Arjunolic acid from Cyclocarya paliurus ameliorates diabetic retinopathy through AMPK/mTOR/HO-1 regulated autophagy pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclocarya paliurus (CP) is a traditional Chinese herb and possesses a variety of biological activities including anti-hyperglycemia, anti-hyperlipidemia, antioxidant and anti-inflammation. Arjunolic acid (AA) is an abundant and bioactive ingredient in CP that shows significant protection against many metabolic diseases such as diabetic complication. Diabetic retinopathy (DR) is a serious complication of diabetes and may lead to vision loss. However, the protective effects and underlying mechanisms of AA against DR is not still understood. AIM OF THE STUDY: We aimed to investigate whether AA activates AMPK/mTOR/HO-1 regulated autophagy pathway to alleviate DR. MATERIALS AND METHODS: In the study, the STZ-induced diabetic model of rats was established, and AA with 10 and 30 mg/kg dosages was given orally for ten weeks to investigate their effect on retinal injury of DR. H2O2-induced ARPE-19 cells were applied to evaluate anti-apoptosis and anti-oxidant effect of AA. RESULTS: The results revealed that AA could prevent STZ-induced weight loss and increase the retinal thickness and nuclei counts. The level of HO-1 protein was upregulated both in vivo and in vitro. In addition, AA prevented retinal damage and cell apoptosis through the AMPK-mTOR-regulated autophagy pathway. Furthermore, anti-apoptosis capacity, as well as the expression of HO-1 and LC3 protein, were effectively locked by AMPK inhibitor dorsomorphin dihydrochloride (compound C). CONCLUSIONS: This finding implies that AA may be a promising candidate drug by protecting retinal cells from STZ-induced oxidative stress and inflammation through the AMPK/mTOR/HO-1 regulated autophagy pathway.

Hypoglycemic effect of astragaloside IV via modulating gut microbiota and regulating AMPK/SIRT1 and PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Astragali, the dried root of Astragalus mongholicus Bunge, has long been used in traditional Chinese Medicine to treat diabetes. Astragaloside IV (AS-IV), one of the most active ingredients in the root, has been shown to have anti-diabetes ability; however, its underlying mechanism is still unclear. MATERIALS AND METHODS: In this study, we evaluated the hypoglycemic effect and possible mechanisms of AS-IV in diabetic mice and insulin resistance-HepG2 cells. The components of the intestinal microflora in mice with type 2 diabetes mellitus (T2DM) were determined using high-throughput 16S rRNA gene sequencing. Moreover, the molecular mechanisms of specific members of insulin signaling pathways were analyzed. RESULTS: AS-IV significantly reversed the abnormalities in blood lipids, glucose, insulin resistance, as well as oxidative stress levels in T2DM mice. Histological finding showed that AS-IV could protect the cellular architecture of the liver and pancreas. AS-IV also regulated the abundance and diversity of intestinal flora of T2DM mice in a positive direction and increased butyric acid levels. The active role of AS-IV as an anti-diabetic compound by regulating the AMPK/SIRT1 and PI3K/AKT signaling pathways was revealed using a T2DM model and verified through the intervention of inhibitors using insulin-resistance HepG2 cells. CONCLUSION: Our results suggested that AS-IV may be used as an anti-diabetic drug candidate owing to its effects of regulating gut microbiota and AMPK/SIRT1 and PI3K/AKT signaling pathways.

Long-term programming of skeletal muscle and liver lipid and energy metabolism by resveratrol supplementation to suckling mice.

Metabolic programming by dietary chemicals consumed in early life stages is receiving increasing attention. We here studied long-term effects of mild resveratrol (RSV) supplementation during lactation on muscular and hepatic lipid metabolism in adulthood. Newborn male mice received RSV or vehicle from day 2-20 of age, were weaned onto a chow diet on day 21, and were assigned to either a high-fat diet (HFD) or a normal-fat diet on day 90 of age for 10 weeks. RSV-treated mice showed in adulthood protection against HFD-induced triacylglycerol accumulation in skeletal muscle, enhanced muscular capacities for fat oxidation and mitochondria activity, signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling in muscle, and increased fat oxidation capacities and a decreased capacity for lipogenesis in liver compared with controls. Thus, RSV supplementation in early postnatal life may help preventing later diet-related disorders linked to ectopic lipid accumulation in muscle and liver tissues.

