microglia-as-surrogate-biosensor-to-determine-nanoparticle

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Minneapolis Veterans Affairs Health Care System

University of Minnesota

The use of statins for the prevention or treatment of different neurodegenerative diseases has generated considerable interest albeit with some controversy. Mechanisms of statin-induced neuroprotection are not well understood. Recently, we reported that simvastatin stimulated neuronal gene expression and protein levels of the major antiapoptotic protein Bcl-2 in vivo and in vitro; suppression of Bcl-2 in SH-SY5Y cells reduced simvastatin neuroprotection; effects were independent of cholesterol and other products of the 3-hydroxy-3-methylglutaryl-CoA reductase pathway. Endothelin-1 (ET-1) can increase Bcl-2 abundance via the transcription factor nuclear factor of activated thymocytes (NFATc), and simvastatin was reported to increase ET-1 gene expression. We tested the hypothesis that simvastatin stimulation of Bcl-2 involves up-regulation of ET-1 and binding of NFATc to Bcl-2 promoter sites in SH-SY5Y human neuroblastoma cells. Simvastatin increased both intracellular and secreted ET-1 protein levels. Exogenous ET-1 increased Bcl-2 protein abundance, which was inhibited by ET-1 receptor antagonists. Simvastatin increased translocation of NFATc3 to the nucleus while reducing nuclear NFATc1 and having no effect on NFATc4. Endothelin-1 also increased NFATc3 levels in the nucleus, and this increase was inhibited by ET-1 receptor antagonists. Treatment of cells with simvastatin stimulated binding of NFATc3 to the Bcl-2 promoter. We report novel findings showing that up-regulation of Bcl-2 by simvastatin involves ET-1 and the transcription factor NFATc3. Discovering how statins can selectively alter a specific NFATc isoform that leads to an increase in an antiapoptotic protein will provide a new approach to understanding statin-induced neuroprotection and conditions outside the brain in which apoptosis contributes to pathophysiology.

Simvastatin stimulates production of the antiapoptotic protein Bcl-2 via endothelin-1 and NFATc3 in SH-SY5Y cells.

