Gut Microbiota and Bile Acids Mediate the Clinical Benefits of YH1 in Male Patients with Type 2 Diabetes Mellitus: A Pilot Observational Study.

Our previous clinical trial showed that a novel concentrated herbal extract formula, YH1 (Rhizoma coptidis and Shen-Ling-Bai-Zhu-San), improved blood glucose and lipid control. This pilot observational study investigated whether YH1 affects microbiota, plasma, and fecal bile acid (BA) compositions in ten untreated male patients with type 2 diabetes (T2D), hyperlipidemia, and a body mass index ≥ 23 kg/m2. Stool and plasma samples were collected for microbiome, BA, and biochemical analyses before and after 4 weeks of YH1 therapy. As previous studies found, the glycated albumin, 2-h postprandial glucose, triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels were significantly improved after YH1 treatment. Gut microbiota revealed an increased abundance of the short-chain fatty acid-producing bacteria Anaerostipes and Escherichia/Shigella. Furthermore, YH1 inhibited specific phylotypes of bile salt hydrolase-expressing bacteria, including Parabacteroides, Bifidobacterium, and Bacteroides caccae. Stool tauro-conjugated BA levels increased after YH1 treatment. Plasma total BAs and 7α-hydroxy-4-cholesten-3-one (C4), a BA synthesis indicator, were elevated. The reduced deconjugation of BAs and increased plasma conjugated BAs, especially tauro-conjugated BAs, led to a decreased glyco- to tauro-conjugated BA ratio and reduced unconjugated secondary BAs. These results suggest that YH1 ameliorates T2D and hyperlipidemia by modulating microbiota constituents that alter fecal and plasma BA compositions and promote liver cholesterol-to-BA conversion and glucose homeostasis.

PLA2G6 mutations cause motor dysfunction phenotypes of young-onset dystonia-parkinsonism type 14 and can be relieved by DHA treatment in animal models.

Parkinson's disease (PD), the most common neurodegenerative motor disorder, is currently incurable. Although many studies have provided insights on the substantial influence of genetic factors on the occurrence and development of PD, the molecular mechanism underlying the disease is largely unclear. Previous studies have shown that point mutations in the phospholipase A2 group VI gene (PLA2G6) correlate with young-onset dystonia-parkinsonism type 14 (PARK14). However, limited information is available regarding the pathogenic role of this gene and the mechanism underlying its function. To study the role of PLA2G6 mutations, we first used zebrafish larvae to screen six PLA2G6 mutations and revealed that injection of D331Y, T572I, and R741Q mutation constructs induced phenotypes such as motility defects and reduction in dopaminergic neurons. The motility defects could be alleviated by treatment with L-3, 4-dihydroxyphenylalanine (L-dopa), indicating that these mutations are pathological for PARK14 symptoms. Furthermore, the injection of D331Y and T572I mutation constructs reduced phospholipase activity of PLA2G6 and its lipid metabolites, which confirmed that these two mutations are loss-of-function mutations. Metabolomic analysis revealed that D331Y or T572I mutation led to higher phospholipid and lower docosahexaenoic acid (DHA) levels, indicating that reduced DHA levels are pathological for defective motor functions. Further, a dietary DHA supplement relieved the motility defects in PLA2G6D331Y/D331Y knock-in mice. This result revealed that the D331Y mutation caused defective PLA2G6 phospholipase activity and consequently reduced the DHA level, which is the pathogenic factor responsible for PARK14. The results of this study will facilitate the development of therapeutic strategies for PARK14.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Targeting Ubiquitin Proteasome Pathway with Traditional Chinese Medicine for Treatment of Spinocerebellar Ataxia Type 3.

