In vivo Evaluation of Two Thiazolidin-4-one Derivatives in High Sucrose Diet Fed Pre-diabetic Mice and Their Modulatory Effect on AMPK, Akt and p38 MAP Kinase in L6 Cells

Abstract

We had previously demonstrated the anti-diabetic potential and pancreatic protection of two thiazolidin-4-one derivatives containing nicotinamide moiety (NAT-1 and NAT-2) in STZ-induced diabetic mice. However, due to the limitations of the STZ model, we decided to undertake a detailed evaluation of anti-diabetic potential of the molecules on a high sucrose diet (HSD) fed diabetic mouse model. Further, in vitro mechanistic studies on the phosphorylation of AMPK, Akt and p38 MAP kinase in L6 myotubes and anti-inflammatory studies in RAW264.7 mouse monocyte macrophage cells were performed. 15 months of HSD induced fasting hyperglycaemia and impaired glucose tolerance in mice. Treatment with NAT-1 and NAT-2 (100 mg/kg) for 45 days significantly improved the glucose tolerance and lowered fasting blood glucose levels compared to untreated control. An improvement in the elevated triglycerides and total cholesterol levels, and favorable rise in HDL cholesterol were also observed with test drug treatment. Also, no major changes were observed in the liver (albumin, AST and ALT) and kidney (creatinine and urea) parameters. This was further confirmed in their respective histology profiles which revealed no gross morphological changes. In L6 cells, significant phosphorylation of Akt and p38 MAP kinase proteins were observed with 100 μM of NAT-1 and NAT-2 with no significant changes in phosphorylation of AMPK. The molecules failed to exhibit anti-inflammatory activity as observed by their effect on the generation of ROS and nitrite, and nuclear levels of NF-κB in LPS-stimulated RAW264.7 cells. In summary, the molecules activated Akt and p38 MAP kinase which could have partly contributed to their anti-hyperglycaemic and hypolipidemic activities in vivo.

Keywords: AMPK; Akt; high sucrose diet; mouse model; p38 MAP kinase; thiazolidin-4-one derivatives.

FIGURE 1

The chemical structure of the test compounds. (A) NAT-1: N-[2-(4-Methoxy-phenyl)-4-oxo-thiazolidin-3-yl]-nicotinamide; (B) NAT-2: N-[2-(3,5-Di-tert-butyl-4-hydroxy-phenyl)-4-oxo-thiazolidin-3-yl]-nicotinamide.

FIGURE 2

Effect of 15-month high-sucrose diet on (A) fasting blood glucose; (B) plasma triglyceride levels in mice. All values are expressed as mean + SEM (n = 6 for normal animals and 21 for HSD fed animals). ^∗p < 0.05 compared to normal diet.

FIGURE 3

Effect of 15-month high sucrose diet (HSD) on OGTT after a glucose load (2 g/kg, b.w.), where (A) plot of plasma glucose levels (mg/dl) vs. time interval; (B) area under the curve (AUC; expressed as arbitrary units) of the glucose tolerance curve. The time taken for the plasma glucose levels to come back to their pre-glucose-load values were considered for calculation of AUC. All values are expressed as mean + SEM (n = 5–6). ^ap < 0.05 compared to normal control; ^bp < 0.05 compared to HSD control. In OGTT, after the glucose load in the normal diet fed group, the basal plasma glucose levels were attained in less than 60 min whereas in HSD fed group, the basal levels were was not attained even after 4 h. With metformin and NAT-1 treatment, the basal blood glucose was attained in 60 min and in 2 h by NAT-2 treatment.

FIGURE 4

Effect of NAT-1 and NAT-2 on lipid parameters in plasma of HSD-fed mice. (A) Triglyceride levels; (B) total cholesterol levels; (C) HDL cholesterol levels. All values are expressed as mean ± SEM (n = 5–6). ^ap < 0.05 vs. normal control, ^bp < 0.05 vs. HSD control.

FIGURE 5

Effect of NAT-1 and NAT-2 on liver parameters in plasma of HSD-fed mice. (A) Albumin levels; (B) aspartate aminotransferase (AST) levels; (C) alanine transaminase (ALT) levels. All values are expressed as mean ± SEM (n = 5–6). No significant difference was observed.

FIGURE 6

Effect of NAT-1 and NAT-2 on morphological feature of mice liver. Optical microscopy: H&E (200×). Photomicrographs of representative mice are shown. (A) Normal control; (B) HSD fed control; (C) HSD fed mice treated with metformin; (D) HSD fed mice treated with NAT-1; (E) HSD fed mice treated with NAT-2.

FIGURE 7

Effect of NAT-1 and NAT-2 on kidney parameters in plasma of HSD-fed mice. (A) Creatinine levels; (B) urea levels. All values are expressed as mean ± SEM (n = 5–6). ^ap < 0.05 vs. normal control.

FIGURE 8

Effect of NAT-1 and NAT-2 on morphological feature of mice kidney. Optical microscopy: H&E (200×). Photomicrographs of representative mice are shown. (A) Normal control; (B) HSD fed control; (C) HSD fed mice treated with metformin; (D) HSD fed mice treated with NAT-1; (E) HSD fed mice treated with NAT-2.

FIGURE 9

Effect of NAT-1 and NAT-2 on nuclear levels of NF-κB in LPS stimulated RAW264.7 cells where (A) blot showing nuclear levels of NF-κB; (B) relative density ratio of NF-κB/GAPDH. Values are expressed as mean ± SEM. ^ap < 0.05 vs. normal control.

FIGURE 10

Effect of NAT-1 and NAT-2 on phosphorylation of molecular markers – AMPK, p38 MAP kinase and Akt in L6 cells, where (A) representative blots showing the levels of various markers and their phosphorylated forms tested; (B) ratio of relative densities of phospho-AMPK and AMPK; (C) ratio of relative densities of phospho-p38 MAPK and p38 MAPK; (D) ratio of relative densities of phospho-Akt and Akt. RD represents ratio of band intensity of marker protein to its respective housekeeping protein, i.e., GAPDH. Values are expressed as mean ± SEM. ^ap < 0.05 vs. normal control.