&#x80CC;&#x666F;&#x3002;&#x7EBF;&#x7C92;&#x4F53;&#x7535;&#x5B50;&#x8F6C;&#x8FD0;&#x86CB;&#x767D;&#x8868;&#x8FBE;&#x6539;&#x53D8;&#x8868;&#x660E;&#x9884;&#x5904;&#x7406;&#x5FC3;&#x808C;&#x7EC4;&#x7EC7;&#x4E2D;&#x3002;&#x6211;&#x4EEC;&#x60F3;&#x786E;&#x5B9A;&#x662F;&#x5426;&#x8FD9;&#x4E9B;&#x6539;&#x52A8;&#x4F9D;&#x7136;&#x5B58;&#x5728;&#x5728;&#x7B2C;&#x4E8C;&#x4E2A;&#x7A97;&#x53E3;&#x7684;&#x4FDD;&#x62A4; (SWOP)&#xFF0C;&#x5982;&#x679C;&#x6709;&#xFF0C;&#x662F;&#x5426;&#x6709;&#x5229;&#x7684;&#x80FD;&#x91CF;&#x72B6;&#x6001;&#x5728;&#x968F;&#x540E;&#x7684;&#x7F3A;&#x8840;&#x671F;&#x95F4;&#x63D0;&#x4F9B;&#x4E86;&#x4FBF;&#x5229;&#x3002;&#x65B9;&#x6CD5;&#x3002;&#x5341;&#x56DB;&#x4E2A;&#x732A;&#x672F;&#x4E0E;&#x6CD5;&#x63F4;&#x7F72;&#x52A8;&#x8109;&#x5341; 2 &#x5206;&#x949F;&#x6C14;&#x7403;&#x901A;&#x8D27;&#x81A8;&#x80C0;&#x7684; SWOP &#x8BAE;&#x5B9A;&#x4E66;&#xFF0C;&#x6BCF; 2 &#x5206;&#x949F;&#x518D;&#x704C;&#x6CE8;&#x6240;&#x5206;&#x9694;&#x3002;&#x4E8C;&#x5341;&#x56DB;&#x4E2A;&#x5C0F;&#x65F6;&#x540E;&#xFF0C;&#x7EBF;&#x7C92;&#x4F53;&#x88AB;&#x9694;&#x79BB;&#x7684; SWOP &#x548C;&#x6DF1;&#x6C34;&#x732A;&#x5FC3;&#x548C;&#x4E3A;&#x89E3;&#x5076;&#x8054;&#x86CB;&#x767D; (UCP)-2 &#x5185;&#x5BB9;&#x5206;&#x6790;&#x7684;&#x5370;&#x8FF9;&#x5206;&#x6790;&#xFF0C;&#x86CB;&#x767D;&#x8D28;&#x7EC4;&#x5B66;&#x53D8;&#x5316;&#x7531; iTRAQ &reg; &#x548C;&#x547C;&#x5438;&#x7684;&#x6C27;&#x7535;&#x6781;&#x3002;&#x5728;&#x5E76;&#x884C;&#x7684;&#x4F53;&#x5185;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x9AD8;&#x80FD;&#x6838;&#x82F7;&#x9178;&#x83B7;&#x5F97;&#x8DE8;&#x58C1;&#x6D3B;&#x68C0;&#x4ECE;&#x9EBB;&#x9189;&#x7684; SWOP &#x548C;&#x6DF1;&#x6C34;&#x732A;&#x5728;&#x57FA;&#x7EBF;&#x548C;&#x7F3A;&#x8840;&#x4F4E;&#x6D41;&#x91CF;&#x6301;&#x7EED;&#x671F;&#x95F4;&#x3002;&#x7ED3;&#x679C;&#x3002;&#x76F8;&#x6BD4;&#x6DF1;&#x6C34;&#x7EBF;&#x7C92;&#x4F53;&#xFF0C;&#x6269;&#x589E; SWOP &#x5FC3;&#x810F;&#x7EC4;&#x7EC7;&#x8868;&#x73B0;&#x51FA;&#x66F4;&#x591A;&#x7684;&#x8DDF;&#x5355;&#x4FE1;&#x7528;&#x8BC1; 2 &#x548C;&#x66F4;&#x590D;&#x6742;&#x56DB; &#xFF08;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c &#x6C27;&#x5316;&#x9176;&#xFF09;&#x3001; &#x86CB;&#x767D;&#x8868;&#x8FBE;&#x7684;&#x590D;&#x6742; V &#xFF08;ATPase&#xFF09;&#x3002;&#x4E0E;&#x6DF1;&#x6C34;&#x732A;&#x4F53;&#x5185;&#x6761;&#x4EF6;&#x671F;&#x95F4;&#x3001; &#x58C1;&#x80FD;&#x91CF;&#x5B66; SWOP &#x5FC3;&#xFF0C;&#x81EA;&#x7531;&#x80FD;&#x91CF;&#x7684;&#x4E09;&#x78F7;&#x9178;&#x817A;&#x82F7;&#x6C34;&#x89E3;&#x7684;&#x4F30;&#x8BA1; &#xFF08;&Delta;G(0))&#xFF0C;&#x90FD;&#x5728;&#x57FA;&#x7EBF;&#x76F8;&#x4F3C;&#x4F46;&#x4E0B;&#x8DCC;&#x5E74;&#x5E95;&#x7F3A;&#x8840;&#x4F4E;&#x6D41;&#x91CF;&#x5C0F;&#x4E8E; (-&#x4E0E; 57.0&plusmn;2.1-51.1&plusmn;1.4 kJ/mol &#xFF1B;P &lt; 0.05&#xFF09;&#x3002;&#x7ED3;&#x8BBA;&#x3002;&#x5185;&#x4ECE;&#x9884;&#x5904;&#x7406;&#x7684; SWOP &#x5185;&#x5FC3;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#xFF0C;&#x8DDF;&#x5355;&#x4FE1;&#x7528;&#x8BC1; 2 &#x589E;&#x52A0;&#xFF0C;&#x5728;&#x4E0E;&#x589E;&#x5F3A;&#x590D;&#x6742;&#x7B2C;&#x56DB;&#x7AE0;&#x548C;&#x7B2C;&#x4E94;&#x7AE0;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x97F3;&#x4E50;&#x4F1A;&#xFF0C;&#x5728;&#x4F4E;&#x6D41;&#x91CF;&#x7F3A;&#x8840;&#x671F;&#x95F4;&#x4F20;&#x6388;&#x6709;&#x5229;&#x7684;&#x80FD;&#x91CF;&#x72B6;&#x6001;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x7684;&#x652F;&#x6301;&#x53EF;&#x80FD;&#x4F1A;&#x51CF;&#x5C11;&#x6C27;&#x5316;&#x635F;&#x4F24;&#x7684;&#x7EBF;&#x7C92;&#x4F53;&#x9002;&#x5E94;&#x5E76;&#x4E0D;&#x80FD;&#x76F4;&#x63A5;&#x51CF;&#x5C11;&#x80FD;&#x91CF;&#x7684;&#x6574;&#x4F53;&#x6548;&#x7387;&#x671F;&#x95F4;&#x6301;&#x7EED;&#x7684;&#x6C27;&#x5265;&#x593A;&#x7684;&#x6982;&#x5FF5;&#x3002;

