Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “ATP”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca{sup 2+}-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca{sup 2+} transport by SERCA controls key processes in human cells such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca{sup 2+} dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e.g., lipid components, SERCA preparation, and activity assay requirements). ATP-dependent Ca{sup 2+} transport by SERCA in single GUV was detected directly using confocal fluorescence microscopy with the Ca{sup 2+} indicator Fluo-5F. The GUV reconstitution system was validated for functional screening of Ca{sup 2+} transport using thapsigargin (TG), a small-molecule inhibitor of SERCA currently in clinical trials as a prostate cancer prodrug. The GUV system overcomes the problem of inhibitory Ca{sup 2+} accumulation for SERCA in native and reconstituted small unilamellar vesicles (SUV). We propose that charge-mediated fusion provides a widely-applicable method for GUV reconstitution of clinically-important membrane transport proteins. We conclude that GUV reconstitution is a technological advancement for evaluating small-molecule effectors of SERCA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage

Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1 and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC. - Highlights: • Tigecycline selectively targets HCC in vitro and in vivo. • Tigecycline enhances HCC cell response to chemotherapeutic drug. • Tigecycline inhibits mitochondrial translation and functions in HCC cells. • Tigecycline induces oxidative stress and damage in HCC cells. • Mitochondrial biogenesis and respiration is higher in HCC than normal liver cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

PINK1 alleviates myocardial hypoxia-reoxygenation injury by ameliorating mitochondrial dysfunction

PTEN inducible kinase-1 (PINK1) mutant induces mitochondrial dysfunction of cells, resulting in an inherited form of Parkinson's disease. However its exact role in the cardiomyocytes is unclear. The present study examined the function of PINK1 in hypoxia-reoxygenation (H/R) induced H9c2 cell damage and its potential mechanism. The H/R model in H9c2 cells was established by 6 h of hypoxia and 12 h of reoxygenation. The CCK8 and LDH assay indicated that the cell viability was obviously reduced after H/R. The expression of PINK1 was decreased in H/R-induced H9c2 cells compared with control group. The vector overexpressing PINK1 was constructed to transfect into H/R-induced H9c2 cells. Our results showed that cell viability was increased, cell apoptosis and caspase 3, cytochrome C (Cyto C) levels were decreased after LV-PINK1 transfection. Furthermore, PINK1 overexpression stabilized electron transport chain (ETC) activity, increased ATP production, mPTP opening and mitochondrial membrane potential (MMP), inhibited ROS-generating mitochondria, implying PINK1 alleviates H/R induced mitochondrial dysfunction in cardiomyocytes. In addition, the TRAP-1 siRNA was transfected into PINK1 treated H9c2 cells after H/R to detected the molecular mechanism of PINK1 protecting cardiomyocytes. The results indicated that silence of TRAP-1 reversed the effects of PINK1 in H/R-induced H9c2 cells. In conclusion, these results suggest that PINK1 overexpression alleviates H/R-induced cell damage of H9c2 cells by phosphorylation of TRAP-1, and that is a valid approach for protection from myocardial I/R injury. - Highlights: • Effects of H/R on cell viability and PINK1 expression in H9c2 cells. • Effects of PINK1 on cell viability in H9c2 cells with H/R model. • Effects of PINK1 on mitochondrial dysfunction in H9c2 cells with H/R model. • PINK1 ameliorates H/R-induced H9c2 cells injury by activating p-TRAP-1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Anthelminthic drug niclosamide sensitizes the responsiveness of cervical cancer cells to paclitaxel via oxidative stress-mediated mTOR inhibition

