The Polymethoxyflavone Sudachitin Modulates the Circadian Clock and Improves Liver Physiology.

SCOPE: Polymethoxylated flavones (PMFs) are a group of natural compounds known to display a wide array of beneficial effects to promote physiological fitness. Recent studies reveal circadian clocks as an important cellular mechanism mediating preventive efficacy of the major PMF Nobiletin against metabolic disorders. Sudachitin is a PMF enriched in Citrus sudachi, and its functions and mechanism of action are poorly understood. METHODS AND RESULTS: Using circadian reporter cells, it shows that Sudachitin modulates circadian amplitude and period of Bmal1 promoter-driven reporter rhythms, and real-time qPCR analysis shows that Sudachitin alters expression of core clock genes, notably Bmal1, at both transcript and protein levels. Mass-spec analysis reveals systemic exposure in vivo. In mice fed with high-fat diet with or without Sudachitin, it observes increased nighttime activity and daytime sleep, accompanied by significant metabolic improvements in a circadian time-dependent manner, including respiratory quotient, blood lipid and glucose profiles, and liver physiology. Focusing on liver, RNA-sequencing and metabolomic analyses reveal prevalent diurnal alteration in both gene expression and metabolite accumulation. CONCLUSION: This study elucidates Sudachitin as a new clock-modulating PMF with beneficial effects to improve diurnal metabolic homeostasis and liver physiology, suggesting the circadian clock as a fundamental mechanism to safeguard physiological well-being.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer's Disease Model.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder and the most common cause of dementia. Various pathogenic mechanisms have been proposed to contribute to disease progression, and recent research provided evidence linking dysregulated circadian rhythms/sleep and energy metabolism with AD. Previously, we found that the natural compound Nobiletin (NOB) can directly activate circadian cellular oscillators to promote metabolic health in disease models and healthy aging in naturally aged mice. In the current study, using the amyloid-ß AD model APP/PS1, we investigated circadian, metabolic and amyloid characteristics of female mice and the effects of NOB. Female APP/PS1 mice showed reduced sleep bout duration, and NOB treatment exhibited a trend to improve it. While glucose tolerance was unchanged, female APP/PS1 mice displayed exaggerated oxygen consumption and CO2 production, which was mitigated by NOB. Likewise, cold tolerance in APP/PS1 was impaired relative to WT, and interestingly was markedly enhanced in NOB-treated APP/PS1 mice. Although circadian behavioral rhythms were largely unchanged, real-time qPCR analysis revealed altered expression of several core clock genes by NOB in the cerebral cortex, notably Bmal1, Npas2, and Rora. Moreover, NOB was also able to activate various clock-controlled metabolic genes involved in insulin signaling and mitochondrial function, including Igf1, Glut1, Insr, Irs1, Ucp2, and Ucp4. Finally, we observed that NOB attenuated the expression of several AD related genes including App, Bace1, and ApoE, reduced APP protein levels, and strongly ameliorated Aß pathology in the cortex. Collectively, these results reveal novel genotype differences and importantly beneficial effects of a natural clock-enhancing compound in biological rhythms and related pathophysiology, suggesting the circadian clock as a modifiable target for AD.

Cardiolipin Synthesis in Skeletal Muscle Is Rhythmic and Modifiable by Age and Diet.

