Kenneth N. Maclean

CCAAT/enhancing binding protein β deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis†

Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, inflammation, and oxidative stress. To investigate whether the transcription factor CCAAT/Enhancer binding protein (C/EBPβ) is involved in the development of NASH, C57BL/6J wild‐type (WT) or C/EBPβ knockout (C/EBPβ−/−) mice were fed either a methionine and choline deficient (MCD) diet or standard chow. These WT mice fed a MCD diet for 4 weeks showed a 2‐ to 3‐fold increase in liver C/EBPβ messenger RNA and protein, along with increased expression of lipogenic genes peroxisome proliferators‐activated receptor γ and Fas. WT mice also showed increased levels of the endoplasmic reticulum stress pathway proteins phosphorylated eukaryotic translation initiation factor α, phosphorylated pancreatic endoplasmic reticulum kinase, and C/EBP homologous protein, along with inflammatory markers phosphorylated nuclear factor κB and phosphorylated C‐jun N‐terminal kinase compared to chow‐fed controls. Cytochrome P450 2E1 protein and acetyl coA oxidase messenger RNA involved in hepatic lipid peroxidation were also markedly increased in WT MCD diet‐fed group. In contrast, C/EBPβ−/− mice fed a MCD diet showed a 60% reduction in hepatic triglyceride accumulation and decreased liver injury as evidenced by reduced serum alanine aminotransferase and aspartate aminotransferase levels, and by H&E staining. Immunoblots and real‐time qPCR data revealed a significant reduction in expression of stress related proteins and lipogenic genes in MCD diet‐fed C/EBPβ−/− mice. Furthermore, circulating TNFα and expression of acute phase response proteins CRP and SAP were significantly lower in C/EBPβ−/− mice compared to WT mice. Conversely, C/EBPβ over‐expression in livers of WT mice increased steatosis, nuclear factor‐κB, and endoplasmic reticulum stress, similar to MCD diet‐fed mice. Conclusion: Taken together, these data suggest a previously unappreciated molecular link between C/EBPβ, hepatic steatosis and inflammation and suggest that increased C/EBPβ expression may be an important factor underlying events leading to NASH. (HEPATOLOGY 2007;45:1108–1117.)

Perinatal choline supplementation improves cognitive functioning and emotion regulation in the Ts65Dn mouse model of Down syndrome

In addition to mental retardation, individuals with Down syndrome (DS) also develop the neuropathological changes typical of Alzheimers disease (AD) and the majority of these individuals exhibit dementia. The Ts65Dn mouse model of DS exhibits key features of these disorders, including early degeneration of cholinergic basal forebrain (CBF) neurons and impairments in functions dependent on the two CBF projection systems; namely, attention and explicit memory. Herein, we demonstrate that supplementing the maternal diet with excess choline during pregnancy and lactation dramatically improved attentional function of the adult trisomic offspring. Specifically, the adult offspring of choline-supplemented Ts65Dn dams performed significantly better than unsupplemented Ts65Dn mice on a series of 5 visual attention tasks, and in fact, on some tasks did not differ from the normosomic (2N) controls. A second area of dysfunction in the trisomic animals, heightened reactivity to committing an error, was partially normalized by the early choline supplementation. The 2N littermates also benefited from increased maternal choline intake on 1 attention task. These findings collectively suggest that perinatal choline supplementation might significantly lessen cognitive dysfunction in DS and reduce cognitive decline in related neurodegenerative disorders such as AD.

Integrated Stress Response Modulates Cellular Redox State via Induction of Cystathionine γ-Lyase CROSS-TALK BETWEEN INTEGRATED STRESS RESPONSE AND THIOL METABOLISM

Background: The integrated stress response (ISR) maintains cellular homeostasis during aberrant protein folding (ER stress). Results: The ISR enhances glutathione synthesis through up-regulation of cystathionine γ-lyase via the eIF2α-ATF4 pathway. Conclusion: Cells undergoing the ISR induce cystathionine γ-lyase, thereby maintaining cellular homeostasis. Significance: These findings link the cells response to ER stress and redox homeostasis through the ISR. The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine β-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4−/−) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4−/− MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4−/− MEFs could be rescued from l-Hcy-induced apoptosis by β-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4−/− MEFs showed little or no CSE protein but did express cystathionine β-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4+/+ MEFs. Consistent with ATF4−/− MEFs, CSE−/− MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE+/+ MEFs. Liver and kidney GSH levels were also reduced in CSE−/− mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