Drug repurposing represents an alternative therapeutic strategy to cancer treatment. The potent anti-cancer activities of a FDA-approved anthelminthic drug niclosamide have been demonstrated in various cancers. However, whether niclosamide is active against cervical cancer is unknown. In this study, we investigated the effects of niclosamide alone and its combination with paclitaxel in cervical cancer in vitro and in vivo. We found that niclosamide significantly inhibited proliferation and induced apoptosis of a panel of cervical cancer cell lines, regardless of their cellular origin and genetic pattern. Niclosamide also inhibited tumor growth in cervical cancer xenograft mouse model. Importantly, niclosamide significantly enhanced the responsiveness of cervical cancer cell to paclitaxel. We further found that niclosamide induced mitochondrial dysfunctions via inhibiting mitochondrial respiration, complex I activity and ATP generation, which led to oxidative stress. ROS scavenge agent N-acetyl-L-cysteine (NAC) completely reversed the effects of niclosamide in increasing cellular ROS, inhibiting proliferation and inducing apoptosis, suggesting that oxidative stress induction is the mechanism of action of niclosamide in cervical cancer cells. In addition, niclosamide significantly inhibited mammalian target of rapamycin (mTOR) signaling pathway in cervical cancer cells and its inhibitory effect on mTOR is modulated by oxidative stress. Our work suggests that niclosamide is a useful addition to the treatment armamentarium for cervical cancer and induction of oxidative stress may be a potential therapeutic strategy in cervical cancer. - Highlights: • Niclosamide is active against cervical cancer cells in vitro and in vivo. • Niclosamide sensitizes cervical cancer cell response to paclitaxel. • Niclosamide induces mitochondrial dysfunction and oxidative damage. • Niclosamide inhibits mTOR signaling in an oxidative stress-dependent manner.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Antibiotic bedaquiline effectively targets growth, survival and tumor angiogenesis of lung cancer through suppressing energy metabolism

Highlights: • Bedaquiline targets lung cancer in vitro and in vivo. • Bedaquiline potently inhibits angiogenesis in vitro and in vivo. • Bedaquiline inhibits mitochondrial respiration and glycolysis. • Bedaquiline causes energy crisis and oxidative damage. Tumor angiogenesis plays essential roles during lung cancer progression and metastasis. Therapeutic agent that targets both tumor cell and vascular endothelial cell may achieve additional anti-tumor efficacy. We demonstrate that bedaquiline, a FDA-approved antibiotic drug, effectively targets lung cancer cells and angiogenesis. Bedaquiline dose-dependently inhibits proliferation and induces apoptosis of a panel of lung cancer cell lines regardless of subtypes and molecular heterogeneity. Bedaquiline also inhibits capillary network formation of human lung tumor associated-endothelial cell (HLT-EC) on Matrigel and its multiple functions, such as spreading, proliferation and apoptosis, even in the presence of vascular endothelial growth factor (VEGF). We further demonstrate that bedaquiline acts on lung cancer cells and HLT-EC via inhibiting mitochondrial respiration and glycolysis, leading to ATP reduction and oxidative stress. Consistently, oxidative damage on DNA, protein and lipid were detected in cells exposed to bedaquiline. Importantly, the results obtained in in vitro cell culture are reproducible in in vivo xenograft lung cancer mouse model, confirming that bedaquiline suppresses lug tumor growth and angiogenesis, and increases oxidative stress. Our findings demonstrating that energy depletion is effectively against lung tumor cells and angiogenesis. Our work also provide pre-clinical evidence to repurpose antibiotic bedaquiline for lung cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Angiotensin-converting enzyme 2 regulates mitochondrial function in pancreatic β-cells

Highlights: • ACE2 improved glucose stimulated insulin secretion (GSIS) and mitochondrial membrane potential in INS-1 cells. • PCR array demonstrated that ACE2 up-regulated 67 mitochondria-related genes in INS-1 cells. • ACE2 deletion suppressed mitochondrial oxidation and calcium influx in islets. • ACE2 over-expression in db/db mice promoted mitochondrial oxidation and calcium influx in islets. Mitochondrial metabolism plays an essential role in the regulation of insulin release and glucose homeostasis. Evidence demonstrated that the angiotensin-converting enzyme 2 (ACE2) participates in the regulation of glucose metabolism, however, its role in mitochondrial metabolism remains unclear. The purpose of our study was to determine if ACE2 can regulate mitochondrial function in pancreatic β-cells. We found that ACE2 over-expression restored glucose-stimulated insulin secretion (GSIS) and mitochondrial membrane potential (MMP) in the presence of H{sub 2}O{sub 2} in INS-1 cells. PCR array demonstrated that ACE2 over-expression up-regulated 67 mitochondria-related genes in INS-1 cells. In pancreatic islets, ACE2 ablation attenuated intracellular calcium influx with a decrease in GSIS. Ace2{sup −/y} mice islets exhibited impaired mitochondrial respiration and lower production of ATP, along with decreased expression of genes involved in mitochondrial oxidation. In islets from db/db mice, ACE2 over-expression increased intracellular calcium influx and restored impaired mitochondrial oxidation, potentially causing an increase in GSIS. These results shed light on the potential roles of ACE2 in mitochondrial metabolism, moreover, may improve our understanding of diabetes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

