Shaolong Yang

Glucosamine improves cardiac function following trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-κB signaling

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.

Differential alterations in cardiovascular responses during the progression of polymicrobial sepsis in the mouse.

Although the mouse has been extensively used to study immune consequences of sepsis and other genetic anomalies, the changes in various cardiovascular parameters such as cardiac output, organ perfusion, as well as oxygen utilization have not been characterized in this species during sepsis. To determine this, polymicrobial sepsis was induced in male adult C3H/NeN mice by cecal ligation and puncture (CLP, two punctures with a 22-gauge needle). The animals were then resuscitated with normal saline subcutaneously. At 5 or 24 h after CLP (time points previously shown to be within the hyperdynamic and hypodynamic stage of sepsis, respectively, in the rat), cardiac output and blood flow in major organs were determined using a well-established radioactive microsphere method, and stroke volume and total peripheral resistance were calculated. In addition, oxygen delivery and consumption were determined. The results indicate that cardiac output, stroke volume, oxygen delivery and consumption, and blood flow in the liver, small intestine, spleen, and kidneys increased significantly at 5 h after CLP. This was associated with significantly decreased total peripheral resistance. In contrast, total peripheral resistance increased and the other above-mentioned parameters, as well as mean arterial pressure, decreased significantly at 24 h after the onset of sepsis. Thus, the cardiovascular response to polymicrobial sepsis in the mouse is characterized by an early hyperdynamic phase (i.e., 5 h after CLP) followed by a late hypodynamic phase (24 h post-CLP). Since the radioactive microsphere technique provides a reliable method for determining various hemodynamic parameters in the mouse, the correlation between the cardiovascular response and immune or potentially genetic alterations can be examined in this species during the progression of sepsis.

Inhibition of cardiac PGC-1α expression abolishes ERβ agonist-mediated cardioprotection following trauma-hemorrhage

PGC‐1α (peroxisome proliferator‐acti‐vated receptor [PPARγ] coactivator‐1α) activates PPARα and mitochondrial transcription factor A (Tfam), which regulate proteins, fatty acid and ATP metabolism (i.e., FAT/CD36, MCAD, and COX I). Recently we found that the salutary effects of estradiol (E2) on cardiac function following trauma‐hemorrhage (T‐H) are mediated via estrogen receptor (ER)β. In this study we tested the hypothesis that ERβ‐mediated cardioprotection is induced via up‐regulation of PGC‐1α through PPARα or Tfam‐dependent pathway. Male rats underwent T‐H and received ERα agonist propylpyra‐zole‐triol (PPT), ERβ agonist diarylpropionitrile (DPN), E2, or vehicle. Another group was treated with antisense PGC‐1α oligonucleotides prior to administration of DPN. E2 and DPN treatments attenuated the decrease in cardiac mitochondrial ATP, abrogated the T‐H‐induced lipid accumulation, and normalized PGC‐1α, PPARα, FAT/CD36, MCAD, Tfam, and COX I after T‐H. In contrast, PPT administration did not abrogate lipid accumulation. Moreover, in PPT‐treated animals mitochondrial ATP remained significantly lower than those observed in DPN‐ or E2‐treated animals. Prior administration of antisense PGC‐1α prevented DPN‐mediated cardioprotection and increase in ATP levels and Tfam but not in PPARα following T‐H. These findings suggest that the salutary effects of E2 on cardiac function following T‐H are mediated via ERβ up‐regulation of PGC‐1 α through Tfam‐dependent pathway.—Hsieh, Y.‐C., Choudhry, M. A., Yu, H.‐P., Shimizu, T., Yang, S., Suzuki, T., Chen, J., Bland, K. I., Chaudry, I. H. Inhibition of cardiac PGC‐1 α expression abolishes ERβ agonist‐mediated cardioprotection following trauma‐hemorrhage. FASEB J. 20, 1109–1117 (2006)

Glucosamine administration during resuscitation improves organ function after trauma hemorrhage.

