Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice.

BACKGROUND: CDGSH iron-sulfur domain-containing protein 2 (CISD2), a pro-longevity gene, mediates healthspan in mammals. CISD2 is down-regulated during aging. Furthermore, a persistently high level of CISD2 promotes longevity and ameliorates an age-related skin phenotype in transgenic mice. Here we translate the genetic evidence into a pharmaceutical application using a potent CISD2 activator, hesperetin, which enhances CISD2 expression in HEK001 human keratinocytes from an older person. We also treated naturally aged mice in order to study the activator's anti-aging efficacy. METHODS: We studied the biological effects of hesperetin on aging skin using, firstly, a cell-based platform, namely a HEK001 human keratinocyte cell line established from an older person. Secondly, we used a mouse model, namely old mice at 21-month old. In the latter case, we investigate the anti-aging efficacy of hesperetin on ultraviolet B (UVB)-induced photoaging and naturally aged skin. Furthermore, to identify the underlying mechanisms and potential biological pathways involved in this process we carried out transcriptomic analysis. Finally, CISD2 knockdown HEK001 keratinocytes and Cisd2 knockout mice were used to study the Cisd2-dependent effects of hesperetin on skin aging. RESULTS: Four findings are pinpointed. Firstly, in human skin, CISD2 is mainly expressed in proliferating keratinocytes from the epidermal basal layer and, furthermore, CISD2 is down-regulated in the sun-exposed epidermis. Secondly, in HEK001 human keratinocytes from an older person, hesperetin enhances mitochondrial function and protects against reactive oxygen species-induced oxidative stress via increased CISD2 expression; this enhancement is CISD2-dependent. Additionally, hesperetin alleviates UVB-induced damage and suppresses matrix metalloproteinase-1 expression, the latter being a major indicator of UVB-induced damage in keratinocytes. Thirdly, transcriptomic analysis revealed that hesperetin modulates a panel of differentially expressed genes that are associated with mitochondrial function, redox homeostasis, keratinocyte function, and inflammation in order to attenuate senescence. Intriguingly, hesperetin activates two known longevity-associated regulators, namely FOXO3a and FOXM1, in order to suppress the senescence-associated secretory phenotype. Finally, in mouse skin, hesperetin enhances CISD2 expression to ameliorate UVB-induced photoaging and this occurs via a mechanism involving CISD2. Most strikingly, late-life treatment with hesperetin started at 21-month old and lasting for 5 months, is able to retard skin aging and rejuvenate naturally aged skin in mice. CONCLUSIONS: Our results reveal that a pharmacological elevation of CISD2 expression at a late-life stage using hesperetin treatment is a feasible approach to effectively mitigating both intrinsic and extrinsic skin aging and that hesperetin could act as a functional food or as a skincare product for fighting skin aging.

Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination.

Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.

Hesperetin promotes longevity and delays aging via activation of Cisd2 in naturally aged mice.

BACKGROUND: The human CISD2 gene is located within a longevity region mapped on chromosome 4q. In mice, Cisd2 levels decrease during natural aging and genetic studies have shown that a high level of Cisd2 prolongs mouse lifespan and healthspan. Here, we evaluate the feasibility of using a Cisd2 activator as an effective way of delaying aging. METHODS: Hesperetin was identified as a promising Cisd2 activator by herb compound library screening. Hesperetin has no detectable toxicity based on in vitro and in vivo models. Naturally aged mice fed dietary hesperetin were used to investigate the effect of this Cisd2 activator on lifespan prolongation and the amelioration of age-related structural defects and functional decline. Tissue-specific Cisd2 knockout mice were used to study the Cisd2-dependent anti-aging effects of hesperetin. RNA sequencing was used to explore the biological effects of hesperetin on aging. RESULTS: Three discoveries are pinpointed. Firstly, hesperetin, a promising Cisd2 activator, when orally administered late in life, enhances Cisd2 expression and prolongs healthspan in old mice. Secondly, hesperetin functions mainly in a Cisd2-dependent manner to ameliorate age-related metabolic decline, body composition changes, glucose dysregulation, and organ senescence. Finally, a youthful transcriptome pattern is regained after hesperetin treatment during old age. CONCLUSIONS: Our findings indicate that a Cisd2 activator, hesperetin, represents a promising and broadly effective translational approach to slowing down aging and promoting longevity via the activation of Cisd2.