FOXJ1 promotes bladder cancer cell growth and regulates Warburg effect

Forkhead Box J1 (FOXJ1) which belongs to Fox gene family, plays complex and crucial roles in processes of development, organogenesis, regulation of the immune system, as well as progression of several malignancies. However, how FOXJ1 functions in bladder cancer remains unclear. Here, we report that FOXJ1 is upregulated in most bladder cancer patients, and predicts poor clinical outcomes. FOXJ1 facilitates bladder cancer cell proliferation and colony formation. FOXJ1 knockdown suppresses bladder tumor growth in nude mice. Mechanistically, FOXJ1 enhances glycolysis by increasing glucose uptake, lactate production and extracellular acidification rate (ECAR), and decreasing ATP generation and oxygen consumption rate (OCR) in bladder cancer cells. Our findings provide clues regarding the role of FOXJ1 as a tumor inducer in bladder cancer and an enhancer in glycolysis. Targeting FOXJ1 could be a potential therapeutic strategy in bladder cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Thermostable DNA helicase improves the sensitivity of digital PCR

Highlights: • A thermostable helicase (Tk-Upf1) unwound duplex DNA with a 5′ to 3′ polarity. • Tk-Upf1 eliminated noise DNAs derived from primer mis-annealing in PCR. • The thermostable helicase improved sensitivity of SNPs detection in digital PCR. DNA/RNA helicases, which catalyze the unwinding of duplex nucleic acids using the energy of ATP hydrolysis, contribute to various biological functions involving DNA or RNA. Euryarchaeota-specific helicase Tk-EshA (superfamily 2) from the hyperthermophilic archaeon Thermococcus kodakarensis has been used to decrease generation of mis-amplified products (noise DNAs) during PCR. In this study, we focused on another type (superfamily 1B) of helicase, Tk-Upf1 (TK0178) from T. kodakarensis, and compared its effectiveness in PCR and digital PCR with that of Tk-EshA. For this purpose, we obtained Tk-Upf1 as a recombinant protein and assessed its enzymatic characteristics. Among various double-stranded DNA (dsDNA) substrates (forked, 5′ overhung, 3′ overhung, and blunt-ended duplex), Tk-Upf1 had the highest unwinding activity toward 5′ overhung DNAs. Noise DNAs were also eliminated in the presence of Tk-Upf1 at concentrations 10-fold lower than those required to yield a comparable reduction with Tk-EshA. When a 5′ or 3′ overhung mis-annealed primer was included as a competitive primer along with specific primers, noise DNAs derived from the mis-annealed primer were eliminated in the presence of Tk-Upf1. In digital PCR, addition of Tk-EshA or Tk-Upf1 increased fluorescent intensities and improved separation between common and risk allele clusters, indicating that both helicases functioned as signal enhancers. In comparison with Tk-EshA, a smaller amount of Tk-Upf1 was required to improve the performance of digital PCR.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

GhoT of the GhoT/GhoS toxin/antitoxin system damages lipid membranes by forming transient pores

Highlights: • Determined that toxin GhoT disrupts membranes by forming transient pores. • Determined that membrane charge affects the activity of GhoT. • Determined through molecular dynamic simulations why GhoT F38R is inactive. GhoT is a bacterial toxin of the type V toxin/antitoxin system that allows Escherichia coli to reduce its metabolism in response to oxidative and bile stress. GhoT functions by increasing membrane permeability and reducing both ATP levels and the proton motive force. However, how GhoT damages the inner membrane has not been elucidated. Here we investigated how GhoT damages membranes by studying its interaction with lipid bilayers and determined that GhoT does not cause macroscopic disruption of the lipid bilayer to increase membrane permeability to the dye carboxyfluorescein. Using circular dichroism, we found that GhoT forms an alpha helical structure in lipid bilayers that agrees with the structure predicted by the I-TASSER protein structure prediction program. The structure generated using I-TASSER was used to conduct coarse-grained molecular dynamics simulations, which indicate that GhoT damages the cell membrane, as a multimer, by forming transient transmembrane pores.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Glucagon-like peptide-1 contributes to increases ABCA1 expression by downregulating miR-758 to regulate cholesterol homeostasis

