Joanne M. Ajmo

Resveratrol alleviates alcoholic fatty liver in mice

Alcoholic fatty liver is associated with inhibition of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), two critical signaling molecules regulating the pathways of hepatic lipid metabolism in animals. Resveratrol, a dietary polyphenol, has been identified as a potent activator for both SIRT1 and AMPK. In the present study, we have carried out in vivo animal experiments that test the ability of resveratrol to reverse the inhibitory effects of chronic ethanol feeding on hepatic SIRT1-AMPK signaling system and to prevent the development of alcoholic liver steatosis. Resveratrol treatment increased SIRT1 expression levels and stimulated AMPK activity in livers of ethanol-fed mice. The resveratrol-mediated increase in activities of SIRT1 and AMPK was associated with suppression of sterol regulatory element binding protein 1 (SREBP-1) and activation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha). In parallel, in ethanol-fed mice, resveratrol administration markedly increased circulating adiponectin levels and enhanced mRNA expression of hepatic adiponectin receptors (AdipoR1/R2). In conclusion, resveratrol treatment led to reduced lipid synthesis and increased rates of fatty acid oxidation and prevented alcoholic liver steatosis. The protective action of resveratrol is in whole or in part mediated through the upregulation of a SIRT1-AMPK signaling system in the livers of ethanol-fed mice. Our study suggests that resveratrol may serve as a promising agent for preventing or treating human alcoholic fatty liver disease.

Involvement of mammalian sirtuin 1 in the action of ethanol in the liver

Chronic ethanol feeding causes liver steatosis in animal models by upregulating the sterol regulatory element-binding protein 1 (SREBP-1), which subsequently increases the synthesis of hepatic lipid. SREBP-1 activity is regulated by reversible acetylation at specific lysine residues. The present study tests the hypothesis that activation of SREBP-1 by ethanol may be mediated by mammalian sirtuin 1 (SIRT1), a NAD(+)-dependent class III protein deacetylase. The effects of ethanol on SIRT1 were determined in cultured rat hepatoma cells and in the livers of ethanol-fed mice. In rat H4IIEC3 cells, we observed that ethanol exposure induced SREBP-1c lysine acetylation and SREBP-1c transcriptional activity. The effect of ethanol was abolished by expression of wild-type SIRT1 or by treatment with resveratrol, a known potent SIRT1 agonist. Conversely, knocking down SIRT1 by the small silencing SIRT1 plasmid SIRT1shRNA or expression of a SIRT1 mutant, SIRT1(H363Y), did not negate the ethanol effect. These findings suggest that the effect of ethanol on SREBP-1 is mediated, at least in part, through SIRT1 inhibition. Consistent with the in vitro findings, chronic ethanol feeding substantially downregulated hepatic SIRT1 in mice. Inhibition of hepatic SIRT1 activity was associated with an increase in the acetylated active nuclear form of SREBP-1c in the livers of ethanol-fed mice. Our results indicate an essential role for SIRT1 in mediating the effects of ethanol on SREBP-1 and hepatic lipid metabolism, as well as the development of alcoholic fatty liver. Hence, SIRT1 may represent a novel therapeutic target for treatment of human alcoholic fatty liver disease.

Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced TNF-α production in cultured macrophage cell lines

Dysregulation of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of alcoholic liver injury. Sirtuin 1 (SIRT1) is an NAD(+)-dependent class III protein deacetylase that is known to be involved in regulating production of proinflammatory cytokines including TNF-alpha. In the present study, we examined the role of SIRT1 signaling in TNF-alpha generation stimulated by either lipopolysaccharide (LPS), acetaldehyde (AcH), or acetate (two major metabolites of ethanol) in two cultured macrophage cell lines. In both rat Kupffer cell line 1 (RKC1) and murine RAW 264.7 macrophages, treatment with either LPS, AcH, or acetate caused significant decreases in SIRT1 transcription, translation, and activation, which essentially demonstrated an inverse relationship with TNF-alpha levels. LPS, AcH, and acetate each provoked the release of TNF-alpha from RKC1 cells, whereas coincubation with resveratrol (a potent SIRT1 agonist) inhibited this effect. Conversely, addition of sirtinol (a known SIRT1 inhibitor) or knocking down SIRT1 by the small silencing SIRT1 plasmid (SIRT1shRNA) augmented TNF-alpha release, suggesting that impairment of SIRT1 may contribute to TNF-alpha secretion. Further mechanistic studies revealed that inhibition of SIRT1 by LPS, AcH, or acetate was associated with a marked increase in the acetylation of the RelA/p65 subunit of nuclear transcription factor (NF-kappaB) and promotion of NF-kappaB transcriptional activity. Taken together, our findings suggest that SIRT1-NF-kappaB signaling is involved in regulating LPS- and metabolites-of-ethanol-mediated TNF-alpha production in rat Kupffer cells and in murine macrophages. Our study provides new insights into understanding the molecular mechanisms underlying the development of alcoholic steatohepatitis.

