The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease.

BACKGROUND: Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. METHODS: For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. RESULTS: Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. CONCLUSION: Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress.

CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury.

Macrophages are an essential component of the immune response to ischaemic injury and play an important role in promoting inflammation and its resolution, which is necessary for tissue repair. The type I transmembrane glycoprotein CD163 is exclusively expressed on macrophages, where it acts as a receptor for haemoglobin:haptoglobin complexes. An extracellular portion of CD163 circulates in the blood as a soluble protein, for which no physiological function has so far been described. Here we show that during ischaemia, soluble CD163 functions as a decoy receptor for TWEAK, a secreted pro-inflammatory cytokine of the tumour necrosis factor family, to regulate TWEAK-induced activation of canonical nuclear factor-κB (NF-κB) and Notch signalling necessary for myogenic progenitor cell proliferation. Mice with deletion of CD163 have transiently elevated levels of TWEAK, which stimulate muscle satellite cell proliferation and tissue regeneration in their ischaemic and non-ischaemic limbs. These results reveal a role for soluble CD163 in regulating muscle regeneration after ischaemic injury.

Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent.

Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

H2S protects against pressure overload-induced heart failure via upregulation of endothelial nitric oxide synthase.

BACKGROUND: Cystathionine γ-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload-induced heart failure. METHODS AND RESULTS: Transverse aortic constriction was performed in wild-type, CSE knockout, and cardiac-specific CSE transgenic mice. In addition, C57BL/6J or CSE knockout mice received a novel H2S donor (SG-1002). Mice were followed up for 12 weeks with echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels after transverse aortic constriction. CSE knockout mice exhibited significantly greater cardiac dilatation and dysfunction than wild-type mice after transverse aortic constriction, and cardiac-specific CSE transgenic mice maintained cardiac structure and function after transverse aortic constriction. H2S therapy with SG-1002 resulted in cardioprotection during transverse aortic constriction via upregulation of the vascular endothelial growth factor-Akt-endothelial nitric oxide synthase-nitric oxide-cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. CONCLUSIONS: Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure, and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability.

Selective ß2-adrenoreceptor stimulation attenuates myocardial cell death and preserves cardiac function after ischemia-reperfusion injury.

OBJECTIVE: ß(2)-adrenoreceptor activation has been shown to protect cardiac myocytes from cell death. We hypothesized that acute ß(2)-adrenoreceptor stimulation, using arformoterol (ARF), would attenuate myocardial ischemia/reperfusion (R) injury via NO synthase activation and cause a subsequent increase in NO bioavailability. METHODS AND RESULTS: Male C57BL/6J and endothelial NO synthase (eNOS) knockout mice were subjected to 45 minutes of myocardial ischemia and 24 hours of R. ARF or vehicle was administered 5 minutes before R. Serum troponin-I was measured, and infarct size per area-at-risk was evaluated at 24 hours of R. Echocardiography was performed at baseline and 2 weeks after R. Myocardial cAMP, protein kinase A, eNOS/Akt phosphorylation status, and NO metabolite levels were assayed. ARF (1 µg/kg) reduced infarct size per area-at-risk by 53.1% (P<0.001 versus vehicle) and significantly reduced troponin-I levels (P<0.001 versus vehicle). Ejection fraction was significantly preserved in ARF-treated hearts compared with vehicle hearts at 2 weeks of R. Serum cAMP and nuclear protein kinase A C-α increased 5 and 15 minutes after ARF injection, respectively (P<0.01). ARF increased Akt phosphorylation at Thr(308) (P<0.001) and Ser(473) (P<0.01), and eNOS phosphorylation at Ser(1177) (P<0.01). ARF treatment increased heart nitrosothiol levels (P<0.001) at 15 min after injection. ARF failed to reduce infarct size in eNOS(-/-) mice. CONCLUSIONS: Our results indicate that ß(2)-adrenoreceptor stimulation activates cAMP, protein kinase A, Akt, and eNOS and augments NO bioavailability. Activation of this prosurvival signaling pathway attenuates myocardial cell death and preserves cardiac function after ischemia/reperfusion.

Betaine treatment attenuates chronic ethanol-induced hepatic steatosis and alterations to the mitochondrial respiratory chain proteome.

Introduction. Mitochondrial damage and disruption in oxidative phosphorylation contributes to the pathogenesis of alcoholic liver injury. Herein, we tested the hypothesis that the hepatoprotective actions of betaine against alcoholic liver injury occur at the level of the mitochondrial proteome. Methods. Male Wister rats were pair-fed control or ethanol-containing liquid diets supplemented with or without betaine (10 mg/mL) for 4-5 wks. Liver was examined for triglyceride accumulation, levels of methionine cycle metabolites, and alterations in mitochondrial proteins. Results. Chronic ethanol ingestion resulted in triglyceride accumulation which was attenuated in the ethanol plus betaine group. Blue native gel electrophoresis (BN-PAGE) revealed significant decreases in the content of the intact oxidative phosphorylation complexes in mitochondria from ethanol-fed animals. The alcohol-dependent loss in many of the low molecular weight oxidative phosphorylation proteins was prevented by betaine supplementation. This protection by betaine was associated with normalization of SAM : S-adenosylhomocysteine (SAH) ratios and the attenuation of the ethanol-induced increase in inducible nitric oxide synthase and nitric oxide generation in the liver. Discussion/Conclusion. In summary, betaine attenuates alcoholic steatosis and alterations to the oxidative phosphorylation system. Therefore, preservation of mitochondrial function may be another key molecular mechanism responsible for betaine hepatoprotection.

Cytoprotective actions of hydrogen sulfide in ischaemia-reperfusion injury.

Hydrogen sulfide (H(2)S) has been known as a highly toxic gas for several centuries. There have been considerable advances made in the H(2)S field regarding its physiological role; however, there is much more work that needs to be done. The biosynthesis of H(2)S has been attributed to three endogenous enzymes: cystathionine ß-synthase (CBS), cystathionine γ-lyase (CGL or CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). These enzymes require further investigation to more fully elucidate the cellular expression profile, regulation and precise role of these critical enzymes in the production of H(2)S. In recent years, H(2)S has been demonstrated to have cytoprotective effects in multiple organ systems. In particular, it has been demonstrated that the administration of H(2)S either prior to ischaemia or at reperfusion significantly ameliorates myocardial and hepatic ischaemia-reperfusion injury. Therefore, this review focuses on the cardioprotective and hepatoprotective role of H(2)S. In addition, the review provides a summary of several known molecular targets of H(2)S protection.

Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection.

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

Assessment of mitochondrial dysfunction arising from treatment with hepatotoxicants.

Mitochondrial dysfunction from toxicants is recognized as a causative factor in the development of numerous liver diseases including steatohepatitis, cirrhosis, and cancer. Toxicant-mediated damage to mitochondria result in depressed ATP production, inability to maintain proper cellular calcium homeostasis, and increased reactive oxygen species production. These disruptions contribute to hepatocellular death and lead to liver pathology. Herein, we describe a series of basic and advanced methodologies that can be incorporated into research projects aimed to understand the role of mitochondrial dysfunction in toxicant-induced hepatotoxicity. Protocols are provided for isolation of liver mitochondria, assessment of respiratory function, measurement of mitochondrial calcium uptake, and reactive oxygen species production, as well as characterization of the mitochondrial protein thiol proteome using 2D gel electrophoresis. Data obtained from these methods can be integrated into a logical and mechanistic framework to advance understanding of the role of mitochondrial dysfunction in the pathogenesis of toxicant-induced liver diseases.