Abnormal regulation of lipid metabolism is associated with type 2 diabetes mellitus (T2DM). GLP-1 as a new treatment for T2DM, has unique effects in modulating cholesterol homeostasis. However, the mechanism of this effect is largely missing. The aim of this study was to determine the effects of GLP-1 on cholesterol-induced lipotoxicity in hepatocytes and examine the underlying mechanisms. The cell viability was determined, and caspase-3 was used to detect the effects of GLP-1 on cholesterol-induced apoptosis. The alterations of miR-758 and ATP-binding cassette transporter A1 (ABCA1) resulting from cholesterol incubation or GLP-1 were detected by qRT-PCR and Western blot assays. Overexpression of miR-758 abrogated the GLP-1-mediated ABCA1 expression, and conversely, down-regulation of miR-758 aggravated GLP-1's action and revealed significant promotion effects. BODIPY-Cholesterol efflux assay revealed that treatment with miR-758 inhibitor significantly enhanced ABCA1-dependent cholesterol efflux, resulting in reduced total cholesterol. Furthermore, Oil red O staining and cholesterol measurement were used to detect lipid accumulation. As a result, cholesterol significantly attenuated cell viability, promoted cell apoptosis, and facilitated lipid accumulation, and these effects were reversed by GLP-1. This study provides evidence that, in HepG2 cells, GLP-1 may affect cholesterol homeostasis by regulating the expression of miR-758 and ABCA1. These data can inform the development of biomarkers for miR-758, and potentially other drugs, on the key pathways of lipid metabolism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar

Highlights: • Overexpression of PtABCC1 enhanced Hg tolerance in Arabidopsis and poplar. • Overexpression of PtABCC1 enhanced Hg accumulation in Arabidopsis and poplar. • PtABCC1 was the first identified poplar ABC transporter for phytoremediation of Hg. Mercury (Hg) is a highly biotoxic heavy metal that contaminates the environment. Phytoremediation is a green technology for environmental remediation and is used to clean up Hg contaminated soil in recent years. In this study, we isolated an ATP-binding cassette (ABC) transporter gene PtABCC1 from Populus trichocarpa and overexpressed it in Arabidopsis and poplar. The transgenic plants conferred higher Hg tolerance than wild type (WT) plants, and overexpression of PtABCC1 could lead to 26–72% or 7–160% increase of Hg accumulation in Arabidopsis or poplar plants, respectively. These results demonstrated that PtABCC1 plays a crucial role in enhancing tolerance and accumulation to Hg in plants, which provides a promising way for phytoremediation of Hg contamination.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification of aberrant circular RNA expression and its potential clinical value in primary great saphenous vein varicosities

Highlights: • This study identified 232 significantly aberrant circRNAs in primary great saphenous vein varicosities (PGSVVs). • A circRNA-miRNA-gene co-expression network was created. • Hsa{sub c}irc{sub 0}006427, hsa{sub c}irc{sub 0}089810 and hsa{sub c}irc{sub 0}005267 might be potential biological markers for PGSVVs. This study aimed to identify aberrant circRNA expression in primary great saphenous vein varicosities (PGSVVs) and investigate its potential clinical significance. High-throughput sequencing was applied to analyze the differential expression of circRNAs in three paired vein samples of PGSVVs and a control group. Computational bioinformatics analyses, including gene ontology (GO) analysis and encyclopedia of genes and genomes (KEGG) pathway analysis, were performed to further enrich microRNA data. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the selected aberrant circRNAs in all vein samples, and a receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic and potential clinical significance. A total of 232 circRNAs were significantly aberrantly expressed in PGSVVs, including 105 and 127 upregulated and downregulated circRNAs, respectively. The predicted top 10 downstream miRNAs of the significantly altered circRNAs are hsa-miR-103a-2-5p, hsa-miR-141-5p, hsa-miR-3692-5p, hsa-miR-4659a-3p, hsa-miR-4659b-3p, hsa-miR-4691-5p, hsa-miR-4778-3p, hsa-miR-6738-3p, hsa-miR-6792-3p and hsa-miR-6873-3p. GO analysis revealed that the most enriched and meaningful biological process, cellular component, and molecular function terms were related to the regulation of catabolic processes, the cytoplasm and ATP binding, respectively. The most enriched and meaningful pathways revealed by KEGG pathway analysis were related to “axon guidance” “vitamin digestion and absorption” and “NF-Kappa B signaling pathway”. Hsa{sub c}irc{sub 0}006427 (P = 0.007), hsa{sub c}irc{sub 0}089810 (P = 0.044) and hsa{sub c}irc{sub 0}005267 (P = 0.049) were significantly downregulated in the PGSVV group relative to their expression levels in the control group, and the area under the ROC curve was 0.9091, 0.8025 and 0.8148, respectively. This study demonstrated that circRNAs were differentially expressed in PGSVVs, and highlights the crucial roles of circRNAs in the pathogenesis of PGSVVs and indicates that hsa{sub c}irc{sub 0}006427, hsa{sub c}irc{sub 0}089810 and hsa{sub c}irc{sub 0}005267 might be potential diagnostic and clinical biological markers.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The ER membrane insertase Get1/2 is required for efficient mitophagy in yeast