&#x8868;&#x8FBE;&#x6539;&#x53D8;&#x7EBF;&#x7C92;&#x4F53;&#x7535;&#x5B50;&#x8F6C;&#x8FD0;&#x86CB;&#x767D;&#x548C;&#x6539;&#x5584;&#x5FC3;&#x808C;&#x80FD;&#x91CF;&#x72B6;&#x6001;&#x671F;&#x95F4;&#x665A;&#x7F3A;&#x8840;&#x9884;&#x5904;&#x7406;&#x3002;

Background. Altered expression of mitochondrial electron transport proteins has been shown in preconditioned myocardial tissue. We wish to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Methods. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQÂ® and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Results. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased UCP-2 and a greater expression of complex IV (cytochrome c oxidase) and complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (Î”G(0)), were similar at baseline but decreased less by the end of low-flow ischemia (-57.0Â±2.1 versus -51.1Â±1.4 kJ/mol; P<0.05). Conclusions. Within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

Altered Expression of Mitochondrial Electron Transport Proteins and Improved Myocardial Energetic State During Late Ischemic Preconditioning.

Arri&egrave;re-plan. Une expression alt&eacute;r&eacute;e des prot&eacute;ines de transport des &eacute;lectrons mitochondriale a &eacute;t&eacute; d&eacute;montr&eacute;e dans le tissu myocardique pr&eacute;conditionn&eacute;. Nous voulons d&eacute;terminer si ces alt&eacute;rations persistent dans la deuxi&egrave;me fen&ecirc;tre de Protection (SWOP) et dans l&#39;affirmative, si un &eacute;tat &eacute;nerg&eacute;tique favorable est favoris&eacute; au cours de l&#39;isch&eacute;mie ult&eacute;rieur. M&eacute;thodes. Quatorze porcs ont subi un protocole SWOP avec dix inflations 2 minutes ballonnet dans l&#39;art&egrave;re LAD, s&eacute;par&eacute;s par la reperfusion de 2 minutes. Vingt-quatre heures plus tard, les mitochondries ont &eacute;t&eacute; isol&eacute;es de coeurs de cobaye SWOP et SHAM et analys&eacute;s pour la teneur en prot&eacute;ines d&eacute;couplante (UCP) -2 par analyse par western blot, prot&eacute;omiques change par iTRAQ &reg; et la respiration par une &eacute;lectrode &agrave; oxyg&egrave;ne. En parall&egrave;le des &eacute;tudes in vivo, des nucl&eacute;otides ont &eacute;t&eacute; obtenues par biopsie transmurale de porcs anesth&eacute;si&eacute;s de SWOP et SHAM au d&eacute;part et au cours de l&#39;isch&eacute;mie soutenue de faible d&eacute;bit. R&eacute;sultats. Par rapport aux mitochondries SHAM, ex vivo tissu cardiaque SWOP d&eacute;montr&eacute; UCP-2 accrue et une plus grande expression du complexe IV (cytochrome c oxydase) et des prot&eacute;ines complexes de V (ATPase). En comparaison avec des porcs SHAM dans des conditions in vivo, transmurale &eacute;nerg&eacute;tique dans les coeurs SWOP, selon l&#39;estimation de l&#39;&eacute;nergie libre d&#39;hydrolyse de l&#39;ATP (&Delta;G(0)), ressemblaient au d&eacute;part mais moins &agrave; la fin de l&#39;isch&eacute;mie de faible d&eacute;bit diminu&eacute; (- 57.0&plusmn;2.1 contre - 51.1&plusmn;1.4 kJ/mol ; P &lt; 0,05). Conclusions. Dans les mitochondries isol&eacute;es de coeurs SWOP pr&eacute;conditionn&eacute;s, UCP-2 est augment&eacute;e et de concert avec des prot&eacute;ines complexes IV et V renforc&eacute;es, conf&egrave;re un &eacute;tat &eacute;nerg&eacute;tique favorable durant l&#39;&eacute;tiage isch&eacute;mie. Ces donn&eacute;es supportent l&#39;id&eacute;e que mitochondriales adaptations qui peuvent r&eacute;duire les dommages oxydants ne r&eacute;duisent pas l&#39;efficacit&eacute; globale de l&#39;&eacute;nerg&eacute;tique au cours de la privation d&#39;oxyg&egrave;ne soutenue.

Expression alt&eacute;r&eacute;e de prot&eacute;ines mitochondriale de Transport d&#39;&eacute;lectrons et l&#39;&eacute;tat &eacute;nerg&eacute;tique myocardique am&eacute;lior&eacute; au cours de la fin de pr&eacute;conditionnement isch&eacute;mique.