ABSTRACT Stress-induced hyperglycemia is necessary for maximal rates of survival after severe hemorrhage; however, the responsible mechanisms are not clear. One consequence of hyperglycemia is an increase in hexosamine biosynthesis, which leads to increases in levels of O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins. This modification has been shown to lead to improved survival of isolated cells after stress. In view of this, we hypothesized that glucosamine (GlcNH2), which more selectively increases the levels of O-GlcNAc administration after shock, will have salutary effects on organ function after trauma hemorrhage (TH). Fasted male rats that underwent midline laparotomy were bled to a mean arterial blood pressure of 40 mmHg for 90 min and then resuscitated with Ringer lactate (four times the shed blood volume). Administration of 2.5 mL of 150 mmol L−1 GlcNH2 midway during resuscitation improved cardiac output 2-fold compared with controls that received 2.5 mL of 150 mmol L−1 NaCl. GlcNH2 also improved perfusion of various organs systems, including kidney and brain, and attenuated the TH-induced increase in serum levels of IL-6 (902 ± 224 vs. 585 ± 103 pg mL−1) and TNF-&agr; (540 ± 81 vs. 345 ± 110 pg mL−1) (values are mean ± SD). GlcNH2 administration resulted in significant increase in protein-associated O-GlcNAc in the heart and brain after TH. Thus, GlcNH2 administered during resuscitation improves recovery from TH, as assessed by cardiac function, organ perfusion, and levels of circulating inflammatory cytokines. This protection correlates with enhanced levels of nucleocytoplasmic protein O-GlcNAcylation and suggests that increased O-GlcNAc could be the mechanism that links stress-induced hyperglycemia to improved outcomes.

The role of Kupffer cell α2-adrenoceptors in norepinephrine-induced TNF-α production

Although previous studies have demonstrated that plasma levels of the proinflammatory cytokine tumor necrosis factor-K (TNF-K) increase during early sepsis, the precise mechanism responsible for its upregulation remains to be elucidated. Since recent studies have shown that the gut is an important source of norepinephrine (NE) release during early sepsis and enterectomy prior to the onset of sepsis attenuates TNF-K production, we hypothesized that gut-derived NE plays a major role in upregulating TNF-K via the activation of K2-adrenoceptors on Kupffer cells. To confirm that NE increases TNF-K synthesis and release, Kupffer cells were isolated from normal rats and incubated with NE (20 or 50 nM) or another K2adrenergic agonist clonidine (50 nM) without addition of Escherichia coli endotoxin. Supernatant levels of TNF-K were then measured. In additional animals, intraportal infusion of NE (20 WM) with or without the specific K2-adrenergic antagonist yohimbine (1 mM) at a rate of 13 Wl/min was carried out for 2 h. Plasma and Kupffer cell levels of TNF-K were assayed thereafter. Moreover, the effects of NE and yohimbine on TNF-K production was further examined using an isolated perfused liver preparation. The results indicate that both NE and clonidine increased TNF-K release by approximately 4^7fold in the isolated cultured Kupffer cells. Similarly, intraportal infusion of NE in vivo or in isolated livers increased TNF-K synthesis and release which was inhibited by co-infusion of yohimbine. Furthermore, the increased cellular levels of TNF-K in Kupffer cells after in vivo administration of NE was also blocked by yohimbine. These results, taken together, suggest that gut-derived NE upregulates TNF-K production in Kupffer cells through an K2-adrenergic pathway, which appears to be responsible at least in part for the increased levels of circulating TNF-K observed during early sepsis as well as other pathophysiologic conditions such as trauma, hemorrhagic shock, or gut ischemia/reperfusion. fl 2001 Elsevier Science B.V. All rights reserved.

The protective effects of PUGNAc on cardiac function after trauma-hemorrhage are mediated via increased protein O-GlcNAc levels.

We have previously shown that administration of glucosamine after trauma-hemorrhage (TH) improved cardiac output and organ perfusion, and this was associated with increased levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins in the heart and brain. An alternative means of increasing O-GlcNAc levels is by inhibition of O-linked N-acetylglucosaminidase, which catalyzes the removal of N-acetylglucosamine from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc). The goal of this study, therefore, was to determine whether PUGNAc administration after TH also improves recovery of organ perfusion and function. Fasted male rats were bled to and maintained at a mean arterial blood pressure of 40 mmHg for 90 min, followed by fluid resuscitation. Intravenous administration of PUGNAc (200 &mgr;mol/kg body weight) 30 min after the onset of resuscitation significantly improved cardiac output compared with the vehicle controls (12.3 ± 1.3 mL/min per 100 g body weight vs. 25.5 ± 2.0 mL/min per 100 g body weight; P < 0.05), decreased total peripheral resistance (6.6 ± 0.8 mmHg/mL per minute per 100 g body weight vs. 3.7 ± 0.3 mmHg/mL per minute per 100 g body weight; P < 0.05), and increased perfusion of critical organ systems, including the kidney and liver, determined at 2 h after the end of resuscitation. Treatment with PUGNAc also attenuated the TH-induced increase in plasma IL-6 levels (864 ± 112 pg/mL vs. 392 ± 188 pg/mL; P < 0.05) and TNF-&agr; levels (216 ± 21 pg/mL vs. 94 ± 11 pg/mL; P < 0.05) and significantly increased O-GlcNAc levels in the heart, liver, and kidney. Thus, PUGNAc, like glucosamine, improves cardiac function and organ perfusion and reduced the level of circulating IL-6 and TNF-&agr; after TH. The similar effects of glucosamine and PUGNAc support the notion that the protection associated with both interventions is mediated via increased protein O-GlcNAc levels.