Mitophagy is an evolutionarily conserved autophagy pathway that selectively eliminates mitochondria to control mitochondrial quality and quantity. Although mitophagy is thought to be crucial for cellular homeostasis, how this catabolic process is regulated remains largely unknown. Here we demonstrate that mitophagy during prolonged respiratory growth is strongly impaired in yeast cells lacking Get1/2, a transmembrane complex mediating insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane. Under the same conditions, loss of Get1/2 caused only slight defects in other types of selective and bulk autophagy. In addition, mitophagy and other autophagy-related processes are mostly normal in cells lacking Get3, a cytosolic ATP-driven chaperone that promotes delivery of TA proteins to the Get1/2 complex. We also found that Get1/2-deficient cells exhibited wildtype-like induction and mitochondrial localization of Atg32, a protein essential for mitophagy. Notably, Get1/2 is important for Atg32-independent, ectopically promoted mitophagy. Together, we propose that Get1/2-dependent TA protein(s) and/or the Get1/2 complex itself may act specifically in mitophagy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CBX3 promotes proliferation and regulates glycolysis via suppressing FBP1 in pancreatic cancer

More and more evidence has demonstrated that Chromobox protein homolog 3(CBX3) has an important role in carcinogenesis by regulating several mechanisms, such as heterochromatin formation, gene silencing, DNA replication and repair. However, its role in pancreatic cancer has seldom been discussed. In the present study, we silenced CBX3 expression in pancreatic cancer cell lines and identified the positive roles of CBX3 in cancer cell proliferation. Furthermore, we demonstrated that silencing CBX3 in pancreatic cancer cells inhibited aerobic glycolysis, the basis for providing cancer cells with building blocks for macromolecule synthesis and ATP that required. To search for the underlying molecular mechanism, we turned to examine the impact of CBX3 on the expression of FBP1, a negative regulator of aerobic glycolysis in pancreatic cancer and indicated that CBX3 negatively regulated FBP1 expression. Silencing FBP1 expression attenuated the decrease in glycolytic capacity that caused by CBX3 knockdown in pancreatic cancer cells. Taken together, these data reveal that CBX3 serves as a positive regulator of aerobic glycolysis via suppressing of the FBP1 in pancreatic cancer cells. Disrupting the CBX3-FBP1 signaling axis would be effective to treat pancreatic cancer and prevent aerobic glycolysis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CART peptide activates the Nrf2/HO-1 antioxidant pathway and protects hippocampal neurons in a rat model of Alzheimer's disease