Nephroprotective activity of Aframomum melegueta seeds extract against diclofenac-induced acute kidney injury: A mechanistic study.

ETHNOPHARMACOLOGICAL RELEVANCE: In Africa, Aframomum species have been traditionally used to treat illnesses such as inflammation, hypertension, diarrhea, stomachache and fever. Moreover, Aframomum melegueta seed extracts (AMSE) are used in traditional medicine to relieve stomachaches and inflammatory diseases. AIM: Chronic administration of diclofenac (DIC) has been reported to cause acute kidney injury (AKI), which is a serious health condition. The nephroprotective effect of AMSE is yet to be elucidated. Accordingly, this study aims to investigate the phytoconstituents of standardized AMSE, evaluate its nephroprotective effects against DIC-induced AKI in rats, and elaborate its underlying molecular mechanisms. MATERIALS AND METHODS: The quantitative estimation of major AMSE constituents and profiling of its secondary metabolites were conducted via RP-HPLC and LC-ESI/Triple TOF/MS, respectively. Next, DIC (50 mg/kg)-induced AKI was achieved in Sprague-Dawley rats and DIC-challenged rats were administered AMSE (100 and 200 mg/kg) orally. All treatments were administered for five consecutive days. Blood samples were collected and the sera were used for estimating creatinine, urea and, kidney injury molecule (KIM)-1 levels. Kidney specimens were histopathologically assessed and immunohistochemically examined for c-Myc expression. A portion of the kidney tissue was homogenized and examined for levels of oxidative stress markers (MDA and GSH). Heme oxygenase (HO)-1, TNF-α, IL-6, Bax, Bcl2 and caspase-3 renal levels were quantified by ELISA. Moreover, the protein expression levels of NF-Ò¡B p65 was quantified using Western blot analysis, whereas mRNA expression levels of AMPK, SIRT-1, nuclear factor erythroid-2-related factor (Nrf2) and STAT3 were detected using qRT-PCR in the remaining kidney tissues. RESULTS: Standardized AMSE was shown to primarily contain 6-gingerol, 6-shogaol and 6-paradol among the 73 compounds that were detected via LC-ESI/Triple TOF/MS including phenolic acids, hydroxyphenylalkanes, diarylheptanoids and fatty acids. Relative to DIC-intoxicated rats, AMSE modulated serum creatinine, urea, KIM-1, renal MDA, TNF-α, IL-6, Bax, and caspase-3 levels. AMSE has also improved renal tissue architecture, enhanced GSH and HO-1 levels, and upregulated renal Nrf2, AMPK, and SIRT-1 mRNA expression levels. Furthermore, AMSE suppressed NF-Ò¡B p65 protein and STAT3 mRNA expression, and further reduced c-Myc immunohistochemical expression in renal tissues. Overall, our findings revealed that AMSE counteracted DIC-induced AKI via its antioxidant, anti-inflammatory, and antiapoptotic activities. Moreover, AMSE activated Nrf2/HO1 and AMPK/SIRT1, and inhibited NF-Ò¡B/STAT3 signaling pathways. Therefore, AMSE is a promising agent for inhibiting DIC-induced nephrotoxicity.