Circadian clocks regulate metabolic processes in a tissue-specific manner, which deteriorates during aging. Skeletal muscle is the largest metabolic organ in our body, and our previous studies highlight a key role of circadian regulation of skeletal muscle mitochondria in healthy aging. However, a possible circadian regulation of cardiolipin (CL), the signature lipid class in the mitochondrial inner membrane, remains largely unclear. Here, we show that CL levels oscillate during the diurnal cycle in C2C12 myotubes. Disruption of the Ror genes, encoding the ROR nuclear receptors in the secondary loop of the circadian oscillator, in C2C12 cells was found to dampen core circadian gene expression. Importantly, several genes involved in CL synthesis, including Taz and Ptpmt1, displayed rhythmic expression which was disrupted or diminished in Ror-deficient C2C12 cells. In vivo studies using skeletal muscle tissues collected from young and aged mice showed diverse effects of the clock and aging on the oscillatory expression of CL genes, and CL levels in skeletal muscle were enhanced in aged mice relative to young mice. Finally, consistent with a regulatory role of RORs, Nobiletin, a natural agonist of RORs, was found to partially restore transcripts levels of CL synthesis genes in aged muscle under a dietary challenge condition. Together, these observations highlight a rhythmic CL synthesis in skeletal muscle that is dependent on RORs and modifiable by age and diet.

Coordinate Regulation of Cholesterol and Bile Acid Metabolism by the Clock Modifier Nobiletin in Metabolically Challenged Old Mice.

Cholesterol and bile acid (BA) homeostasis plays a central role in systemic metabolism. Accumulating evidence suggests a key regulatory function of the circadian clock, our biological timer, in lipid metabolism, particularly cholesterol and bile acid flux. Previously, we showed that Nobiletin (NOB), a natural compound targeting the ROR (Retinoic acid receptor-related orphan receptor) nuclear receptors in the circadian oscillator, strongly protects lipid homeostasis, including normal serum cholesterol levels in high-fat (HF) fed mice at both young and old ages. In this study, we further examined the role of NOB in cholesterol metabolism in HF-fed aged mice, and found that NOB lowered the serum LDL/VLDL cholesterol levels and consequently the LDL/HDL ratio. BA levels in the serum were markedly reduced in the HF.NOB group, and examination of additional hepatic markers further indicate a protective role of NOB in the liver. At the molecular level, whereas HF feeding downregulated hepatic expression of several ROR target genes involved in bile acid synthesis, NOB treatment (HF.NOB) was able to rescue it. In accordance, fecal BA excretion was enhanced by NOB, and microbial 16S sequencing revealed alteration of several taxa known to be involved in secondary BA production in the gut. Together, these results demonstrate concerted effects of the clock-modulating compound NOB in cholesterol and BA metabolism, suggesting pharmacological manipulation of the clock as a novel therapeutic strategy against metabolic disorders and age-related decline.

Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge.

Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.

Period2 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Myricetin improves endurance capacity and mitochondrial density by activating SIRT1 and PGC-1α.

Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

The Small Molecule Nobiletin Targets the Molecular Oscillator to Enhance Circadian Rhythms and Protect against Metabolic Syndrome.

Dysregulation of circadian rhythms is associated with metabolic dysfunction, yet it is unclear whether enhancing clock function can ameliorate metabolic disorders. In an unbiased chemical screen using fibroblasts expressing PER2::Luc, we identified Nobiletin (NOB), a natural polymethoxylated flavone, as a clock amplitude-enhancing small molecule. When administered to diet-induced obese (DIO) mice, NOB strongly counteracted metabolic syndrome and augmented energy expenditure and locomotor activity in a Clock gene-dependent manner. In db/db mutant mice, the clock is also required for the mitigating effects of NOB on metabolic disorders. In DIO mouse liver, NOB enhanced clock protein levels and elicited pronounced gene expression remodeling. We identified retinoid acid receptor-related orphan receptors as direct targets of NOB, revealing a pharmacological intervention that enhances circadian rhythms to combat metabolic disease via the circadian gene network.

Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated Clock(Δ19/+) heterozygous (Clk/+) mice, known to display lengthened periodicity and dampened amplitude, as a model of partially perturbed clocks. Interestingly, Clk/+ mice exhibited improved glycemic control and resistance to circadian period lengthening under high-fat diet (HFD). Furthermore, BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD. Pharmacological and molecular studies showed that BMAL1 turnover entailed proteasomal and autophagic activities, and CLOCKΔ19 attenuated both processes. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT in HFD. Finally, transcriptome analysis revealed reprogramming of clock-controlled metabolic genes in Clk/+ mice. Our results demonstrate a novel role of autophagy in circadian regulation and reveal an unforeseen plasticity of circadian and metabolic networks.

