Activation of the hexosamine biosynthesis pathway and protein O-GlcNAcylation modulate hypertrophic and cell signaling pathways in cardiomyocytes from diabetic mice - PubMed (original) (raw)

Activation of the hexosamine biosynthesis pathway and protein O-GlcNAcylation modulate hypertrophic and cell signaling pathways in cardiomyocytes from diabetic mice

Susan A Marsh et al. Amino Acids. 2011 Mar.

Abstract

Patients with diabetes have a much greater risk of developing heart failure than non-diabetic patients, particularly in response to an additional hemodynamic stress such as hypertension or infarction. Previous studies have shown that increased glucose metabolism via the hexosamine biosynthesis pathway (HBP) and associated increase in O-linked-β-N-acetylglucosamine (O-GlcNAc) levels on proteins contributed to the adverse effects of diabetes on the heart. Therefore, in this study we tested the hypothesis that diabetes leads to impaired cardiomyocyte hypertrophic and cell signaling pathways due to increased HBP flux and O-GlcNAc modification on proteins. Cardiomyocytes isolated from type 2 diabetic db/db mice and non-diabetic controls were treated with 1 μM ANG angiotensin II (ANG) and 10 μM phenylephrine (PE) for 24 h. Activation of hypertrophic and cell signaling pathways was determined by assessing protein expression levels of atrial natriuretic peptide (ANP), α-sarcomeric actin, p53, Bax and Bcl-2 and phosphorylation of p38, ERK and Akt. ANG II and PE significantly increased levels of ANP and α-actin and phosphorylation of p38 and ERK in the non-diabetic but not in the diabetic group; phosphorylation of Akt was unchanged irrespective of group or treatment. Constitutive Bcl-2 levels were lower in diabetic hearts, while there was no difference in p53 and Bax. Activation of the HBP and increased protein O-GlcNAcylation in non-diabetic cardiomyocytes exhibited a significantly decreased hypertrophic signaling response to ANG or PE compared to control cells. Inhibition of the HBP partially restored the hypertrophic signaling response of diabetic cardiomyocytes. These results suggest that activation of the HBP and protein O-GlcNAcylation modulates hypertrophic and cell signaling pathways in type 2 diabetes.

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Figures

Fig. 1

Fig. 1

Expression of a ANP, b α-actin and c TRPC1 protein in cardiomyocytes isolated from non-diabetic control (control) and diabetic (db/db) hearts following 24 h treatment with ANG (1 μM) or PE (10 μM). Upper panels are representative immunoblots and the lower panels are mean densitometric data from three individual experiments normalized to calsequestrin. *P < 0.05 versus control untreated group

Fig. 2

Fig. 2

Expression of phosphorylated (P) and total (T) a p38, b ERK and c Akt protein in cardiomyocytes isolated from non-diabetic control (control) and diabetic (db/db) hearts following 24 h treatment with ANG (1 μM) or PE (10 μM). Upper panels are representative immunoblots and the lower panels are mean densitometric data from three individual experiments of phosphorylated proteins normalized to their respective total protein. *P < 0.05 versus control untreated group

Fig. 3

Fig. 3

a Immunoblots for ANP, α-actin and calsequestrin in untreated cardiomyocytes and cardiomyocytes following 24 h treatment with ANG (1 μM) or PE (10 μM). Cardiomyocytes isolated from db/db animals (d) were also treated with the GFAT inhibitors azaserine (5 μM) or Don (20 μM). Mean densitometric data for b ANP and c α-actin expression normalized to calsequestrin from three individual experiments. Data presented as mean ± SE of five individual experiments. *P < 0.05 versus db/db group induced with ANG or PE

Fig. 4

Fig. 4

a Representative anti-_O_-GlcNAc immunoblots of whole heart homogenates from non-diabetic mice (control; n = 3) and three diabetic (db/db; n = 3) mice; b mean intensity of _O_-GlcNAc proteins determined by densitometric analysis with levels normalized to calsequestrin for bands 1–5 as indicated; c representative anti-_O_-GlcNAc immunoblots from cardiomyocytes treated with high glucose (25 mM; +HG), glucosamine (5 mM; +GlcN) or PUGNAc (100 μM; +PUGNAc) for 24 h; d mean intensity of _O_-GlcNAc proteins determined by densitometric analysis with levels normalized to calsequestrin for all bands and bands 1–5 as indicated. *P < 0.05 versus control or untreated

Fig. 5

Fig. 5

a Immunoblots for ANP, α-actin and calsequestrin in control non-diabetic cardiomyocytes following 24 h treatment with ANG (1 μM) or PE (10 μM) in the presence of high glucose (25 mM; +HG), glucosamine (5 mM; +GlcN) or PUGNAc (100 μM; +PUGNAc). Mean densitometric data for b ANP and c α-actin expression normalized to calsequestrin from three individual experiments. *P < 0.05 versus 5 mM glucose

Fig. 6

Fig. 6

a Immunoblots for ANP, α-actin and calsequestrin in control non-diabetic cardiomyocytes following 24 h treatment with ANG (1 μM) or PE (10 μM) in the presence of normal glucose (5 mM), high glucose (25 mM; HG) or

D

-mannitol (25 mM). Mean densitometric data for b ANP and c α-actin expression normalized to calsequestrin from three individual experiments. *P < 0.05 versus 5 mM glucose

Fig. 7

Fig. 7

Expression levels and mean densitometric data of pro-apoptotic proteins p53 and Bax, and the anti-apoptotic protein Bcl-2 in the hearts of non-diabetic (Con) and diabetic (db/db) mouse whole heart homogenates. *P < 0.05 versus Con

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References

    1. Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation. 2007;115:3213–3223. - PubMed
    1. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414:813–820. - PubMed
    1. Champattanachai V, Marchase RB, Chatham JC. Glucosamine protects neonatal cardiomyocytes from ischemia–reperfusion injury via increased protein-associated O-GlcNAc. Am J Physiol Cell Physiol. 2007;292:C178–C187. - PubMed
    1. Chatham JC, Marchase RB. The role of protein O-linked beta-N-acetylglucosamine in mediating cardiac stress responses. Biochim Biophys Acta. 2010;1800:57–66. - PMC - PubMed
    1. Chatham JC, Forder JR, McNeill JH, editors. The Heart in Diabetes. Kluwer Academic Publishers; Norwell: 1996.

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