Effect of Inonotus obliquus (Fr.) Pilat extract on the regulation of glycolipid metabolism via PI3K/Akt and AMPK/ACC pathways in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Inonotus obliquus (Fr.) Pilat is a mushroom belonging to the family Hymenochaetaceae. It is popularly called the Chaga mushroom in Russian folk medicine and has been used as a traditional medicine to treat diabetes mellitus in Eastern European and Asian countries. However, its effects on glycolipid metabolism disorders and underlying molecular mechanism of action remain unclear. AIM OF THE STUDY: I. obliquus contains abundant functional components, which provide potential medicinal value. The purpose of this study was to investigate compositions of I. obliquus extract with a high-pressure water extraction method, and investigate the anti-type 2 diabetic effects of I. obliquus extract and the possible underlying mechanisms involved. MATERIALS AND METHODS: The I. obliquus was extracted by a high-pressure water extraction method, and tested its main components by special assay kit and instrumental analysis. Type 2 diabetic C57BL/6 mice were induced by high-fat diet with low-dose STZ injection, and were daily gavaged with different doses of I. obliquus extract for 8 weeks. Glycemic, blood lipid profile, and histopathology of liver and pancreas were assessed. Underlying mechanisms related to glycemic control in liver were further performed. RESULTS: The I. obliquus extract main compounds were ß-Glucans, triterpenoids and polyphenol by determination. Oral administration of 250 mg/kg and 500 mg/kg I. obliquus extract significantly alleviated blood glucose and insulin resistance. Moreover, 250 mg/kg and 500 mg/kg of I. obliquus extract increased liver glycogen content and high-density lipoprotein cholesterol (HDL-C) levels while decreased total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels. Furthermore, the protein expression levels of phosphatidylinositol-3 kinase (PI3K), p-protein kinase B (Akt), p-adenosine monophosphate activated protein kinase (AMPK), and p-acetyl-CoA carboxylase (ACC) were upregulated, whereas sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) were downregulated after supplement with 250 mg/kg and 500 mg/kg of I. obliquus extract. Interestingly, I. obliquus extract was a dose-effect relationship within a certain range. 250 mg/kg had obvious anti-diabetes effect, and the effect of 500 mg/kg dose was the same as that of metformin. CONCLUSION: I. obliquus extract ameliorated insulin resistance and lipid metabolism disorders in diabetic mice. The hypoglycemic and hypolipidemic properties of I. obliquus extract were supposedly exerted via the regulation of the PI3K/Akt and AMPK/ACC signaling pathways.

Teaghrelin Protects SH-SY5Y Cells against MPP+-Induced Neurotoxicity through Activation of AMPK/SIRT1/PGC-1α and ERK1/2 Pathways.

The prevalence and incidence of Parkinson's disease (PD), an age-related neurodegenerative disease, are higher among elderly people. Independent of etiology, dysfunction and loss of dopaminergic neurons are common pathophysiological changes in PD patients with impaired motor and non-motor function. Currently, preventive or therapeutic treatment for combating PD is limited. The ghrelin axis and ghrelin receptor have been implicated in the preservation of dopaminergic neurons and have potential implications in PD treatment. Teaghrelin, a compound originating from Chin-Shin Oolong tea, exhibits ghrelin agonist activity. In this study, the neuroprotective potential of teaghrelin against PD was explored in a cell model in which human neuroblastoma SH-SY5Y cells were treated with the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+). Upon MPP+ exposure, SH-SY5Y cells exhibited decreased mitochondrial complex I activity and apoptotic cell death. Teaghrelin activated AMP-activated protein kinase (AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways to antagonize MPP+-induced cell death. Herein, we propose that teaghrelin is a potential candidate for the therapeutic treatment of PD.

Optimization of nucleotide sugar supply for polysaccharide formation via thermodynamic buffering.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

Electroacupuncture Upregulated Ghrelin in Rats with Functional Dyspepsia via AMPK/TSC2/Rheb-Mediated mTOR Inhibition.