Hintergrund. Ver&auml;nderte Expression der mitochondriale Elektronentransport Proteine wurde in vorkonditionierten myokardialem Gewebe gezeigt. Wir wollen, ob diese Ver&auml;nderungen im zweiten Fenster Schutz (SWOP) beibehalten, und wenn ja, zu bestimmen, ob ein g&uuml;nstiger energetischer Zustand w&auml;hrend nachfolgende Isch&auml;mie erleichtert wird. Methoden. Vierzehn Schweine unterzog sich ein SWOP-Protokoll mit zehn 2-Minuten-Ballon-Inflationen in der LAD-Arterie, die durch 2 Minuten Reperfusion getrennt. Vierundzwanzig Stunden sp&auml;ter Mitochondrien wurden isoliert von SWOP und SHAM-Schwein-Herzen und f&uuml;r uncoupling Protein (UCP)-2 Inhalt von western-Blot-Analyse analysiert, Proteomic &auml;ndert durch iTRAQ &reg; und Atmung durch eine Sauerstoff-Elektrode. In parallelen in-vivo-Studien energiereiche Nukleotide durch Transmural Biopsie bei narkotisierten SWOP und SHAM Schweine an der Baseline und w&auml;hrend nachhaltige Low-Flow-Isch&auml;mie Ringversuch Ergebnisse. Im Vergleich mit SHAM Mitochondrien, ex Vivo nachgewiesen SWOP Herzgewebe erh&ouml;hte UCP-2 und einem st&auml;rkeren Ausdruck Komplex IV (Cytochrom C-Oxidase) und komplexen Proteine, V (ATPase). Im Vergleich zu SHAM Schweine w&auml;hrend der in-vivo-Bedingungen, Transmural Energetik in SWOP-Herzen, als gesch&auml;tzt durch die freie Energie der ATP-Hydrolyse (&Delta;G(0)), &auml;hnlich bei Studienbeginn waren aber verringert weniger Ende Low-Flow-Isch&auml;mie (- 57.0&plusmn;2.1 im Vergleich - 51.1&plusmn;1.4 kJ/Mol; P &lt; 0,05). Schlussfolgerungen. In isolierten Mitochondrien von vorkonditionierten SWOP-Herzen UCP-2 wird erh&ouml;ht und im Konzert mit verst&auml;rkten Komplex IV und V-Proteine, vermittelt einen guten energetischen Zustand w&auml;hrend der Low-Flow-Isch&auml;mie. Diese Daten unterst&uuml;tzen die Vorstellung, dass die mitochondriale Anpassungen, die oxidativen Sch&auml;den zu verringern, k&ouml;nnen nicht die Gesamteffizienz der Energetik w&auml;hrend der anhaltenden Sauerstoffmangel reduzieren.

Ge&auml;nderte Ausdruck von mitochondriale Elektronentransport-Proteinen und verbesserte myokardialen energetischen Zustand w&auml;hrend der sp&auml;ten isch&auml;mischen Vorkonditionierung.

Sullo sfondo. Alterata espressione delle proteine di trasporto degli elettroni mitocondriale &egrave; stato dimostrato nel tempo tessuto miocardico. Vogliamo determinare se queste alterazioni persistono nella seconda finestra di protezione (SWOP) e se &egrave; cos&igrave;, se uno stato energico favorevole &egrave; facilitato durante l&#39;ischemia successiva. Metodi. Quattordici suini sub&igrave; un protocollo SWOP con dieci inflazioni 2 minuti palloncino nell&#39;arteria LAD, ciascuno separato da riperfusione 2 minuti. Ventiquattro ore pi&ugrave; tardi, i mitocondri sono stati isolati dai cuori di maiale SWOP e SHAM e analizzati per proteina disgiungente (UCP) -2 contenuta da analisi western blot, proteomic cambia da iTRAQ &reg; e respirazione da un elettrodo di ossigeno. In paralleli studi in vivo, nucleotidi ad alta energie sono stati ottenuti da biopsie transmurali da suini SWOP e SHAM anestetizzati al basale e durante l&#39;ischemia a basso flusso continuo. Risultati. Confrontato con i mitocondri SHAM, ex vivo tessuto cardiaco SWOP dimostrato maggiore UCP-2 e una maggiore espressione del complesso IV (citocromo c ossidasi) e proteine complesse V (ATPasi). Rispetto ai maiali SHAM durante le condizioni in vivo, transmurale energetica nei cuori SWOP, come stimato di energia libera di idrolisi dell&#39;ATP (&Delta;G(0)), erano simili al basale, ma meno entro la fine di ischemia a basso flusso &egrave; diminuito (- 57.0&plusmn;2.1 versus - 51.1&plusmn;1.4 kJ/mol; P &lt; 0,05). Conclusioni. All&#39;interno di mitocondri isolati da tempo cuori SWOP, UCP-2 &egrave; aumentato e in concerto con proteine maggiore complesso IV e V, conferisce uno stato energico favorevole durante l&#39;ischemia a basso flusso. Questi dati supportano la nozione che mitocondriali degli adattamenti che possono ridurre il danno ossidante non riducono l&#39;efficienza complessiva dell&#39;energetica durante la privazione dell&#39;ossigeno sostenuta.