Ammonia-lowering activities and carbamoyl phosphate synthetase 1 (Cps1) induction mechanism of a natural flavonoid.

OBJECTIVE: Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism. MATERIALS/METHODS: C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1. RESULTS: NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPß. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition. CONCLUSION: NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms.

Transmissible microbial and metabolomic remodeling by soluble dietary fiber improves metabolic homeostasis.

Dietary fibers are increasingly appreciated as beneficial nutritional components. However, a requisite role of gut microbiota in fiber function and the overall impact of fibers on metabolomic flux remain unclear. We herein showed enhancing effects of a soluble resistant maltodextrin (RM) on glucose homeostasis in mouse metabolic disease models. Remarkably, fecal microbiota transplantation (FMT) caused pronounced and time-dependent improvement in glucose tolerance in RM recipient mice, indicating a causal relationship between microbial remodeling and metabolic efficacy. Microbial 16S sequencing revealed transmissible taxonomic changes correlated with improved metabolism, notably enrichment of probiotics and reduction of Alistipes and Bacteroides known to associate with high fat/protein diets. Metabolomic profiling further illustrated broad changes, including enrichment of phenylpropionates and decreases in key intermediates of glucose utilization, cholesterol biosynthesis and amino acid fermentation. These studies elucidate beneficial roles of RM-dependent microbial remodeling in metabolic homeostasis, and showcase prevalent health-promoting potentials of dietary fibers.

Manipulating the circadian and sleep cycles to protect against metabolic disease.

Modernization of human society parallels an epidemic of metabolic disorders including obesity. Apart from excess caloric intake, a 24/7 lifestyle poses another important challenge to our metabolic health. Recent research under both laboratory and epidemiological settings has indicated that abnormal temporal organization of sleep and wakeful activities including food intake is a significant risk factor for metabolic disease. The circadian clock system is our intrinsic biological timer that regulates internal rhythms such as the sleep/wake cycle and also responses to external stimuli including light and food. Initially thought to be mainly involved in the timing of sleep, the clock, and/or clock genes may also play a role in sleep architecture and homeostasis. Importantly, an extensive body of evidence has firmly established a master regulatory role of the clock in energy balance. Together, a close relationship between well-timed circadian/sleep cycles and metabolic health is emerging. Exploiting this functional connection, an important holistic strategy toward curbing the epidemic of metabolic disorders (e.g., obesity) involves corrective measures on the circadian clock and sleep. In addition to behavioral and environmental interventions including meal timing and light control, pharmacological agents targeting sleep and circadian clocks promise convenient and effective applications. Recent studies, for example, have reported small molecules targeting specific clock components and displaying robust beneficial effects on sleep and metabolism. Furthermore, a group of clock-amplitude-enhancing small molecules (CEMs) identified via high-throughput chemical screens are of particular interest for future in vivo studies of their metabolic and sleep efficacies. Elucidating the functional relationship between clock, sleep, and metabolism will also have far-reaching implications for various chronic human diseases and aging.

Central mechanisms of adiposity in adult female mice with androgen excess.

OBJECTIVE: Androgen excess in women is associated with visceral adiposity. However, little is known on the mechanism through which androgen promotes visceral fat accumulation. METHODS: To address this issue, female mice to chronic androgen excess using 5α-dihydrotestosterone (DHT) and studied the regulation of energy homeostasis was exposed. RESULTS: DHT induced a leptin failure to decrease body weight associated with visceral adiposity but without alterations in leptin anorectic action. This paralleled leptin's failure to upregulate brown adipose tissue expression of uncoupling protein-1, associated with decreased energy expenditure (EE). DHT decreased hypothalamic proopiomelanocortin (pomc) mRNA expression and increased POMC intensity in neuronal bodies of the arcuate nucleus while simultaneously decreasing the intensity of POMC projections to the dorsomedial hypothalamus (DMH). This was associated with a failure of the melanocortin 4 receptor agonist melanotan-II to suppress body weight. CONCLUSION: Taken together, these data indicate that androgen excess promotes visceral adiposity with reduced POMC neuronal innervation in the DMH, reduced EE but without hyperphagia.