BACKGROUND: Gastrointestinal motility disorder is an important pathological basis for functional dyspepsia (FD). Epigastric ache and discomfort are the main symptoms of FD, and ghrelin deficiency is closely related to the occurrence and development of FD. While electroacupuncture (EA) alleviated the symptoms of FD patients and improved their quality of life, there is a lack of sufficient mechanistic evidence to support these beneficial effects. METHODS: An in vivo FD model was established in wild-type and mammalian target of rapamycin (mTOR) knockout (-/-) rats. FD rats were subjected to EA with or without mTOR agonists or inhibitors. Gastric emptying and intestinal propulsion were assessed, and pathological changes in the hypothalamus, gastric antrum, and small intestine were examined histologically. In addition, ghrelin expression and AMPK/TSC2/Rheb/mTOR activation were detected by quantitative reverse transcription polymerase chain reaction and western blot. RESULTS: EA alone or in combination with mTOR inhibitors improved gastrointestinal function in FD rats by increasing the rates of intestinal propulsion and gastric emptying, and pathological changes in the hypothalamus, gastric antrum, and small intestine were alleviated. This may be related to the significant upregulation of ghrelin expression and the effective activation of the AMPK/TSC2/Rheb/mTOR signaling pathway. Interestingly, EA also improved gastrointestinal function and ghrelin expression in mTOR (-/-) KO FD rats. CONCLUSION: Altering the level of ghrelin by regulating AMPK/TSC2/Rheb-mediated mTOR inhibition is an important way through which EA treats FD. The complex EA-mediated regulatory mechanisms of the brain-gut axis still require further exploration.

Mechanistic evaluation of AMPK/SIRT1/FXR signaling axis, inflammation, and redox status in thioacetamide-induced liver cirrhosis: The role of Cichorium intybus linn (chicory)-supplemented diet.

Liver cirrhosis is a scene profitable to the advance of hepatocellular carcinoma (HCC). The current work was engrossed to weigh the potential role of Cichorium intybus linn against thioacetamide (TAA)-induced liver cirrhosis and their probable underlying biochemical and molecular mechanisms. farnesoid-X-receptor (FXR) expression, proliferating cell nuclear antigen (PCNA) immunoreactivity, and activated AMP protein kinase (pAMPK), sirtuin-1 (SIRT1), and interleukin-6 (IL6) levels were estimated in hepatic tissue by real-time PCR, immunohistochemistry, and immunoassay, respectively. C. intybus linn supplementation caused a significant improvement in serum liver enzymes, albumin, bilirubin levels, tissues redox status and hepatic histological features in addition to decreased IL6 level, hydroxylproline content, and PCNA immunoreactivity. On contrary, increased pAMPK/SIRT1 levels and upregulated FXR gene expression were observed. C. intybus linn could feasibly protect against TAA-induced hepatic damage, fibrosis, and cirrhosis by relieving oxidative stress and by interruption of the inflammatory pathway via AMPK/SIRT1/FXR signaling. PRACTICAL APPLICATIONS: No specific therapies are available until now to target the underlying mechanisms for protection against liver diseases. Herbal protection is widely available and cheap with no side effect. Cichorium intybus linn, a natural supplement, is proved in this current work to have the potential of being hepatoprotectant, antioxidant, and anti-inflammatory agents, thus reducing the risk of hepatic cirrhosis.

In vitro influence of selenium on the proliferation of and steroidogenesis in goat luteinized granulosa cells.