Serotonin receptor HTR6-mediated mTORC1 signaling regulates dietary restriction-induced memory enhancement.

Dietary restriction (DR; sometimes called calorie restriction) has profound beneficial effects on physiological, psychological, and behavioral outcomes in animals and in humans. We have explored the molecular mechanism of DR-induced memory enhancement and demonstrate that dietary tryptophan-a precursor amino acid for serotonin biosynthesis in the brain-and serotonin receptor 5-hydroxytryptamine receptor 6 (HTR6) are crucial in mediating this process. We show that HTR6 inactivation diminishes DR-induced neurological alterations, including reduced dendritic complexity, increased spine density, and enhanced long-term potentiation (LTP) in hippocampal neurons. Moreover, we find that HTR6-mediated mechanistic target of rapamycin complex 1 (mTORC1) signaling is involved in DR-induced memory improvement. Our results suggest that the HTR6-mediated mTORC1 pathway may function as a nutrient sensor in hippocampal neurons to couple memory performance to dietary intake.

Cisd2 is essential to delaying cardiac aging and to maintaining heart functions.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart's electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction.

Rejuvenation: Turning Back Time by Enhancing CISD2.

The aging human population with age-associated diseases has become a problem worldwide. By 2050, the global population of those who are aged 65 years and older will have tripled. In this context, delaying age-associated diseases and increasing the healthy lifespan of the aged population has become an important issue for geriatric medicine. CDGSH iron-sulfur domain 2 (CISD2), the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928), plays a pivotal role in mediating lifespan and healthspan by maintaining mitochondrial function, endoplasmic reticulum integrity, intracellular Ca2+ homeostasis, and redox status. Here, we summarize the most up-to-date publications on CISD2 and discuss the crucial role that this gene plays in aging and age-associated diseases. This review mainly focuses on the following topics: (1) CISD2 is one of the few pro-longevity genes identified in mammals. Genetic evidence from loss-of-function (knockout mice) and gain-of-function (transgenic mice) studies have demonstrated that CISD2 is essential to lifespan control. (2) CISD2 alleviates age-associated disorders. A higher level of CISD2 during natural aging, when achieved by transgenic overexpression, improves Alzheimer's disease, ameliorates non-alcoholic fatty liver disease and steatohepatitis, and maintains corneal epithelial homeostasis. (3) CISD2, the expression of which otherwise decreases during natural aging, can be pharmaceutically activated at a late-life stage of aged mice. As a proof-of-concept, we have provided evidence that hesperetin is a promising CISD2 activator that is able to enhance CISD2 expression, thus slowing down aging and promoting longevity. (4) The anti-aging effect of hesperetin is mainly dependent on CISD2 because transcriptomic analysis of the skeletal muscle reveals that most of the differentially expressed genes linked to hesperetin are regulated by hesperetin in a CISD2-dependent manner. Furthermore, three major metabolic pathways that are affected by hesperetin have been identified in skeletal muscle, namely lipid metabolism, protein homeostasis, and nitrogen and amino acid metabolism. This review highlights the urgent need for CISD2-based pharmaceutical development to be used as a potential therapeutic strategy for aging and age-associated diseases.

Upregulation of Cisd2 attenuates Alzheimer's-related neuronal loss in mice.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2), a protein that declines in an age-dependent manner, mediates lifespan in mammals. Cisd2 deficiency causes accelerated aging and shortened lifespan, whereas persistent expression of Cisd2 promotes longevity in mice. Alzheimer's disease (AD) is the most prevalent form of senile dementia and is without an effective therapeutic strategy. We investigated whether Cisd2 upregulation is able to ameliorate amyloid ß (Aß) toxicity and prevent neuronal loss using an AD mouse model. Our study makes three major discoveries. First, using the AD mouse model (APP/PS1 double transgenic mice), the dosage of Cisd2 appears to modulate the severity of AD phenotypes. Cisd2 overexpression (∼two-fold) significantly promoted survival and alleviated the pathological defects associated with AD. Conversely, Cisd2 deficiency accelerated AD pathogenesis. Secondly, Cisd2 overexpression protected against Aß-mediated mitochondrial damage and attenuated loss of neurons and neuronal progenitor cells. Finally, an increase in Cisd2 shifted the expression profiles of a panel of genes that are dysregulated by AD toward the patterns observed in wild-type mice. These findings highlight Cisd2-based therapies as a potential disease-modifying strategy for AD. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Rejuvenating the Aging Heart by Enhancing the Expression of the Cisd2 Prolongevity Gene.

