analyzed by one-way ANOVA followed by Duncan's multiple range post hoc test. The statistically different values (p < 0.05) are denoted with different case letters (b–d) as follows: b compared vs. diabetic control group (a); c compared vs. glimepiride; d compared vs. metformin.