Developmental androgen excess disrupts reproduction and energy homeostasis in adult male mice.

Polycystic ovary syndrome is a common endocrine disorder in females of reproductive age and is believed to have a developmental origin in which gestational androgenization programs reproductive and metabolic abnormalities in offspring. During gestation, both male and female fetuses are exposed to potential androgen excess. In this study, we determined the consequences of developmental androgenization in male mice exposed to neonatal testosterone (NTM). Adult NTM displayed hypogonadotropic hypogonadism with decreased serum testosterone and gonadotropin concentrations. Hypothalamic KiSS1 neurons are believed to be critical to the onset of puberty and are the target of leptin. Adult NTM exhibited lower hypothalamic Kiss1 expression and a failure of leptin to upregulate Kiss1 expression. NTM displayed an early reduction in lean mass, decreased locomotor activity, and decreased energy expenditure. They displayed a delayed increase in subcutaneous white adipose tissue amounts. Thus, excessive neonatal androgenization disrupts reproduction and energy homeostasis and predisposes to hypogonadism and obesity in adult male mice.

Developmental androgen excess programs sympathetic tone and adipose tissue dysfunction and predisposes to a cardiometabolic syndrome in female mice.

Among women, the polycystic ovarian syndrome (PCOS) is considered a form of metabolic syndrome with reproductive abnormalities. Women with PCOS show increased sympathetic tone, visceral adiposity with enlarged adipocytes, hypoadiponectinemia, insulin resistance, glucose intolerance, increased inactive osteocalcin, and hypertension. Excess fetal exposure to androgens has been hypothesized to play a role in the pathogenesis of PCOS. Previously, we showed that neonatal exposure to the androgen testosterone (NT) programs leptin resistance in adult female mice. Here, we studied the impact of NT on lean and adipose tissues, sympathetic tone in cardiometabolic tissues, and the development of metabolic dysfunction in mice. Neonatally androgenized adult female mice (NTF) displayed masculinization of lean tissues with increased cardiac and skeletal muscle as well as kidney masses. NTF mice showed increased and dysfunctional white adipose tissue with increased sympathetic tone in both visceral and subcutaneous fat as well as increased number of enlarged and insulin-resistant adipocytes that displayed altered expression of developmental genes and hypoadiponectinemia. NTF exhibited dysfunctional brown adipose tissue with increased mass and decreased energy expenditure. They also displayed decreased undercarboxylated and active osteocalcin and were predisposed to obesity during chronic androgen excess. NTF showed increased renal sympathetic tone associated with increased blood pressure, and they developed glucose intolerance and insulin resistance. Thus, developmental exposure to testosterone in female mice programs features of cardiometabolic dysfunction, as can be observed in women with PCOS, including increased sympathetic tone, visceral adiposity, insulin resistance, prediabetes, and hypertension.

Early-life exposure to testosterone programs the hypothalamic melanocortin system.

