Glucosamine improves cardiac function following trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-{kappa}B signaling

Abstract

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-alpha and IL-6 mRNA, IkappaB-alpha phosphorylation, NF-kappaB, NF-kappaB DNA binding activity, ICAM-1, and MPO activity. LPS (2 microg/ml) increased the levels of IkappaB-alpha phosphorylation, TNF-alpha, ICAM-1, and NF-kappaB in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in IkappaB-alpha phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS-induced activation of NF-kappaB. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-kappaB pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock.

Fig. 1.

Glucosamine treatment increases cardiac O-linked N-acetylglucosamine (O-GlcNAc) levels and attenuates NF-κB activation following trauma-hemorrhage (T-H) and resuscitation. A: immunoblots of O-GlcNAc, phospho (P)- and total (T)-IκB-α and GAPDH. B: mean densitometric analysis of immunoblots of CTD110 normalized to GAPDH and ratio of P- and T-IκB-α (P/T-IκB-α). C: immunoblots of nuclear extracts of NF-κB and histone-1. D: mean densitometric analysis of nuclear NF-κB protein levels normalized to histone-1. E: NF-κB DNA-binding activity (n = 5). Sham and S + G groups, rats subjected to sham surgery ± glucosamine treatment; T-H and T-H-G groups, rats subjected to T-H ± glucosamine treatment during resuscitation. Data are presented as means ± SE with n = 5 rats in each group. *P < 0.05 vs. sham; #P < 0.05 vs. T-H.

Fig. 2.

Glucosamine treatment attenuates cardiac TNF-α mRNA (A), IL-6 mRNA (B), ICAM-1 expression (C), and myeloperoxidase (MPO; D) activity following T-H and resuscitation. Data are presented as means ± SE with n = 5 rats in each group. RQ, relative quantification. *P < 0.05 vs. sham; #P < 0.05 vs. T-H.

Fig. 3.

Glucosamine treatment of cultured adult rat cardiomyocytes attenuates LPS-induced production of TNF-α (A) and IL-6 (B). Freshly isolated adult cardiomyocytes were treated with glucosamine 30 min before LPS (1 μg/ml); TNF-α and IL-6 levels in the media were determined 6 h after LPS administration. *P < 0.05 vs. control (Con); #P < 0.05 vs. LPS treated (n = 4–7 experiments in each group).

Fig. 4.

Glucosamine treatment increases O-GlcNAc levels and attenuates LPS-induced NF-κB activation in neonatal rat ventricular myocytes (NRVMs). A: cytoplasmic protein levels of O-GlcNAc, P- and T-IκB-α, ICAM-1, TNF-α, and GAPDH. O, O-GlcNAc transferase (OGT) adenovirus. B: nuclear protein levels of O-GlcNAc, NF-κB, and histone-1. NRVMs were treated with glucosamine 30 min before LPS (2 μg/ml) for 6 h. Left: typical immunoblots. Right: mean densitometric analysis normalized to GAPDH or histone-1. Data are normalized to control groups and presented as means ± SE of n = 4–7 experiments in each group. *P < 0.05 vs. Con; #P < 0.05 vs. LPS treated.

Fig. 5.

OGT overexpression increases O-GlcNAc levels and attenuates LPS-induced NF-κB activation in NRVMs. A: cytoplasmic protein levels of O-GlcNAc, OGT, P- and T-IκB-α, ICAM-1, TNF-α, and GAPDH. B: nuclear protein levels of O-GlcNAc, OGT, NF-κB, and histone-1. NRVMs were transfected with OGT adenovirus 48 h before LPS (2 μg/ml) for 6 h. Left: typical immunoblots. Right: mean densitometric analysis normalized to GAPDH or histone-1. Data are normalized to control groups and presented as means ± SE of n = 4–7 experiments in each group. *P < 0.05 vs. Con; #P < 0.05 vs. LPS treated.

Fig. 6.

Decreased OGT expression in NRVMs reduced O-GlcNAc levels and enhanced LPS-induced increase in IκB-α phosphorylation. A: immunoblots of O-GlcNAc, OGT, P- and T-IκB-α. B: mean densitometric analysis of O-GlcNAc and OGT normalized to GAPDH. C: mean densitometric analysis of P/T-IκB-α. Cells were transfected with either OGT siRNA (si-OGT) or scrambled siRNA (si-Neg) 48 h before treatment with LPS (2 μg/ml) for 6 h. Data are normalized to Si-Neg group and presented as means ± SE of n = 5 experiments per group. *P < 0.05 vs. Con; #P < 0.05 vs. LPS treated.

Fig. 7.

Glucosamine treatment of RAW 264.7 cells increased cardiac O-GlcNAc levels and attenuated LPS-induced P-IκB-α and inducible nitric oxide synthase (iNOS) protein expression. Cells were pretreated with 5 mM glucosamine for 30 min and before treatment with LPS (0.1 μg/ml, 6 h). C, control; L, LPS treated.