Nine autosomal dominant spinocerebellar ataxias (SCAs) are caused by an abnormal expansion of CAG trinucleotide repeats that encodes a polyglutamine (polyQ) tract within different genes. Accumulation of aggregated mutant proteins is a common feature of polyQ diseases, leading to progressive neuronal dysfunction and degeneration. SCA type 3 (SCA3), the most common form of SCA worldwide, is characterized by a CAG triplet expansion in chromosome 14q32.1 ATXN3 gene. As accumulation of the mutated polyQ protein is a possible initial event in the pathogenic cascade, clearance of aggregated protein by ubiquitin proteasome system (UPS) has been proposed to inhibit downstream detrimental events and suppress neuronal cell death. In this study, Chinese herbal medicine (CHM) extracts were studied for their proteasome-activating, polyQ aggregation-inhibitory and neuroprotective effects in GFPu and ATXN3/Q 75 -GFP 293/SH-SY5Y cells. Among the 14 tested extracts, 8 displayed increased proteasome activity, which was confirmed by 20S proteasome activity assay and analysis of ubiquitinated and fused GFP proteins in GFPu cells. All the eight extracts displayed good aggregation-inhibitory potential when tested in ATXN3/Q 75 -GFP 293 cells. Among them, neuroprotective effects of five selected extracts were shown by analyses of polyQ aggregation, neurite outgrowth, caspase 3 and proteasome activities, and ATXN3-GFP, ubiquitin, BCL2 and BAX protein levels in neuronal differentiated ATXN3/Q 75 -GFP SH-SY5Y cells. Finally, enhanced proteasome function, anti-oxidative activity and neuroprotection of catalpol, puerarin and daidzein (active constituents of Rehmannia glutinosa and Pueraria lobata) were demonstrated in GFPu and/or ATXN3/Q 75 -GFP 293/SH-SY5Y cells. This study may have therapeutic implication in polyQ-mediated disorders.

Dietary Leucine Supplement Ameliorates Hepatic Steatosis and Diabetic Nephropathy in db/db Mice.

Dietary leucine supplementation has been explored for the therapeutic intervention of obesity and obesity-induced metabolic dysfunctions. In this study, we aim to examine the effects of dietary leucine supplementation in db/db mice. Mice were treated with or without leucine (1.5% w/v) in drinking water for 12 weeks. The leucine supplement was found to reduce insulin resistance and hepatic steatosis in db/db mice. Using Nuclear Magnetic Resonance (NMR)-based lipidomics, we found that the reduction of hepatic triglyceride synthesis was correlated with attenuated development of fatty liver. In addition, diabetic nephropathy (DN) was also ameliorated by leucine. Using liquid chromatography⁻time-of-flight mass spectrometry (LC-TOF MS)-based urine metabolomics analysis, we found that the disturbance of the tricarboxylic acid (TCA) cycle was reversed by leucine. The beneficial effects of leucine were probably due to AMP-activated protein kinase (AMPK) activation in the liver and kidneys of db/db mice. Thus, dietary leucine supplementation may potentially be a nutritional intervention to attenuate hepatic steatosis and early DN in type II diabetes.

Anti-enterovirus 71 activities of Melissa officinalis extract and its biologically active constituent rosmarinic acid.

Enterovirus 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European and Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, and viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive oxygen species (ROS) generation; and translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus to cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely to suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP A1 translocation and EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO and its constituent RA possess anti-EV71 activities, and may serve as a candidate drug for therapeutic and prophylactic uses against EV71 infection.

Antiviral activities of Schizonepeta tenuifolia Briq. against enterovirus 71 in vitro and in vivo.

No effective drug is currently available for treatment of enterovirus 71 (EV71) infection. Schizonepeta tenuifolia Briq. (ST) has been used as a herbal constituent of traditional Chinese medicine. We studied whether the aqueous extract of Schizonepeta tenuifolia Briq (STE) has antiviral activity. STE inhibited replication of EV71, as evident by its ability to diminish plaque formation and cytopathic effect induced by EV71, and to inhibit the synthesis of viral RNA and protein. Moreover, daily single-dose STE treatment significantly improved the survival of EV71-infected mice, and ameliorated the symptoms. Mechanistically, STE exerts multiple effects on enteroviral infection. Treatment with STE reduced viral attachment and entry; the cleavage of eukaryotic translation initiation factor 4 G (eIF4G) by EV71 protease, 2Apro; virus-induced reactive oxygen species (ROS) formation; and relocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from the nucleus to the cytoplasm. It was accompanied by a decline in EV71-associated hyperphosphorylation of p38 kinase and EPS15. It is plausible that STE may inhibit ROS-induced p38 kinase activation, and subsequent hnRNP A1 relocation and EPS15-mediated membrane trafficking in infected cells. These findings suggest that STE possesses anti-EV71 activities, and may serve as health food or candidate antiviral drug for protection against EV71.

Aqueous extract of Glycyrrhiza inflata inhibits aggregation by upregulating PPARGC1A and NFE2L2-ARE pathways in cell models of spinocerebellar ataxia 3.

Spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, and 17 and dentatorubropallidoluysian atrophy, as well as Huntington disease, are a group of neurodegenerative disorders caused by a CAG triplet-repeat expansion encoding a long polyglutamine (polyQ) tract in the respective mutant proteins. The cytoplasmic and nuclear aggregate formation, a pathological hallmark of polyQ diseases, is probably the initial process triggering the subsequent pathological events. Compromised oxidative stress defense capacity and mitochondrial dysfunction have emerged as contributing factors to the pathogenesis of polyQ diseases. The roots of licorice (Glycyrrhiza species) have long been used as an herbal medicine. In this study, we demonstrate the aggregate-inhibitory effect of Glycyrrhiza inflata herb extract and its constituents licochalcone A and ammonium glycyrrhizinate (AMGZ) in both 293 and SH-SY5Y ATXN3/Q75 cells, SCA3 cell models. The reporter assay showed that G. inflata herb extract, licochalcone A, and AMGZ could enhance the promoter activity of peroxisome proliferator-activated receptor γ, coactivator 1α (PPARGC1A), a known regulator of mitochondrial biogenesis and antioxidative response genes. G. inflata extract, licochalcone A, and AMGZ upregulated PPARGC1A expression and its downstream target genes, SOD2 and CYCS, in the 293 ATXN3/Q75 cell model. The expression of nuclear factor erythroid 2-related factor 2 (NFE2L2), the principal transcription factor that binds to antioxidant-responsive elements (AREs) to promote ARE-dependent gene expression when the cells respond to oxidative stress, and its downstream genes, HMOX1, NQO1, GCLC, and GSTP1, was also increased by G. inflata herb extract, licochalcone A, and AMGZ. Knockdown of PPARGC1A increased aggregates in ATXN3/Q75 cells and also attenuated the aggregate-inhibiting effect of the tested compounds. G. inflata extract and its constituents significantly elevated GSH/GSSG ratio and reduced reactive oxidative species in ATXN3/Q75 cells. The study results suggest that the tested agents activate PPARGC1A activity and NFE2L2-ARE signaling to increase mitochondrial biogenesis, decrease oxidative stress, and reduce aggregate formation in SCA3 cellular models.

Mineralocorticoid receptor antagonist spironolactone prevents chronic corticosterone induced depression-like behavior.

High level of serum corticosteroid is frequently associated with depression, in which a notable HPA (hypothalamus-pituitary-adrenal) axis hyperactivity is often observed. There are two types of corticosteroid receptors expressed in the hippocampus that provide potent negative feedback regulation on the HPA axis but dysfunction during depression, i.e. the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The balance between hippocampal MR and GR during chronic stress plays an important role in the occurrence of depression. The aim of this study is to explore if chronic corticosterone administration would induce depression-like behavior and affect the expression and function of hippocampal MR and GR, in addition to assess whether manipulation of corticosteroid receptors would modulate depressive behaviors. Hence, mice were treated with corticosterone (40 mg/kg) for 21 days followed by assessment in a battery of depression-like behaviors. The results show that chronic corticosterone-treated animals displayed an increased immobility time in a forced-swimming test, decreased preference to sucrose solution and novel object recognition performance, and enhanced hippocampal serotonin but decreased MR expression in both hippocampus and hypothalamus. On the other hand, co-administration of MR antagonist, spironolactone (25mg/kg, i.p. × 7 days) in corticosteroid-treated animals reduced immobility time in a forced-swimming test and improved performance in a novel object recognition test. In conclusion, we demonstrate that chronic corticosterone treatment triggers several depression-like behaviors, and in parallel, down-regulates MR expression in the hippocampus and hypothalamus. Administration of an MR antagonist confers an anti-depressant effect in chronic corticosterone-treated animals.

Biochemical disorders associated with antiproliferative effect of dehydroepiandrosterone in hepatoma cells as revealed by LC-based metabolomics.