In mammals, males consume more food, which is considered a masculinized behavior, but the underlying mechanism of this sex-specific feeding behavior is unknown. In mice, neonatal testosterone (NT) is critical to masculinize the developing brain, leading to sex differences in reproductive physiology. The proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus (ARC) are critical to suppress energy intake and POMC innervation of hypothalamic feeding circuits develops to a large extent neonatally. We hypothesized that NT programs the masculinization of energy intake by programming POMC neurons. We tested this hypothesis by comparing control females and control males (CMs) with female mice neonatally androgenized with testosterone (NTFs). We show that increased food intake in CMs is associated with reduced POMC expression and decreased intensity of neuronal projections from POMC neurons within the ARC compared with control females. We found that NTFs display a masculinized energy intake and ARC POMC expression and innervation as observed in CMs, which can be mimicked by neonatal exposure to the androgen receptor agonist dihydrotestosterone (DHT). NTFs also exhibit hyperleptinemia and a decreased ability of leptin to up-regulate POMC, suppress food intake, and prevent adipose tissue accumulation, independent of signal transducer and activator of transcription 3. However, this leptin resistance is specific to NTFs, is not a consequence of masculinization, and is reproduced by neonatal exposure to estrogen but not DHT. Thus, NT programs a sexual differentiation of POMC neurons in female mice via DHT but also predisposes to leptin resistance and obesity in an estrogen-dependent manner.

T-2 toxin initially activates caspase-2 and induces apoptosis in U937 cells.

T-2 toxin, which belongs to a group of mycotoxins synthesized by Fusarium fungi that are widely encountered as natural contaminants, induced apoptosis with distinct morphological and biological features in U937 cells. The concentration of more than 10nM T-2 toxin affected cell viability, induced nuclear and DNA fragmentation and caspase-3 activation. Caspase-2, -3, -8, and -9 were activated during T-2 toxin-induced apoptosis. T-2 toxin neither inhibited mitochondrial respiratory chain complexes I-IV in isolated mitochondria nor decreased ATP levels in U937 cells. Both enzyme activity assay and Western blot analysis revealed that T-2 toxin activated caspase-2 earlier than caspase-3, -8, and -9. Caspase-2 inhibitor (VDVAD-CHO/fmk) and caspase-8 inhibitor (IETD-CHO/fmk) completely blocked the T-2 toxin-induced process of procaspase-3, while caspase-9 inhibitor (LEHD-CHO/fmk) did so less effectively. Caspase-2 inhibitor entirely blocked T-2 toxin-induced caspase-8, and -9 activation. These results clearly indicate that activation of caspase-2 is essential to T-2 toxin-induced apoptosis and that apoptotic signals are mainly transmitted via caspase-8 and caspase-3 rather than mitochondrial pathway.

The important role of caspase-10 in sodium butyrate-induced apoptosis.

Butyrate, a short chain fatty acid, exhibits a wide variety of biological effects including the inhibition of cell growth, change of cellular morphology and the induction of apoptosis. Sodium butyrate-induced apoptosis has been reported to associate with the up-regulation of pro-apoptotic Bax expression, and the down-regulation of anti-apoptotic Bcl-2 and Bcl-XL expressions. However, in some cases, butyrate has also been shown to cause apoptosis without change in Bcl-2, Bcl-XL and/or Bax. This study investigates the detailed mechanisms of sodium butyrate-induced apoptosis. The effect of sodium butyrate was analyzed in the induction of caspase activities, formation of caspase active forms and mRNA levels in human breast cancer cell line MRK-nu-1. Induction of activities of caspase-3, -10 and, to some extent, -8 and formation of DNA fragmentation were observed with sodium butyrate in a dose- and/or time-dependent manner. The levels of caspase-10 mRNA expression markedly increased in a time-dependent manner by the treatment of sodium butyrate, whereas caspase-8 mRNA expression was not changed. Inhibitors of caspase-8 and caspase-10 reduced caspase-3 activity and subsequent DNA fragmentation induced by sodium butyrate. These caspase inhibitors also inhibited the cleavage of pro-caspase-3 to the active forms indicated by Western blotting analysis. Pyrrolidine dithiocarbamate also inhibited the induction of caspase-10 mRNA expression and caspase-3 activation. Contrary to other reports, levels of Bcl-2, Bcl-XL and Bax mRNA expressions were not distinctly changed by even 5 mM sodium butyrate treatment. Our results suggest that sodium butyrate may trigger apoptosis via the induction of the caspase-10 expression.