In this study, we investigated the effects of Selenium (Se) on the proliferation of and steroidogenesis in goat luteinized granulosa cells (LGCs) and elucidated the mechanisms underlying these effects. Our results showed that proliferating cell nuclear antigen (PCNA), Akt, and phosphoinositide 3-kinase (PI3K) were expressed mainly in ovarian oocytes and granulosa cells (GCs). We observed that 5 ng/mL Se significantly stimulated LGC proliferation, which could be attributed to increases in PCNA, cyclin-dependent kinase 1 (CDK1), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK; Thr172), and phosphorylated Akt (p-Akt; Ser473) and decreases in p21 (P < 0.05). Se treatment also significantly increased estradiol (E2) production, which could be, at least partially, due to increased levels of 3ß-hydroxysteroid dehydrogenase(3ß-HSD), steroidogenic acute regulatory protein (StAR), p-Akt (Ser473), and cyclic adenosine monophosphate (cAMP) (P < 0.05); however, follicle-stimulating hormone (FSH) significantly enhanced the production of E2, progesterone (P4) and cAMP (P < 0.05). Moreover, Se treatment stimulated proliferation and the synthesis of E2 and cAMP in the presence of FSH (P < 0.05). Additionally, the expression of antioxidant-related genes [glutathione peroxidase (GSH-Px) and superoxide dismutase 2 (SOD2)] and the activity of GSH-Px and SOD were progressively elevated by Se treatment (P < 0.05). These data suggested that Se plays an important role in the proliferation of and steroidogenesis in LGC by activating the PI3K/Akt and AMPK pathways, thereby increasing the expression of its downstream cell-cycle- and steroid-synthesis-related genes, as well as regulating cellular oxidative stress.

Zanthoxylum alkylamides activate phosphorylated AMPK and ameliorate glycolipid metabolism in the streptozotocin-induced diabetic rats.

This study aimed to evaluate the effects of Zanthoxylum alkylamides on the glycolipid metabolism of rats with streptozotocin (STZ)-induced diabetes. Diabetic rats were given daily oral treatments of 2, 4, or 8 mg/kg bw alkylamides for 28 days. Alkylamides significantly decreased fasting blood glucose and fructosamine content, as well as relieved organ enlargement caused by diabetes. The serum and liver triglyceride, malondialdehyde, and free fatty-acid contents of rats with STZ-induced diabetes were significantly reduced. Total cholesterol in the liver also significantly decreased. Quantitative polymerase chain reaction (Q-PCR) and Western blot detected insignificantly increased (P > 0.05) mRNA expression levels of adenosine monophosphate-activated protein kinase (AMPK) in the skeletal muscle of diabetic rats. However, AMPK and p-AMPK (Thr172) protein expression levels significantly increased. The mRNA and protein expression levels of silencing information regulator 1 significantly increased. The mRNA expression levels of acetyl-CoA-carboxylase (ACC) and protein p-ACC (Ser79) also increased. The mRNA and protein expression levels of glucose transporter type 4 (GLUT4) were significantly upregulated in the skeletal muscle cell membranes of diabetic rats. Results indicated that alkylamides activated the AMPK-signaling pathway. Thus, inhibiting ACC activity reduced fatty-acid synthesis. The rapid translocation of GLUT4 mediated increased glucose transport rate and reduced blood glucose. Therefore, alkylamides can ameliorate glucose and lipid metabolism disorders in diabetic rats by activating the AMPK pathway.

Limb girdle myasthenia with digenic RAPSN and a novel disease gene AK9 mutations.

Though dysfunction of neuromuscular junction (NMJ) is associated with congenital myasthenic syndrome (CMS), the proteins involved in neuromuscular transmission have not been completely identified. In this study, we aimed to identify a novel CMS gene in a consanguineous family with limb-girdle type CMS. Homozygosity mapping of the novel CMS gene was performed using high-density single-nucleotide polymorphism microarrays. The variants in CMS gene were identified by whole-exome sequencing (WES) and Sanger sequencing. A 20 MB-region of homozygosity (ROH) was mapped on chromosome 6q15-21. This was the only ROH that present in all clinically affected siblings and absent in all clinically unaffected siblings. WES showed a novel variant of AK9 gene located in this ROH. This variant was a start-gain mutation and introduced a cryptic 5'-UTR signal in intron 5 of the AK9 gene. The normal splicing signal would be interfered by the cryptic translation signal leading to defective splicing. Another 25 MB-ROH was found on chromosome 11p13-q12 in all siblings. WES showed a homozygous RAPSN pathogenic variant in this ROH. Since RAPSN-associated limb-girdle type CMS was only manifested in AK9 homozygous variant carriers, the disease phenotype was of digenic inheritance, and was determined by the novel disease modifier AK9 which provides NTPs for N-glycosylation. This is the first time that this specific genotype-phenotype correlation is reported. Importantly, the AK9-associated nucleotide deficiency may replete by dietary supplements. Since AK9 is a disease modifier, enhancing N-glycosylation by increasing dietary nucleotides may be a new therapeutic option for CMS patients.