Experimental evidence for therapeutic potential of taurine in the treatment of nonalcoholic fatty liver disease

The incidence of obesity is now at epidemic proportions and has resulted in the emergence of nonalcoholic fatty liver disease (NAFLD) as a common metabolic disorder that can lead to liver injury and cirrhosis. Excess sucrose and long-chain saturated fatty acids in the diet may play a role in the development and progression of NAFLD. One factor linking sucrose and saturated fatty acids to liver damage is dysfunction of the endoplasmic reticulum (ER). Although there is currently no proven, effective therapy for NAFLD, the amino sulfonic acid taurine is protective against various metabolic disturbances, including alcohol-induced liver damage. The present study was undertaken to evaluate the therapeutic potential of taurine to serve as a preventative treatment for diet-induced NAFLD. We report that taurine significantly mitigated palmitate-mediated caspase-3 activity, cell death, ER stress, and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high-sucrose diet, dietary taurine supplementation significantly reduced hepatic lipid accumulation, liver injury, inflammation, plasma triglycerides, and insulin levels. The high-sucrose diet resulted in an induction of multiple components of the unfolded protein response in the liver consistent with ER stress, which was ameliorated by taurine supplementation. Treatment of mice with the ER stress-inducing agent tunicamycin resulted in liver injury, unfolded protein response induction, and hepatic lipid accumulation that was significantly ameliorated by dietary supplementation with taurine. Our results indicate that dietary supplementation with taurine offers significant potential as a preventative treatment for NAFLD.

Oxidative Stress and the ER Stress Response in a Murine Model for Early-Stage Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

Cystathionine β-synthase is coordinately regulated with proliferation through a redox-sensitive mechanism in cultured human cells and Saccharomyces cerevisiae

Cystathionine β‐synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration. Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells. In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth‐arrested cultures or those growing non‐fermentatively on ethanol or glycerol had ∼3‐fold less activity. CBS activity assays and Western blotting indicated that growth‐specific regulation of CBS is evolutionarily conserved in a range of human cell lines. CBS activity was found to be maximal during proliferation and was reduced two‐ to five‐fold when cells became quiescent at confluence. In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum‐starvation, nutrient depletion, or the induction of differentiation. Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation. The intracellular level of the CBS regulator compound, S‐adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation‐specific regulation of human CBS. Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo.

Deletion mutagenesis of human cystathionine β-synthase: Impact on activity, oligomeric status, and S-adenosylmethionine regulation

Cystathionine β-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5′-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coliindicated that residues 497–543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathioneS-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine β-synthase contains two hydrophobic motifs designated “CBS domains.” Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation byS-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated “human only” (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6 months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Neural Stem Cells Reduce Hippocampal Tau and Reelin Accumulation in Aged Ts65Dn Down Syndrome Mice

Tau accumulation, in the form of neurofibrillary tangles (NFT), is an early neuropathological characteristic of Alzheimers disease (AD) and early onset AD frequently seen in Down syndrome (DS). We investigated the presence of tau accumulation in the brains of aging DS mice using the Ts65Dn mouse model. All aged mice appeared to have substantial clusters of extracellular granules that were positive for tau and reelin, but not for amyloid-β or APP. These clusters were found primarily in CA1 of the hippocampus. In addition, the aged trisomic DS mice had a significantly greater accumulation of extracellular tau/reelin granular deposits compared to disomic littermates. These granules were similar to those described by others who also found extracelluar proteinous granules in the brains of non-DS mice engineered to model aging and/or AD. When neural stem cells (NSC) were implanted unilaterally into the hippocampus of the Ts65Dn mice, the tau/reelin-positive granules were significantly reduced in both trisomic and disomic mice. Our findings indicate that changes in tau/reelin-positive granules could be used as an index for neuropathological assessment in aging DS and AD. Furthermore, changes in granule density could be used to test the efficacy of novel treatments, such as NSC implantation. Lastly, it is speculated that the unique abilities of NSC to migrate and express growth factors might be a contributing factor to reducing tau/reelin accumulation in aging DS and AD.

Cystathionine beta-synthase null homocystinuric mice fail to exhibit altered hemostasis or lowering of plasma homocysteine in response to betaine treatment

Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585–1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (−/−) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (−/−) mice and restored fertility to female cbs (−/−) mice but did so without significantly lowering Hcy levels. Surviving cbs (−/−) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (−/−) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (−/−) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (−/−) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.