Adiponectin and alcoholic fatty liver disease

Worldwide, one of the most prevalent forms of chronic disease is alcoholic fatty liver, which may progress to more severe forms of liver injury including steatohepatitis, fibrosis, and cirrhosis. The molecular mechanisms by which ethanol consumption causes accumulation of hepatic lipid are multiple and complex. Chronic ethanol exposure is thought to cause enhanced hepatic lipogenesis and impaired fatty acid oxidation by inhibiting key hepatic transcriptional regulators such as AMP‐activated kinase (AMPK), sirtuin 1 (SIRT1), PPAR‐gamma coactivator alpha (PGC‐1α), peroxisome proliferator‐activated receptor alpha (PPARα), and sterol regulatory element‐binding protein 1 (SREBP‐1). Adiponectin is an adipose‐derived hormone with a variety of beneficial biological functions. Increasing evidence suggests that altered adiponectin production in adipose tissue and impaired expression of hepatic adiponectin receptors (AdipoRs) are associated with the development of alcoholic liver steatosis in several rodent models. More importantly, studies have demonstrated a protective role of adiponectin against alcoholic liver steatosis. The hepato‐protective effect of adiponectin is largely mediated by the coordination of multiple signaling pathways in the liver, leading to enhanced fat oxidation, reduced lipid synthesis and prevention of hepatic steatosis. This review begins with an assessment of the current understanding of the role of adiponectin and its receptors in the regulation of lipid homeostasis in liver, with emphasis on their relationship to the development of alcoholic liver steatosis. Following sections will review hepatic signaling molecules involved in the protective actions of adiponectin against alcoholic fatty liver and summarize the current knowledge of regulatory mechanisms of adiponectin expression and secretion in response to chronic ethanol exposure. We will conclude with a discussion of potential strategies for treating human alcoholic fatty liver disease (AFLD), including nutritional and pharmacological modulation of adiponectin and its receptors.

Deletion of SIRT1 from Hepatocytes in Mice Disrupts Lipin-1 Signaling and Aggravates Alcoholic Fatty liver

BACKGROUND & AIMS Sirtuin (SIRT1) is a nicotinamide adenine dinucleotide-dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors. Ethanol exposure disrupts SIRT1 activity and contributes to alcoholic liver disease in rodents, but the exact pathogenic mechanism is not clear. We compared mice with liver-specific deletion of Sirt1 (Sirt1LKO) mice with their LOX littermates (controls). METHODS We induced alcoholic liver injury in male Sirt1LKO and control mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Liver and serum samples were collected. We also measured messenger RNA levels of SIRT1, SFRS10, and lipin-1β and lipin-1α in liver samples from patients with alcoholic hepatitis and individuals without alcoholic hepatitis (controls). RESULTS On the ethanol-containing diet, livers of Sirt1LKO mice accumulated larger amounts of hepatic lipid and expressed higher levels of inflammatory cytokines than control mice; serum of Sirt1LKO mice had increased levels of alanine aminotransferase and aspartate aminotransferase. Hepatic deletion of SIRT1 exacerbated ethanol-mediated defects in lipid metabolism, mainly by altering the function of lipin-1, a transcriptional regulator of lipid metabolism. In cultured mouse AML-12 hepatocytes, transgenic expression of SIRT1 prevented fat accumulation in response to ethanol exposure, largely by reversing the aberrations in lipin-1 signaling induced by ethanol. Liver samples from patients with alcoholic hepatitis had reduced levels of SIRT1 and a higher ratio of Lpin1β/α messenger RNAs than controls. CONCLUSIONS In mice, hepatic deletion of Sirt1 promotes steatosis, inflammation, and fibrosis in response to ethanol challenge. Ethanol-mediated impairment of hepatic SIRT1 signaling via lipin-1 contributes to development of alcoholic steatosis and inflammation. Reagents designed to increase SIRT1 regulation of lipin-1 can be developed to treat patients with alcoholic fatty liver disease.