Alterata espressione delle proteine di trasporto degli elettroni mitocondriale e miglioramento dello stato energetico del miocardio durante il tardo precondizionamento ischemico.

&#x80CC;&#x666F;&#x3002;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x96FB;&#x5B50;&#x4F1D;&#x9054;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x306F;&#x524D;&#x51E6;&#x7406;&#x4ED8;&#x304D;&#x306E;&#x5FC3;&#x7B4B;&#x7D44;&#x7E54;&#x3067;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4FDD;&#x8B77;&#x7B2C; 2 &#x30A6;&#x30A3;&#x30F3;&#x30C9;&#x30A6; (SWOP) &#x3067;&#x3053;&#x308C;&#x3089;&#x306E;&#x5909;&#x66F4;&#x3092;&#x6C38;&#x7D9A;&#x5316;&#x3059;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3001;&#x304A;&#x3088;&#x3073;&#x5834;&#x5408;&#x306F;&#x3001;&#x6709;&#x5229;&#x306A;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30C3;&#x30B7;&#x30E5;&#x306A;&#x72B6;&#x614B;&#x304C;&#x5F8C;&#x7D9A;&#x306E;&#x865A;&#x8840;&#x6642;&#x4FC3;&#x9032;&#x3055;&#x308C;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x5224;&#x65AD;&#x3057;&#x305F;&#x3044;&#x3068;&#x601D;&#x3044;&#x307E;&#x3059;&#x3002;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3002;14 &#x8C5A; SWOP &#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB; 10 2 &#x5206;&#x6C17;&#x7403;&#x30A4;&#x30F3;&#x30D5;&#x30EC; LAD &#x52D5;&#x8108;&#x3092;&#x65BD;&#x884C;&#x3057;&#x305F;, &#x305D;&#x308C;&#x305E;&#x308C; 2 &#x5206;&#x518D;&#x704C;&#x6D41;&#x306B;&#x3088;&#x308B;&#x5206;&#x96E2;&#x3057;&#x307E;&#x3059;&#x3002;20-4 &#x6642;&#x9593;&#x5F8C;&#x3001;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2; SWOP &#x3068;&#x30B7;&#x30E3;&#x30E0;&#x306E;&#x8C5A;&#x5FC3;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3055;&#x308C;&#x3001;&#x897F;&#x90E8;&#x306E;&#x3057;&#x307F;&#x306E;&#x5206;&#x6790;&#xFF09; &#x3092;&#x8131;&#x5171;&#x5F79;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; (UCP)-2 &#x30B3;&#x30F3;&#x30C6;&#x30F3;&#x30C4; &#x30D7;&#x30ED;&#x30C6;&#x30AA;&#x30FC;&#x30E0;&#x5909;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066; iTRAQ &reg; &#x3068;&#x9178;&#x7D20;&#x96FB;&#x6975;&#x306B;&#x3088;&#x308B;&#x547C;&#x5438;&#x3002;&#x4E26;&#x5217;&#x306E; in vivo &#x3067;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x9AD8;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x306E;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x8CAB;&#x751F;&#x691C;&#x3067;&#x9EBB;&#x9154;&#x306E; SWOP &#x3068;&#x30B7;&#x30E3;&#x30E0;&#x8C5A;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x6642;&#x3068;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x4F4E;&#x6D41;&#x91CF;&#x865A;&#x8840;&#x6642;&#x304B;&#x3089;&#x5F97;&#x3089;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#x3002;&#x30B7;&#x30E3;&#x30E0; &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2; ex vivo &#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x5897;&#x52A0; UCP 2 &#x3068;&#x8907;&#x96D1;&#x306A; IV (&#x30C1;&#x30C8;&#x30AF;&#x30ED;&#x30E0;&#x9178;&#x5316;&#x9175;&#x7D20;) &#x304A;&#x3088;&#x3073;&#x8907;&#x96D1;&#x306A; V (atp &#x30A2;&#x30FC;&#x30BC;) &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x5927;&#x304D;&#x3044;&#x767A;&#x73FE; SWOP &#x5FC3;&#x81D3;&#x7D44;&#x7E54;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x30B7;&#x30E3;&#x30E0;&#x8C5A; in vivo &#x3067;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E2D;&#x306B;&#x3001;&#x8CAB;&#x30A8;&#x30CA;&#x30FC;&#x30B8;&#x30A7;&#x30C6;&#x30A3;&#x30AF;&#x30B9; SWOP &#x5FC3;&#x3068;&#x3057;&#x3066;&#x306E; ATP &#x52A0;&#x6C34;&#x5206;&#x89E3;&#x81EA;&#x7531;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x63A8;&#x5B9A;&#x3068;&#x306E;&#x6BD4;&#x8F03; (&Delta;G(0))&#x3001;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x985E;&#x4F3C;&#x3057;&#x3066;&#x3044;&#x305F;&#x304C;&#x5C11;&#x306A;&#x3044;&#x4F4E;&#x6D41;&#x91CF;&#x865A;&#x8840;&#x306E;&#x6700;&#x5F8C;&#x3067;&#x6E1B;&#x5C11; (- &#x5BFE; 57.0&plusmn;2.1 - 51.1&plusmn;1.4 kJ/mol;P &lt; 0.05)&#x3002;&#x7D50;&#x8AD6;&#x3002;&#x5206;&#x96E2;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2; &#x30D7;&#x30EC;&#x30B3;&#x30F3;&#x30C7;&#x30A3;&#x30B7;&#x30E7;&#x30F3; SWOP &#x5FC3;&#x304B;&#x3089;&#x5185; UCP 2 &#x5897;&#x52A0;&#x3057;&#x3001;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x8907;&#x96D1;&#x306A; IV &#x3068; V &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30B3;&#x30F3;&#x30B5;&#x30FC;&#x30C8;&#x3067;&#x306F;&#x3001;&#x4F4E;&#x6D41;&#x91CF;&#x865A;&#x8840;&#x6642;&#x306E;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30C3;&#x30B7;&#x30E5;&#x306A;&#x72B6;&#x614B;&#x304C;&#x826F;&#x597D;&#x306E;&#x4ED8;&#x4E0E;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF; &#x30AA;&#x30AD;&#x30B7;&#x30C0;&#x30F3;&#x30C8;&#x306E;&#x640D;&#x50B7;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x9069;&#x5FDC;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x8AD6;&#x306E;&#x5168;&#x4F53;&#x7684;&#x306A;&#x52B9;&#x7387;&#x4E2D;&#x306B;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x9178;&#x7D20;&#x306E;&#x5265;&#x596A;&#x3092;&#x4F4E;&#x4E0B;&#x3055;&#x305B;&#x306A;&#x3044;&#x3053;&#x3068;&#x6982;&#x5FF5;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;