Differential alterations in systemic and regional oxygen delivery and consumption during the early and late stages of sepsis.

Background: Studies have indicated that regional changes in oxygen utilization during sepsis cannot be predicted from the changes in the whole body oxygen delivery (DO 2 ) and consumption (VO 2 ). The aim of this study, therefore, was to determine whether differential alterations in systemic and regional DO 2 and VO 2 occur during the early and late stages of sepsis. Methods: Adult male Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 hours (i.e., the early, hyperdynamic phase of sepsis) or 20 hours (i.e., the late, hypodynamic phase) after CLP, cardiac output, and organ blood flow were measured by radioactive microspheres. Systemic and regional DO 2 and VO 2 were determined and plasma levels of lactate were measured. Results: Cardiac output and blood flow to the liver, small intestine, and kidneys increased at 5 hours and decreased at 20 hours after CLP. Although both systemic DO 2 and VO 2 increased at 5 hours after CLP, systemic DO 2 but not VO 2 decreased at 20 hours. At 5 hours after CLP, intestinal and renal DO 2 increased. However, DO 2 in all the tested organs decreased at 20 hours after CLP. VO 2 increased in the liver, small intestine, and kidneys at 5 hours after CLP but decreased only in the liver and small intestine at 20 hours after the onset of sepsis. Moreover, plasma lactate levels increased at the late stage of sepsis. Conclusion: Because hepatic and intestinal VO 2 but not systemic and renal VO 2 decreased at 20 hours after CLP, the liver and small intestine seem to be more vulnerable to the hypoxic insult during the hypodynamic stage of polymicrobial sepsis.

Administration of human inter-α-inhibitors maintains hemodynamic stability and improves survival during sepsis

OBJECTIVES The major forms of human inter-alpha-inhibitor proteins circulating in the plasma are inter-alpha-inhibitor (IalphaI, containing one light peptide chain called bikunin and two heavy chains) and pre-alpha-inhibitor (PalphaI, containing one light and one heavy chain). Although it has been reported that a decrease in IalphaI/PalphaI is correlated with an increased mortality rate in septic patients, it remains unknown whether administration of IalphaI/PalphaI early after the onset of sepsis has any beneficial effects on the cardiovascular response and outcome of the septic animal. The aim of this study, therefore, was to determine whether IalphaI and PalphaI have any salutary effects on the depressed cardiovascular function, liver damage, and mortality rate after polymicrobial sepsis. DESIGN Prospective, controlled, randomized animal study. SETTING A university research laboratory. SUBJECTS Male adult rats were subjected to polymicrobial sepsis by cecal ligation and puncture or sham operation followed by the administration of normal saline (i.e., resuscitation). MEASUREMENTS AND MAIN RESULTS At 1 hr after cecal ligation and puncture, human IalphaI/PalphaI at a dose of 30 mg/kg body weight or vehicle (normal saline, 1 mL/rat) were infused intravenously over a period of 30 mins. At 20 hrs after cecal ligation and puncture (i.e., the late, hypodynamic stage of sepsis), cardiac output was measured by using a dye dilution technique, and blood samples were collected for assessing oxygen content. Oxygen delivery, consumption, and extraction ratio were determined. Plasma concentrations of liver enzymes alanine aminotransferase and aspartate aminotransferase as well as lactate and tumor necrosis factor-alpha also were measured. In additional animals, the necrotic cecum was excised at 20 hrs after cecal ligation and puncture with or without IalphaI/PalphaI treatment, and survival was monitored for 10 days thereafter. The results indicate that administration of human IalphaI/PalphaI early after the onset of sepsis maintained cardiac output and systemic oxygen delivery, whereas it increased oxygen consumption and extraction at 20 hrs after cecal ligation and puncture. The elevated concentrations of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-alpha, and lactate were attenuated by IalphaI/PalphaI treatment. In addition, administration of human IalphaI/PalphaI improved the survival rate from 30% to 89% in septic animals at day 10 after cecal ligation and puncture and cecal excision. CONCLUSION Human IalphaI/PalphaI appears to be a useful agent for maintaining hemodynamic stability and improving survival during the progression of polymicrobial sepsis.

