Cardiac metallothionein overexpression rescues diabetic cardiomyopathy in Akt2-knockout mice

Abstract

To efficiently prevent diabetic cardiomyopathy (DCM), we have explored and confirmed that metallothionein (MT) prevents DCM by attenuating oxidative stress, and increasing expression of proteins associated with glucose metabolism. To determine whether Akt2 expression is critical to MT prevention of DCM, mice with either global Akt2 gene deletion (Akt2-KO), or cardiomyocyte-specific overexpressing MT gene (MT-TG) or both combined (MT-TG/Akt2-KO) were used. Akt2-KO mice exhibited symptoms of DCM (cardiac remodelling and dysfunction), and reduced expression of glycogen and glucose metabolism-related proteins, despite an increase in total Akt (t-Akt) phosphorylation. Cardiac MT overexpression in MT-TG/Akt2-KO mice prevented DCM and restored glucose metabolism-related proteins expression and baseline t-Akt phosphorylation. Furthermore, phosphorylation of ERK1/2 increased in the heart of MT-TG/Akt2-KO mice, compared with Akt2-KO mice. As ERK1/2 has been implicated in the regulation of glucose transport and metabolism this increase could potentially underlie MT protective effect in MT-TG/Akt2-KO mice. Therefore, these results show that although our previous work has shown that MT preserving Akt2 activity is sufficient to prevent DCM, in the absence of Akt2 MT may stimulate alternative or downstream pathways protecting from DCM in a type 2 model of diabetes, and that this protection may be associated with the ERK activation pathway.

Keywords: Akt2 knock out; diabetes; glucose metabolism; insulin resistance; metallothionein.

FIGURE 1

Bodyweight, genetic identification and echocardiographic images of mice. Bodyweight (A) was measured. MT (B) and Akt2 (C) was detected by Western blot. Left vol short‐axis M‐mode echocardiography (D) was measured. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control (Akt2). *P <.05 vs the WT; **P <.01 vs the WT; #P <.05 vs MT‐TG; ##P <.01 vs MT‐TG

FIGURE 2

Index for cardiac hypertrophy. Ratio of the heart weight to tibia length (A) was calculated at the end of study. Wheat germ agglutinin (green)‐stained cardiac cross‐sections with quantification of mean cardiomyocyte cross‐sectional area relative fold change (B). Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO

FIGURE 3

Preventive effects of MT‐TG against cardiac fibrosis in Akt2‐KO mice. Cardiac fibrotic remodelling was examined with Sirius red staining (A). Sirius red positive staining was semi‐quantified using a computer imaging analysis system (Image J). Cardiac expression of fibronectin (FN) (B), COL1A1 (C) and TGF‐β (D) as index of fibrotic mediators was detected by Western blot. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control. *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO; #P <.05 vs MT‐TG; ##P <.01 vs MT‐TG

FIGURE 4

Preventive effects of MT‐TG on cardiac inflammation damages in Akt2‐KO mice. Cardiac expression of ICAM‐1 (A), VCAM‐1 (B) and IL‐1β (C), as indexes of inflammation, were detected by a Western blot. Figures A and C are the same gel and membrane using the same β‐Actin. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control. *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO

FIGURE 5

Preventive effects of MT‐TG on cardiac oxidative damage in Akt2‐KO mice. Cardiac expression of 3‐NT (A), 4‐HNE (B), as indexes of oxidative stress damages and of antioxidant enzymes CAT(C), SOD2(D), were detected by Western blot. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control. *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO

FIGURE 6

Effect of MT‐TG on the cardiac Akt2‐mediated glucose metabolism in Akt2‐KO mice. Cardiac expression and its phosphorylation of t‐Akt (A), Akt1 (B), GSK‐3β (C), GS (D) and GP (E) were detected by immunoblotting. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control. *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO

FIGURE 7

Effect of MT‐TG on cardiac Akt2‐mediated glucose metabolism in Akt2‐KO mice. Heart PAS staining (A), red spots mean glycogen. Cardiac expression and its phosphorylation of AS160 (B), HKII (C), PFKFB2 (D) and ERK1/2 (E) were detected by Western blot. Data are presented as mean ± SD (WT: n = 9 in male; n = 6 in female; MT‐TG: n = 8 in male; n = 5 in female; Akt2‐KO: n = 8 in male; n = 8 in female; MT‐TG/Akt2‐KO: n = 9 in male; n = 9 in female). Quantitation plots are expressed as fold WT control. *P <.05 vs the WT; **P <.01 vs the WT; &P <.05 vs Akt2‐KO; &&P <.01 vs Akt2‐KO

FIGURE 8

Schematic illustration of the assumed mechanisms by which the minimal glucose metabolism is compensated by up‐regulated Akt1 in the heart of Akt2‐KO mice (a) and the normal glucose metabolism is preserved by overexpressed cardiac MT in the heart of Akt2‐KO mice (b). Red line indicates the compensative changes related to glucose metabolism in the heart of Akt2‐KO mice compared to WT mice. Green lines indicate the changes related to glucose metabolism preserved by overexpressed cardiac MT in the heart of MT‐TG/Akt2‐KO mice compared to Akt2‐KO mice. Thicker arrows indicate more impact on the targets. Dashed line presents the possible pathways. IR, insulin receptor