DHEA is known to have chemopreventive and antiproliferative activities, and was initially thought to be mediated by inhibition of G6PD. Our previous study has shown that DHEA may act through interference with energy metabolism. To study the effect of pharmacological dose of DHEA on cellular metabolism, and to further delineate the mechanism underlying its antiproliferative effect, we applied a metabolomic approach to globally profile the changes in metabolites in SK-Hep1 cells underexpressing G6PD (Sk-Gi) and control cells (Sk-Sc) after DHEA treatment. RRLC-TOF-MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. DHEA induced changes in glutathione metabolism, lipid metabolism, s-adenosylmethionine (SAM) metabolism, as well as lysine metabolism. Elevation in level of glutathione disulfide, together with a concomitant decrease in level of reduced glutathione, was indicative of increased oxidative stress. Depletion of carnitine and its acyl derivatives reflected decline in fatty acid catabolism. These changes were associated with mitochondrial malfunction and reduction in cellular ATP content. Cardiolipin (CL) and phosphatidylcholine (PC) levels decreased significantly, suggesting that alterations in lipid composition are causally related to decline in mitochondrial function after DHEA treatment. The decline in cellular SAM content was accompanied by decreased expression of methionine adenosyltransferase genes MAT2A and MAT2B. SAM supplementation partially rescued cells from DHEA-induced growth stagnation. Our findings suggest that DHEA causes perturbation of multiple pathways in cellular metabolism. Decreased SAM production, and cardiolipin depletion and the resulting mitochondrial dysfunction underlie the antiproliferative effect of DHEA.

Resveratrol ameliorates metabolic disorders and muscle wasting in streptozotocin-induced diabetic rats.

Diabetes mellitus (DM) is characterized by dysregulated energy metabolism. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in diabetic animals. However, its overall in vivo effects on energy metabolism and the underlying mechanism require further investigation. In the present study, electrospray ionization-tandem mass spectrometry was employed to characterize the urine and plasma metabolomes of control, streptozotocin-induced DM and RSV-treated DM rats. Using principal component analysis (PCA) and heat map analysis, we discovered significant differences among control and experimental groups. RSV treatment significantly reduced the metabolic abnormalities in DM rats. Compared with the age-matched control rats, the level of carnitine was lower, and the levels of acetylcarnitine and butyrylcarnitine were higher in the urine and plasma of DM rats. RSV treatment ameliorated the deranged carnitine metabolism in DM rats. In addition, RSV treatment attenuated the diabetic ketoacidosis and muscle protein degradation, as evidenced from the attenuation of elevated urinary methyl-histidine and plasma branched-chain amino acids levels in DM rats. The beneficial effects of RSV in DM rats were correlated with activation of hepatic AMP-activated protein kinase and SIRT1 expression, increase of hepatic and muscular mitochondrial biogenesis and inhibition of muscle NF-κB activities. We concluded that RSV possesses multiple beneficial metabolic effects in insulin-deficient DM rats, particularly in improving energy metabolism and reducing protein wasting.

Dehydroepiandrosterone induces growth arrest of hepatoma cells via alteration of mitochondrial gene expression and function.

DHEA is known to have anti-proliferative effect. The mechanism is not completely understood. We investigated the mechanism underlying DHEA-induced growth arrest of hepatoma cells. Growth inhibition was associated with increased G6PD activity, and insensitive to reversal by mevalonate. Thus, DHEA does not act via inhibition of G6PD and HMGR. Instead, growth stagnation was accompanied by reduced expression of nucleus-encoded mitochondrial genes; morphological and functional alterations of mitochondria; and depletion of intracellular ATP. Conversely, pyruvate supplementation alleviated DHEA-induced growth inhibition. It is likely that DHEA suppresses cell growth by altering mitochondrial gene expression, morphology and functions.

Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status.

Glucose-6-phosphate dehydrogenase (G6PD) plays important roles in the maintenance of cellular redox balance. It was not until recently that the importance of G6PD in regulation of cellular growth and apoptosis emerged. In the present study, we found that G6PD-deficient fibroblasts were more susceptible to peroxynitrite-induced cytotoxicity. Treatment with peroxynitrite generator 3-morpholinosydnonimine (SIN-1) hydrochloride caused apoptosis in human fibroblast in a dose-dependent manner. This was preceded by a decrease in the intracellular level of glutathione (GSH) as well as accumulation of p53. The extent of apoptosis and glutathione depletion were greater in G6PD-deficient fibroblasts than in the normal counterpart. Pretreatment with green tea polyphenol epigallocatechin-3-gallate (EGCG) effectively blocked peroxynitrite-induced glutathione depletion, p53 accumulation, and apoptosis in both normal and G6PD-deficient cells. EGCG, administered to cells alone or as pretreatment, caused activation of Akt. The protective effect was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin, and LY294002. Our findings suggest that G6PD deficiency enhances the toxicity of peroxynitrite and that EGCG initiates cell survival signaling via the PI3K/akt pathway.