Resveratrol Improves Cardiac Contractility following Trauma-Hemorrhage by Modulating Sirt1

Mitochondria play a critical role in metabolic homeostasis of a cell. Our recent studies, based on the reported interrelationship between c-Myc and Sirt1 (mammalian orthologue of yeast sir2 [silent information regulator 2]) expression and their role in mitochondrial biogenesis and function, demonstrated a significant downregulation of Sirt1 protein expression and an upregulation of c-Myc following trauma-hemorrhage (T-H). Activators of Sirt1 are known to improve mitochondrial function and the naturally occurring polyphenol resveratrol (RSV) has been shown to significantly increase Sirt1 activity by increasing its affinity to both NAD+ and the acetylated substrate. In this study we tested the salutary effect of RSV following T-H and its influence on Sirt1 expression. Rats were subjected to T-H or sham operation. RSV (8 mg/kg body weight, intravenously) or vehicle was administered 10 min after the onset of resuscitation, and the rats were killed 2 h following resuscitation. Sirtinol, a Sirt1 inhibitor, was administered 5 min prior to RSV administration. Cardiac contractility (±dP/dt) was measured and heart tissue was tested for Sirt1, Pgc-1α, c-Myc, cytosolic cytochrome C expression and ATP level. Left ventricular function, after T-H, was improved (P < 0.05) following RSV treatment, with significantly elevated expression of Sirt1 (P < 0.05) and Pgc-1α (P < 0.05), and decreased c-Myc (P < 0.05). We also observed significantly higher cardiac ATP content, declined cytosolic cytochrome C and decreased plasma tumor necrosis factor-α in the T-H-RSV group. The salutary effect due to RSV was abolished by sirtinol, indicating a Sirtl-mediated effect. We conclude that RSV may be a useful adjunct to resuscitation fluid following T-H.

Down-regulation of hepatic CYP1A2 plays an important role in inflammatory responses in sepsis.

ObjectiveAlthough hepatic cytochrome P-450 protein concentrations are altered following endotoxin shock, changes in P-450 isoforms in sepsis have not been fully investigated. The aim of this study was to determine whether the major P-450 isoform in rat liver (i.e., CYP1A2) is down-regulated during the progression of sepsis and, if so, whether reduction of P-450 enzyme system plays an important role in the inflammatory response. DesignProspective, controlled, and randomized animal study. SettingA university/institute research laboratory. SubjectsMale adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). InterventionsP-450 isoforms in the liver (i.e., CYP1A2 and 4A1) were determined using reverse transcription polymerase chain reaction and Western blot analysis at various time points after CLP. Measurements and Main ResultsThe results indicate that CYP1A2 messenger RNA expression decreased significantly at 10 and 20 hrs whereas its protein concentrations decreased at 20 hrs after the induction of sepsis. In contrast, CYP4A1 messenger RNA and protein concentrations were not altered even at 20 hrs after CLP. In an additional experiment, all P-450 isoforms were inhibited by pretreatment with 1-aminobenzotriazole to determine the effect of cytochrome P-450 blockade on inflammatory responses by assessing proinflammatory cytokines. The results show further increases in serum concentrations of tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6 in aminobenzotriazole-treated animals at 10 hrs after CLP, which was associated with elevated concentrations of circulating lactate and severe morphologic alterations in the liver. These results suggest that the integrity of the cytochrome P-450 enzyme system plays an important role in septic inflammatory response. ConclusionThe major hepatic P-450 isoform CYP1A2 is down-regulated and inhibition of P-450 enzyme system is associated with an exacerbated inflammatory response in sepsis. Treatment with pharmaceutical agents that regulate or are metabolized by P-450 enzymes might be approached cautiously in the septic patient if this holds true in a clinical setting.