Highlights: • CART peptide reduced the accumulation of Aβ in the hippocampus of AD rats. • CART peptide improved the cognitive function of AD rats. • CART peptide attenuated the oxidative stress damage in the hippocampus of AD rats. • CART activated the Nrf2/HO-1 pathway to exert antioxidation. • CART inhibit the apoptosis of hippocampal neurons. The accumulation of amyloid-beta (Aβ) and oxidative stress damage in the brain are recognized as early features of Alzheimer's disease (AD). The cocaine- and amphetamine-regulated transcript (CART) peptide may possibly play an antioxidative role in neurons. The aim of this study was to investigate the potential antioxidant mechanism of CART peptide in a rat model of AD. We microinjected of Aβ{sub 1-42} (2μl/4μg/hemisphere) into rat hippocampus to set a rat model of AD. A pre-microinjection of CART peptide (1μl/0.02μg/hemisphere) into rat hippocampus was administered for five consecutive days before Aβ{sub 1-42} treatment. We found that Aβ{sub 1-42} microinjection led to reduction of endogenous CART level in rat hippocampus. CART pretreatment improved the spatial memory and locomotor ability of AD rats. CART peptide decreased the Aβ{sub 1-42} and Aβ production-associated enzyme BACE1 levels. Moreover, CART peptide attenuated the oxidative stress damage with a concrete manifestation of increased MDA as well as decreased T-SOD, GSH and ATP levels in the hippocampus of Aβ{sub 1-42}-treated rat, which may be causatively implicated the activating of Nrf2/HO-1 signaling pathway. Furthermore, CART peptide attenuated neuronal apoptosis with decreased Bax, caspase-9 and caspase-3 levels and increased Bcl-2 level in rat hippocampus. Our results therefore indicate that CART peptide could serve as an antioxidant in early therapy for AD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Activation of galanin receptor 1 inhibits locus coeruleus neurons via GIRK channels

The noradrenergic neurons of the locus coeruleus (LC) are associated with various brain functions and psychiatric disorders, such as addiction and depression. It has been shown that neuropeptide galanin (GAL) inhibits neuronal excitability in LC, but the mechanisms remain unclear. In the present study, we investigated the ionic and signal transduction mechanisms underlying inhibitory effect of GAL on LC neurons using whole-cell patch clamp recording in rat brain slices. Bath application of GAL decreased the spontaneous firings and induced a dose-dependent hyperpolarization of LC neurons and this effect was attenuated by knockdown of Galr1, but not Galr2, confirming that mainly GALR1 mediates the inhibition effect of GAL. The inhibitory effect of GAL was also blocked by treatments of pertussis toxin (PTX), GTP-γ-s or GDP-β-s, respectively, indicating that the functions of PTX sensitive G{sub i/o} protein are required for GAL-induced hyperpolarization. Moreover, the blockers of GIRK (tertiapin-Q or SCH2 3390 hydrochloride) attenuated the GAL response while blocker of BK/SK/K{sub ATP} channels or TASK-1/3 channels did not affect it significantly, suggesting that GIRK channels play an important role in GAL-induced hyperpolarization in LC neurons. Taken together, the inhibitory effect of GAL on LC neurons is mediated by GALR1 via PTX-sensitive G{sub i/o} proteins, which activate GIRK channels.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression

Highlights: • HDAC6 regulates thermogenesis of brown adipose tissue. • cAMP-PKA signaling is responsible for HDAC6-dependent thermogenic regulation to control UCP1 expression. • HDAC6-dependent regulation of UCP1 affects lipid metabolism in brown adipose tissue. Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Transient activation of AMP-activated protein kinase at G1/S phase transition is required for control of S phase in NIH3T3 cells

Highlights: • AMPK is transiently activated at G1/S phase of NIH3T3 cell cycle. • Temporal activation of AMPK is required for control of S phase. • AMPK activation at G1/S phase depends on intracellular calcium and CaMKKbeta. AMP-activated protein kinase (AMPK) functions as a cellular energy sensor by monitoring the cellular AMP:ATP ratio and plays a central role in cellular and whole-body energy homeostasis. Recent studies have suggested that AMPK also contribute to cell cycle regulation, but its role in this field remains almost elusive. In the present study, we report that AMPKα1 was transiently activated during G{sub 1}/S transition phase in NIH3T3 cells in the absence of any metabolic stress. Inhibition of AMPK activity at G{sub 1}/S transition phase completely blocked cells from entering S phase; in contrast, persistent activation of AMPK at G{sub 1}/S transition phase allowed cells to normally enter S phase, but these cells failed to proceed to G{sub 2}/M phase, stacking at S phase. We further demonstrated that activation of AMPK at G{sub 1}/S transition phase depends on Ca{sup 2+} transients and CaMKKβ activity, but not on energy status. Collectively, these data indicate that temporal regulation of AMPK is required for proper control of S phase in NIH3T3 cells.

60 APPLIED LIFE SCIENCES↗