A platycoside-rich fraction from the root of Platycodon grandiflorum enhances cell death in A549 human lung carcinoma cells via mainly AMPK/mTOR/AKT signal-mediated autophagy induction.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Platycodon grandiflorum (PG), commonly known as Kilkyong in Korea, Jiegeng in China, and Kikyo in Japan, has been extensively used as a traditional anti-inflammatory medicine in Asia for the treatment of respiratory conditions, such as bronchitis, asthma, and tonsillitis. Platycosides isolated from PG are especially well-known for their anti-cancer effects. AIM OF THE STUDY: We investigated the involvement of autophagic cell death and other potential molecular mechanisms induced by the platycoside-containing butanol fraction of PG (PGB) in human lung carcinoma cells. MATERIALS AND METHODS: PGB-induced growth inhibition and cell death were measured using a 5-diphenyl-tetrazolium bromide (MTT) assay. The effects of PGB on autophagy were determined by observing microtubule-associated protein 1 light chain 3 (LC3) redistribution with confocal microscopy. The PGB-mediated regulation of autophagy-associated proteins was investigated using Western blotting analysis. Furthermore, the anti-cancer mechanism of PGB was confirmed using chemical inhibitors. A high-performance liquid chromatography (HPLC)-DAD system was used to analyze the platycosides in PGB. RESULTS: In A549 cells, PGB induced significant autophagic cell death. Specifically, PGB upregulated LC3-II in a time- and dose-dependent manner, and it redistributed LC3 via autophagosome formation in the cytoplasm. PGB treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and subsequently suppressed the AKT/mammalian target of the rapamycin (mTOR) pathway. Furthermore, PGB inhibited cell proliferation by regulating the mitogen-activated protein kinase (MAPK) pathways. In this study, six types of platycosides were identified in the PGB using HPLC. CONCLUSIONS: PGB efficiently induced cancer cell death via autophagy and the modulation of the AMPK/mTOR/AKT and MAPK signaling pathways in A549 cells. Therefore, PGB may be an efficacious herbal anti-cancer therapy.

Protein profiling reveals antioxidant and signaling activities of NAP (Davunetide) in rodent hippocampus exposed to hypobaric hypoxia.

NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAP-mediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2D-GE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAP-supplemented hippocampus after hypoxia. Pathway analysis revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.

Can coenzyme q10 improve clinical and molecular parameters in fibromyalgia?

Fibromyalgia (FM) is a complex disorder that affects up to 5% of the general population worldwide. Its pathophysiological mechanisms are difficult to identify and current drug therapies demonstrate limited effectiveness. Both mitochondrial dysfunction and coenzyme Q10 (CoQ10) deficiency have been implicated in FM pathophysiology. We have investigated the effect of CoQ10 supplementation. We carried out a randomized, double-blind, placebo-controlled trial to evaluate clinical and gene expression effects of forty days of CoQ10 supplementation (300 mg/day) on 20 FM patients. This study was registered with controlled-trials.com (ISRCTN 21164124). An important clinical improvement was evident after CoQ10 versus placebo treatment showing a reduction of FIQ (p<0.001), and a most prominent reduction in pain (p<0.001), fatigue, and morning tiredness (p<0.01) subscales from FIQ. Furthermore, we observed an important reduction in the pain visual scale (p<0.01) and a reduction in tender points (p<0.01), including recovery of inflammation, antioxidant enzymes, mitochondrial biogenesis, and AMPK gene expression levels, associated with phosphorylation of the AMPK activity. These results lead to the hypothesis that CoQ10 have a potential therapeutic effect in FM, and indicate new potential molecular targets for the therapy of this disease. AMPK could be implicated in the pathophysiology of FM.