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat

BackgroundHypoxia-ischemia (H-I) can produce widespread neurodegeneration and deep cerebral white matter injury in the neonate. Resident microglia and invading leukocytes promote lesion progression by releasing reactive oxygen species, proteases and other pro-inflammatory mediators. After injury, expression of the gelatin-degrading matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are thought to result in the proteolysis of extracellular matrix (ECM), activation of cytokines/chemokines, and the loss of vascular integrity. Thus, therapies targeting ECM degradation and progressive neuroinflammation may be beneficial in reducing H-I – induced neuropathy. Minocycline has MMP-inhibitory properties and is both anti-inflammatory and neuroprotective. AG3340 (prinomastat) is an MMP inhibitor with high selectivity for the gelatinases. The purpose of this study was to determine whether these compounds could limit H-I – induced injury when administered at a delayed time point.MethodsSprague-Dawley rats were exposed to H-I at postnatal day 7 (P7), consisting of unilateral carotid artery ligation followed by 90 min exposure to 8% O2. Minocycline, AG3340, or vehicle were administered once daily for 6 days, beginning 24 hours after insult. Animals were sacrificed at P14 for neurohistological assessments. Immunohistochemistry was performed to determine the degree of reactive astrogliosis and immune cell activation/recruitment. Neural injury was detected using the Fluoro-Jade stain, a marker that identifies degenerating cells.ResultsCD11b and glial fibrillary acidic protein (GFAP) immunopositive cells increased in ipsilateral cortex after treatment with vehicle alone, demonstrating microglia/macrophage recruitment and reactive astrogliosis, respectively. Fluoro-Jade staining was markedly increased throughout the fronto-parietal cortex, striatum and hippocampus. Treatment with minocycline or AG3340 inhibited microglia/macrophage recruitment, attenuated astrogliosis and reduced Fluoro-Jade staining when compared to vehicle alone.ConclusionThe selective gelatinase inhibitor AG3340 showed equal efficacy in reducing neural injury and dampening neuroinflammation when compared to the anti-inflammatory compound minocycline. Thus, MMP-2 and MMP-9 may be viable therapeutic targets to treat neonatal brain injury.

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.

Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice

Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis.

Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice.

Lipin‐1 regulates lipid metabolism by way of its function as an enzyme in the triglyceride synthesis pathway and as a transcriptional coregulatory protein and is highly up‐regulated in alcoholic fatty liver disease. In the present study, using a liver‐specific lipin‐1‐deficient (lipin‐1LKO) mouse model, we aimed to investigate the functional role of lipin‐1 in the development of alcoholic steatohepatitis and explore the underlying mechanisms. Alcoholic liver injury was achieved by pair feeding wild‐type and lipin‐1LKO mice with modified Lieber‐DeCarli ethanol‐containing low‐fat diets for 4 weeks. Surprisingly, chronically ethanol‐fed lipin‐1LKO mice showed markedly greater hepatic triglyceride and cholesterol accumulation, and augmented elevation of serum liver enzymes accompanied by increased hepatic proinflammatory cytokine expression. Our studies further revealed that hepatic removal of lipin‐1 in mice augmented ethanol‐induced impairment of hepatic fatty acid oxidation and lipoprotein production, likely by way of deactivation of peroxisome proliferator‐activated receptor γ coactivator‐1alpha, a prominent transcriptional regulator of lipid metabolism. Conclusions: Liver‐specific lipin‐1 deficiency in mice exacerbates the development and progression of experimental alcohol‐induced steatohepatitis. Pharmacological or nutritional modulation of hepatic lipin‐1 may be beneficial for the prevention or treatment of human alcoholic fatty liver disease. (Hepatology 2013; 58:1953–1963)

Versican and brevican are expressed with distinct pathology in neonatal hypoxic-ischemic injury.

The developing brain is uniquely susceptible to injury after exposure to hypoxia‐ischemia (H‐I). Lecticans are developmentally regulated in formative white matter and exert growth‐inhibitory effects in several adult injury models, yet little is known regarding their role in neonatal H‐I injury. The main objectives of this study were to examine the expression profiles of brevican and versican in rat using a standard H‐I model and to determine whether altered expression was associated with distinct components of white and gray matter pathology. The H‐I procedure in postnatal day 7 rats produced progressive injury limited to the ipsilateral hemisphere. Cresyl violet staining revealed severe cavitary infarctions at 14 and 21 days that were absent at 4 days. Cellular damage, as measured by glial fibrillary acidic protein and fractin immunoreactivity, occurred in cortical and subcortical gray matter at all end points. O4 sulfatide immunoreactivity was reduced in the external capsule, hippocampal fimbria, and corpus striatum at 4 days relative to that contralaterally, suggesting the loss of preoligodendrocytes. Brevican expression was reduced in the cortex and hippocampus at 4 days but was markedly elevated at later end points, localizing to regions of cellular damage both in and proximal to the lesion core. However, versican was reduced in the external capsule 4 days after H‐I, a reduction that was sustained up to 21 days in white matter. These data demonstrate unique expression profiles for lecticans after neonatal H‐I, suggesting brevican deposition is elevated in response to progressive gray matter injury, whereas diminished versican expression may be associated with deep cerebral white matter injury.