Metabolic Remodeling with Hepatosteatosis Induced Vascular Oxidative Stress in Hepatic ERK2 Deficiency Mice with High Fat Diets

Abstract

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1β were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.

Keywords: ERK2; endothelial dysfunction; hepatosteatosis; insulin resistance; metabolic syndrome; metabolism.

Figure 1

Typical body appearance and macroscopic or microscopic images of liver. LE2KO showed similar increases in body weight with HFHSD for 20 weeks (A) HFHSD-LE2KO significant increased liver weight (B) and fat deposition (C) compared with HFHSD-Control.

Figure 2

HFHSD-LE2KO decreased phosphorylation of AMPK/ACC and increased mRNA expression of SREBP-1c in liver. (A) Phosphorylation of AMPK (Thr172) and ACC (Ser 79) in the livers of controls and LE2KO on NC or HFHSD, respectively (n = 3 for each group). (B,C) Quantification of phosphorylation of AMPK/AMPK and phosphorylation of ACC/ACC. (D) mRNA expression levels of SREBP1c in the livers of control and LE2KO on NC or HFHSD, respectively (n = 6 for each group). Error bars represent SEM; p values were determined by ANOVA (* p < 0.05, vs. HFHSD-Control).

Figure 3

HFHSD-LE2KO have impaired insulin sensitivity in skeletal muscle. (A) Phosphorylation of AKT (Ser 473) in the soleus muscle of controls and LE2KO on NC or HFHSD, respectively (n = 3 for each group). (B,C) Quantification of phosphorylation of AKT/AKT on NC or HFHSD. Error bars represent SEM; p values were determined by t-test (* p < 0.05, vs. control).

Figure 4

HFHSD-LE2KO display enlargement of adipocytes and changes in serum adipocytokines. (A) Typical microscopic images of adipocytes and (B) quantification of size of adipocyte (n = 8 for each group). (C–E) Serum adiponectin levels were lower and serum leptin and IL-1β levels were markedly higher in HFHSD-LE2KO than HFHSD-Control. (n = 8 for each group). Error bars represent SEM; p values were determined by ANOVA (** p < 0.01, *** p < 0.001 vs. HFHSD-Control).

Figure 5

Serum Amino Acid Profile. (A) Homocystein, (B) asymmetric dimethylarginine (ADMA), (C) Citrulin levels were higher in HFHSD-LE2KO than HFHSD-Control. (D) Arginine level was lower in HFHSD-Control than NC-Control, and (E) Ornithine and (F) Ornithine/Arginine ratio were higher in HFHSD-LE2KO than HFHSD-Control. (n = 8 for each group). Data are mean ± SEM and median (interquartile range). (nmol/mL) p values were determined by ANOVA (* p < 0.05). n = 8 for each group.

Figure 6

HFHSD-LE2KO demonstrate increased aortic superoxide levels and impaired endothelium-dependent relaxation. (A) DHE staining of aorta from HFHFD-Control, HFHSD-LE2KO, and HFHSD-LE2KO with pre-incubated of tempol, apocynin, and CCCP and (B) quantification of DHE fluorescence intensity (n = 5–6 for each group). Vascular relaxation of aortic rings with ACh (C) and SNP (D) on NC or HFHSD for 20 weeks, respectively (n = 8–10 for each group). Results of relaxation are expressed as percentage changes in steady-state level of contraction with 10^−6.5 M phenylephrine. Error bars represent SEM; p values were determined by ANOVA (*** p < 0.001 vs. HFHSD-LE2KO without pre-incubation).