Aging is the major risk factor for cardiovascular disease, which is the leading cause of mortality worldwide among aging populations. Cisd2 is a prolongevity gene that mediates lifespan in mammals. Previously, our investigations revealed that a persistently high level of Cisd2 expression in mice is able to prevent age-associated cardiac dysfunction. This study was designed to apply a genetic approach that induces cardiac-specific Cisd2 overexpression (Cisd2 icOE) at a late-life stage, namely a time point immediately preceding the onset of old age, and evaluate the translational potential of this approach. Several discoveries are pinpointed. Firstly, Cisd2 is downregulated in the aging heart. This decrease in Cisd2 leads to cardiac dysfunction and impairs electromechanical performance. Intriguingly, Cisd2 icOE prevents an exacerbation of age-associated electromechanical dysfunction. Secondly, Cisd2 icOE ameliorates cardiac fibrosis and improves the integrity of the intercalated discs, thereby reversing various structural abnormalities. Finally, Cisd2 icOE reverses the transcriptomic profile of the aging heart, changing it from an older-age pattern to a younger pattern. Intriguingly, Cisd2 icOE modulates a number of aging-related pathways, namely the sirtuin signaling, autophagy, and senescence pathways, to bring about rejuvenation of the heart as it enters old age. Our findings highlight Cisd2 as a novel molecular target for developing therapies targeting cardiac aging.

Discovery of a Metabolic Signature Predisposing High Risk Patients with Mild Cognitive Impairment to Converting to Alzheimer's Disease.

Assessing dementia conversion in patients with mild cognitive impairment (MCI) remains challenging owing to pathological heterogeneity. While many MCI patients ultimately proceed to Alzheimer's disease (AD), a subset of patients remain stable for various times. Our aim was to characterize the plasma metabolites of nineteen MCI patients proceeding to AD (P-MCI) and twenty-nine stable MCI (S-MCI) patients by untargeted metabolomics profiling. Alterations in the plasma metabolites between the P-MCI and S-MCI groups, as well as between the P-MCI and AD groups, were compared over the observation period. With the help of machine learning-based stratification, a 20-metabolite signature panel was identified that was associated with the presence and progression of AD. Furthermore, when the metabolic signature panel was used for classification of the three patient groups, this gave an accuracy of 73.5% using the panel. Moreover, when specifically classifying the P-MCI and S-MCI subjects, a fivefold cross-validation accuracy of 80.3% was obtained using the random forest model. Importantly, indole-3-propionic acid, a bacteria-generated metabolite from tryptophan, was identified as a predictor of AD progression, suggesting a role for gut microbiota in AD pathophysiology. Our study establishes a metabolite panel to assist in the stratification of MCI patients and to predict conversion to AD.

Mitochondria and Calcium Homeostasis: Cisd2 as a Big Player in Cardiac Ageing.

The ageing of human populations has become a problem throughout the world. In this context, increasing the healthy lifespan of individuals has become an important target for medical research and governments. Cardiac disease remains the leading cause of morbidity and mortality in ageing populations and results in significant increases in healthcare costs. Although clinical and basic research have revealed many novel insights into the pathways that drive heart failure, the molecular mechanisms underlying cardiac ageing and age-related cardiac dysfunction are still not fully understood. In this review we summarize the most updated publications and discuss the central components that drive cardiac ageing. The following characters of mitochondria-related dysfunction have been identified during cardiac ageing: (a) disruption of the integrity of mitochondria-associated membrane (MAM) contact sites; (b) dysregulation of energy metabolism and dynamic flexibility; (c) dyshomeostasis of Ca2+ control; (d) disturbance to mitochondria-lysosomal crosstalk. Furthermore, Cisd2, a pro-longevity gene, is known to be mainly located in the endoplasmic reticulum (ER), mitochondria, and MAM. The expression level of Cisd2 decreases during cardiac ageing. Remarkably, a high level of Cisd2 delays cardiac ageing and ameliorates age-related cardiac dysfunction; this occurs by maintaining correct regulation of energy metabolism and allowing dynamic control of metabolic flexibility. Together, our previous studies and new evidence provided here highlight Cisd2 as a novel target for developing therapies to promote healthy ageing.