&#x4E0B;&#x65EC;&#x865A;&#x8840;&#x30D7;&#x30EC;&#x30B3;&#x30F3;&#x30C7;&#x30A3;&#x30B7;&#x30E7;&#x30CB;&#x30F3;&#x30B0; &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x96FB;&#x5B50;&#x4F1D;&#x9054;&#x86CB;&#x767D;&#x8CEA;&#x304A;&#x3088;&#x3073;&#x6539;&#x5584;&#x3055;&#x308C;&#x305F;&#x5FC3;&#x7B4B;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30C3;&#x30B7;&#x30E5;&#x306A;&#x72B6;&#x614B;&#x306E;&#x5909;&#x66F4;&#x3055;&#x308C;&#x305F;&#x767A;&#x73FE;&#x3002;

&#xBC30;&#xACBD;&#xC785;&#xB2C8;&#xB2E4;. &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC804;&#xC790; &#xC804;&#xC1A1; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBCC0;&#xACBD; &#xB41C; &#xC2DD; preconditioned &#xC2EC;&#xADFC; &#xC870;&#xC9C1;&#xC5D0; &#xD45C;&#xC2DC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xAC00; &#xC774;&#xB7EC;&#xD55C; &#xBCC0;&#xACBD;&#xC5D0;&#xB294; &#xB450; &#xBC88;&#xC9F8; &#xCC3D;&#xC758; &#xBCF4;&#xD638; (&#xAD50;&#xD658;) &#xC720;&#xC9C0; &#xC5EC;&#xBD80;&#xC640; &#xB9CC;&#xC57D; &#xADF8;&#xB807;&#xB2E4;&#xBA74;, &#xC720;&#xB9AC;&#xD55C; &#xC5D0;&#xB108;&#xC9C0; &#xC0C1;&#xD0DC; &#xC774;&#xD6C4; &#xAD6D; &#xC18C; &#xBE48; &#xD608; &#xB3D9;&#xC548; &#xCD09;&#xC9C4; &#xC5EC;&#xBD80;&#xB97C; &#xD655;&#xC778; &#xD558; &#xACE0; &#xC2F6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBC29;&#xBC95;&#xC785;&#xB2C8;&#xB2E4;. 14 &#xB3FC;&#xC9C0; &#xBC1B;&#xC558;&#xB2E4; &#xC80A;&#xC740;&#xC774; &#xB3D9;&#xB9E5;&#xC5D0;&#xC11C; 10 2 &#xBD84; &#xD48D;&#xC120; inflations &#xAD50;&#xD658; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;, &#xAC01; 2 &#xBD84; reperfusion&#xC73C;&#xB85C; &#xAD6C;&#xBD84;. 24 &#xC2DC;&#xAC04; &#xD6C4;&#xC5D0;, &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xAD50;&#xD658; &#xBC0F; &#xAC00;&#xC9DC; &#xB3FC;&#xC9C0; &#xB9C8;&#xC74C;&#xC5D0;&#xC11C; &#xACE0;&#xB9BD; &#xB410;&#xB2E4; &#xBC0F; Itraq&#xC5D0; &#xC758;&#xD574; proteomic &#xBCC0;&#xACBD; &#xC11C;&#xCABD; &#xC624; &#xC810; &#xBD84;&#xC11D;&#xC5D0; &#xC758;&#xD574; &#xCF58;&#xD150;&#xCE20; uncoupling &#xB2E8;&#xBC31;&#xC9C8; (UCP)-2&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC11D;, &reg; &#xBC0F; &#xC0B0;&#xC18C; &#xC804;&#xADF9;&#xC5D0; &#xC758;&#xD574; &#xD638;&#xD761;. &#xBCD1;&#xB82C; in vivo &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xACE0; &#xC5D0;&#xB108;&#xC9C0; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC; transmural &#xC0DD; &#xAC80; &#xD558; &#xC5EC; &#xCDE8; &#xAD50;&#xD658; &#xBC0F; &#xAC00;&#xC9DC; &#xB3FC;&#xC9C0; &#xAE30;&#xC900;&#xC120; &#xBC0F; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xB0AE;&#xC740; &#xD750;&#xB984; &#xD5C8; &#xD608; &#xC911;&#xC5D0;&#xC11C; &#xAC00;&#xC838;&#xC628;. &#xACB0;&#xACFC;&#xC785;&#xB2C8;&#xB2E4;. &#xAC00;&#xC9DC; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;, ex vivo&#xC5D0; &#xBE44;&#xD574; &#xAD50;&#xD658; &#xC2EC;&#xC7A5; &#xC870;&#xC9C1;&#xC758; &#xC99D;&#xAC00; UCP-2&#xC640; &#xBCF5;&#xC7A1; &#xD55C; IV (&#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c &#xC0B0;&#xD654; &#xD6A8;&#xC18C;)&#xC640; &#xBCF5;&#xC7A1; &#xD55C; V (ATPase) &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD070; &#xC2DD; &#xC2DC;&#xC5F0;. In vivo &#xC0C1;&#xD0DC;&#xC5D0; &#xC788;&#xB294; &#xB3D9;&#xC548; &#xAC00;&#xC9DC; &#xB3FC;&#xC9C0;, transmural &#xB108; &#xC9C0;&#xB860;&#xC73C;&#xB85C; &#xC790;&#xC720; &#xC5D0;&#xB108;&#xC9C0; ATP &#xAC00;&#xC218;&#xBD84;&#xD574;&#xC5D0; &#xC758;&#xD574; &#xC608;&#xC0C1; &#xAD50;&#xD658; &#xB9C8;&#xC74C;&#xC5D0; &#xBE44;&#xD574; (&Delta;G(0)), &#xAE30;&#xC900;&#xC120;&#xC5D0; &#xC720;&#xC0AC; &#xD558;&#xC9C0;&#xB9CC; &#xB0AE;&#xC740; &#xD750;&#xB984; &#xD5C8; &#xD608; &#xB9D0; &#xB35C; &#xAC10;&#xC18C; (-57.0&plusmn;2.1&#xC640;-51.1&plusmn;1.4 kJ/mol; P &lt; 0.05). &#xACB0;&#xB860;&#xC785;&#xB2C8;&#xB2E4;. Preconditioned &#xAD50;&#xD658; &#xB9C8;&#xC74C;&#xC5D0;&#xC11C; &#xACA9;&#xB9AC; &#xB41C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xB0B4;&#xC5D0;&#xC11C; UCP 2 &#xC99D;&#xAC00; &#xD558; &#xACE0; &#xD5A5;&#xC0C1; &#xB41C; &#xBCF5;&#xC7A1; &#xD55C; IV&#xC640; V &#xB2E8;&#xBC31;&#xC9C8;&#xC640; &#xCF58;&#xC11C;&#xD2B8;, &#xB0AE;&#xC740; &#xD750;&#xB984; &#xD5C8; &#xD608; &#xC911; &#xC720;&#xB9AC;&#xD55C; &#xC5D0;&#xB108;&#xC9C0; &#xC0C1;&#xD0DC;&#xB97C; &#xBD80;&#xC5EC; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xC81C; &#xD53C;&#xD574;&#xB97C; &#xC904;&#xC77C; &#xC218; &#xC788;&#xB294; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xAC01; &#xC0C9; &#xD55C; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xC0B0;&#xC18C; &#xBC15;&#xD0C8; &#xD558;&#xB294; &#xB3D9;&#xC548;&#xC5D0; &#xB108; &#xC9C0;&#xB860;&#xC758; &#xC804;&#xBC18;&#xC801;&#xC778; &#xD6A8;&#xC728; &#xC904;&#xC5B4;&#xB4E4;&#xC9C0; &#xC54A;&#xB294; &#xAC1C;&#xB150;&#xC744; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