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.

Thujone, a component of medicinal herbs, rescues palmitate-induced insulin resistance in skeletal muscle.

Thujone is thought to be the main constituent of medicinal herbs that have antidiabetic properties. Therefore, we examined whether thujone ameliorated palmitate-induced insulin resistance in skeletal muscle. Soleus muscles were incubated for < or =12 h without or with palmitate (2 mM). Thujone (0.01 mg/ml), in the presence of palmitate, was provided in the last 6 h of incubation. Palmitate oxidation, AMPK/acetyl-CoA carboxylase (ACC) phosphorylation and insulin-stimulated glucose transport, plasmalemmal GLUT4, and AS160 phosphorylation were examined at 0, 6, and 12 h. Palmitate treatment for 12 h reduced fatty acid oxidation (-47%), and insulin-stimulated glucose transport (-71%), GLUT4 translocation (-40%), and AS160 phosphorylation (-26%), but it increased AMPK (+51%) and ACC phosphorylations (+44%). Thujone (6-12 h) fully rescued palmitate oxidation and insulin-stimulated glucose transport, but only partially restored GLUT4 translocation and AS160 phosphorylation, raising the possibility that an increased GLUT4 intrinsic activity may also have contributed to the restoration of glucose transport. Thujone also further increased AMPK phosphorylation but had no further effect on ACC phosphorylation. Inhibition of AMPK phosphorylation with adenine 9-beta-d-arabinofuranoside (Ara) (2.5 mM) or compound C (50 muM) inhibited the thujone-induced improvement in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. In contrast, the thujone-induced improvement in palmitate oxidation was only slightly inhibited (< or =20%) by Ara or compound C. Thus, while thujone, a medicinal herb component, rescues palmitate-induced insulin resistance in muscle, the improvement in fatty acid oxidation cannot account for this thujone-mediated effect. Instead, the rescue of palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving partial restoration of insulin-stimulated GLUT4 translocation.

Optimized culture conditions for the efficient production of porcine adenylate kinase in recombinant Escherichia coli.

Temperature shift cultivations with amino acid supplementation were optimized to produce porcine adenylate kinase (ADK) in recombinant Escherichia coli harboring a pUC-based recombinant plasmid under the control of the trp promoter. With regard to temperature control, the culture condition was initially maintained at 35 degrees C for cellular growth, but ADK expression was suppressed until the late logarithmic growth phase; subsequently, a temperature shift was applied (from 35 degrees C to 42 degrees C), which resulted in maximal ADK production. In addition, supplementation of amino acids, especially valine and leucine, during the temperature shift stimulated ADK expression from 3.5% to 9.2% and 8.6% of the total protein, respectively. After optimization, 1 g ADK per liter was produced within 16 h of cultivation with a dry cell weight of 21.8 g/l. In this system, there was no loss of the recombinant plasmid during cultivation without selective pressure.

Cpc1, a Chlamydomonas central pair protein with an adenylate kinase domain.

Mutations at CPC1 disrupt assembly of a central pair microtubule-associated complex and alter flagellar beat frequency in Chlamydomonas. Sequences of wild-type genomic clones that complement cpc1, and of corresponding cDNAs, reveal the gene product to be a 205 kDa protein with two predicted functional domains, a single EF hand motif near the C-terminus and an unusual centrally located adenylate kinase domain. Homologs are expressed in mammals (testis and tracheal cilia) as well as ciliated lower eukaryotes. Western blots confirm that Cpc1 is one of six subunits in a 16S central pair-associated complex. Motility defects associated with cpc1 alleles in vivo are partially rescued in vitro by reactivation of axonemes or cell models in saturating concentrations of ATP; thus the Cpc1 complex is essential for maintaining normal ATP concentrations in the flagellum.