Exercise and the Cisd2 Prolongevity Gene: Two Promising Strategies to Delay the Aging of Skeletal Muscle.

Aging is an evolutionally conserved process that limits life activity. Cellular aging is the result of accumulated genetic damage, epigenetic damage and molecular exhaustion, as well as altered inter-cellular communication; these lead to impaired organ function and increased vulnerability to death. Skeletal muscle constitutes ~40% of the human body's mass. In addition to maintaining skeletal structure and allowing locomotion, which enables essential daily activities to be completed, skeletal muscle also plays major roles in thermogenesis, metabolism and the functioning of the endocrine system. Unlike many other organs that have a defined size once adulthood is reached, skeletal muscle is able to alter its structural and functional properties in response to changes in environmental conditions. Muscle mass usually remains stable during early life; however, it begins to decline at a rate of ~1% year in men and ~0.5% in women after the age of 50 years. On the other hand, different exercise training regimens are able to restore muscle homeostasis at the molecular, cellular and organismal levels, thereby improving systemic health. Here we give an overview of the molecular factors that contribute to lifespan and healthspan, and discuss the effects of the longevity gene Cisd2 and middle-to-old age exercise on muscle metabolism and changes in the muscle transcriptome in mice during very old age.

Hepatocellular carcinoma-related cyclin D1 is selectively regulated by autophagy degradation system.

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. CONCLUSION: Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).

Expression of a hepatitis B virus pre-S2 deletion mutant in the liver results in hepatomegaly and hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and has a poor prognosis and a low survival rate; its incidence is on the rise. Hepatitis B virus (HBV) infection is one of the main causes of HCC. A high prevalence of pre-S deletions of HBV surface antigen, which encompass T-cell and/or B-cell epitopes, is found in HBV carriers; antiviral therapy and viral immune escape may cause and select for these HBV mutants. In particular, the presence of pre-S2 deletion mutants is an important risk factor associated with cirrhosis and HCC. We generated Alb-preΔS2 transgenic mice that express a naturally occurring pre-S2 mutant protein containing a 33-nucleotide deletion (preΔS2); the aim was to investigate its effect on hepatocarcinogenesis. After 30 months of follow-up, the liver pathology of the mice fell into four groups: G1, chronic inflammation solely; G2, chronic inflammation and fibrosis; G3, inflammation, fibrosis, and hepatomegaly accompanied by rectal prolapse (4-12%); and G4, hepatomegaly and spontaneous HCC (12-15%). Striking degeneration of the endoplasmic reticulum (ER) was present in the mouse livers at an early stage (4 months old). At 8 months, overt ER stress and the Atf6 pathway of the unfolded protein response (UPR) were induced; at the same time, metabolic pathways associated with mevalonate and cholesterol biogenesis, involving the peroxisomes and the ER, were disturbed. At 20 months and older, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway of the UPR was induced and the Hippo transducer Yap was activated. Together, these ultrastructural aberrations and metabolic disturbance all seem to contribute to the molecular pathogenesis and hepatocarcinogenesis present in the Alb-preΔS2 mice. These findings may contribute to the development of therapies for the liver disorders and HCC associated with pre-S2 deletion mutations among HBV carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice.

CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is a previously uncharacterized novel gene. Significantly, the CISD2 gene is located on human chromosome 4q, where a genetic component for longevity maps. Here we show for the first time that CISD2 is involved in mammalian life-span control. Cisd2 deficiency in mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and these events precede the two earliest manifestations of nerve and muscle degeneration; together, they lead to a panel of phenotypic features suggestive of premature aging. Our study also reveals that Cisd2 is primarily localized in the mitochondria and that mitochondrial degeneration appears to have a direct phenotypic consequence that triggers the accelerated aging process in Cisd2 knockout mice; furthermore, mitochondrial degeneration exacerbates with age, and the autophagy increases in parallel to the development of the premature aging phenotype. Additionally, our Cisd2 knockout mouse work provides strong evidence supporting an earlier clinical hypothesis that WFS is in part a mitochondria-mediated disorder; specifically, we propose that mutation of CISD2 causes the mitochondria-mediated disorder WFS2 in humans. Thus, this mutant mouse provides an animal model for mechanistic investigation of Cisd2 protein function and help with a pathophysiological understanding of WFS2.

EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82.

Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.

Cisd2 modulates the differentiation and functioning of adipocytes by regulating intracellular Ca2+ homeostasis.

CISD2 is a causative gene associated with Wolfram syndrome (WFS). However, it remains a mystery as to how the loss of CISD2 causes metabolic defects in patients with WFS. Investigation on the role played by Cisd2 in specific cell types may help us to resolve these underlying mechanisms. White adipose tissue (WAT) is central to the maintenance of energy metabolism and glucose homeostasis in humans. In this study, adipocyte-specific Cisd2 knockout (KO) mice showed impairment in the development of epididymal WAT (eWAT) in the cell autonomous manner. A lack of Cisd2 caused defects in the biogenesis and function of mitochondria during differentiation of adipocytes in vitro. Insulin-stimulated glucose uptake and secretion of adiponectin by the Cisd2 KO adipocytes were decreased. Moreover, Cisd2 deficiency increased the cytosolic level of Ca(2+) and induced Ca(2+)-calcineurin-dependent signaling that inhibited adipogenesis. Importantly, Cisd2 was found to interact with Gimap5 on the mitochondrial and ER membranes and thereby modulate mitochondrial Ca(2+) uptake associated with the maintenance of intracellular Ca(2+) homeostasis in adipocytes. Thus, it would seem that Cisd2 plays an important role in intracellular Ca(2+) homeostasis, which is required for the differentiation and functioning of adipocytes as well as the regulation of glucose homeostasis in mice.

A biphasic response pattern of lipid metabolomics in the stage progression of hepatitis B virus X tumorigenesis.

Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.

Autophagy suppresses tumorigenesis of hepatitis B virus-associated hepatocellular carcinoma through degradation of microRNA-224.

UNLABELLED: In hepatocellular carcinoma (HCC), dysregulated expression of microRNA-224 (miR-224) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. In this study we determined that autophagy is down-regulated and inversely correlated with miR-224 expression in hepatitis B virus (HBV)-associated HCC patient specimens. These results were confirmed in liver tumors of HBV X gene transgenic mice. Furthermore, miR-224 was preferentially recruited and degraded during autophagic progression demonstrated by real-time polymerase chain reaction and miRNA in situ hybridization electron microscopy after extraction of autophagosomes. Our in vitro study demonstrated that miR-224 played an oncogenic role in hepatoma cell migration and tumor formation through silencing its target gene Smad4. In HCC patients, the expression of low-Atg5, high-miR-224, and low-Smad4 showed significant correlation with HBV infection and a poor overall survival rate. Autophagy-mediated miR-224 degradation and liver tumor suppression were further confirmed by the autophagy inducer amiodarone and miR-224 antagonist using an orthotopic SD rat model. CONCLUSION: A noncanonical pathway links autophagy, miR-224, Smad4, and HBV-associated HCC. These findings open a new avenue for the treatment of HCC.

Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model.

BACKGROUND: Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter. RESULTS: We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples. CONCLUSIONS: The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.

A persistent level of Cisd2 extends healthy lifespan and delays aging in mice.

The CISD2 gene, which is an evolutionarily conserved novel gene, encodes a transmembrane protein primarily associated with the mitochondrial outer membrane. Significantly, the CISD2 gene is located within the candidate region on chromosome 4q where a genetic component for human longevity has been mapped. Previously, we have shown that Cisd2 deficiency shortens lifespan resulting in premature aging in mice. Additionally, an age-dependent decrease in Cisd2 expression has been detected during normal aging. In this study, we demonstrate that a persistent level of Cisd2 achieved by transgenic expression in mice extends their median and maximum lifespan without any apparent deleterious side effects. Cisd2 also ameliorates age-associated degeneration of the skin, skeletal muscles and neurons. Moreover, Cisd2 protects mitochondria from age-associated damage and functional decline as well as attenuating the age-associated reduction in whole-body energy metabolism. These results suggest that Cisd2 is a fundamentally important regulator of lifespan and provide an experimental basis for exploring the candidacy of CISD2 in human longevity.