&#xB2A6;&#xAC8C; &#xD5C8; &#xD608; &#xC131; &#xB298;&#xC5B4;&#xC9C4; &#xC911; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC804;&#xC790; &#xC804;&#xC1A1; &#xB2E8;&#xBC31;&#xC9C8;&#xACFC; &#xC5D0;&#xB108;&#xC9C0; &#xC0C1;&#xD0DC; &#xD5A5;&#xC0C1; &#xB41C; &#xC2EC;&#xADFC;&#xC758; &#xBCC0;&#xACBD; &#xB41C; &#xC2DD;&#xC785;&#xB2C8;&#xB2E4;.

Fondo. Expresi&oacute;n alterada de las prote&iacute;nas de transporte de electrones mitocondrial se ha demostrado en el tejido del miocardio previo. Queremos determinar si estas alteraciones persisten en la segunda ventana de protecci&oacute;n (SWOP) y si es as&iacute;, si un estado energ&eacute;tico favorable se facilita en isquemia subsecuente. M&eacute;todos. Catorce cerdos experimentaron un protocolo SWOP con diez inflaciones de globo de 2 minutos en la arteria da, cada uno separado por reperfusi&oacute;n de 2 minutos. Veinticuatro horas m&aacute;s tarde, las mitocondrias se aislaron de SWOP y SHAM corazones de cerdo y analizados para desacoplar la prote&iacute;na (UCP) -2 contenida por an&aacute;lisis de western blot, prote&oacute;micos cambia por iTRAQ &reg; y la respiraci&oacute;n por un electrodo de ox&iacute;geno. En estudios in vivo paralelos, nucle&oacute;tidos de alta energ&iacute;a se obtuvieron por biopsia transmural de cerdos anestesiados de SWOP y farsa al inicio y durante la isquemia de bajo flujo sostenida. Resultados. En comparaci&oacute;n con las mitocondrias SHAM, ex vivo tejido card&iacute;aco SWOP demostr&oacute; aumento de UCP-2 y una mayor expresi&oacute;n del complejo IV (citocromo c oxidasa) y prote&iacute;nas del complejo V (ATPasa). En comparaci&oacute;n con la farsa cerdos en condiciones in vivo, transmural energ&eacute;tica en corazones SWOP, seg&uacute;n la estimaci&oacute;n de la energ&iacute;a libre de hidr&oacute;lisis de ATP (&Delta;G(0)), eran similares al inicio pero menos al final de la isquemia de bajo flujo disminuido (- 57.0&plusmn;2.1 versus - 51.1&plusmn;1.4 kJ/mol; P &lt; 0.05). Conclusiones. Dentro de las mitocondrias aisladas de corazones SWOP acondicionarse, UCP-2 se incrementa y en concierto con prote&iacute;nas mejoradas del complejo IV y V, imparte un estado energ&eacute;tico favorable durante la isquemia de bajo flujo. Estos datos apoyan la noci&oacute;n que adaptaciones mitocondriales que pueden reducir el da&ntilde;o oxidante no reducen la eficiencia global de energ&iacute;a durante la privaci&oacute;n de ox&iacute;geno sostenido.

Expresi&oacute;n alterada de prote&iacute;nas de transporte de electrones mitocondrial y mejor estado energ&eacute;tico del miocardio durante finales de precondicionamiento isqu&eacute;mico.

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.

Resistance to obesity is becoming an exception rather than the norm, and understanding mechanisms that lead some to remain lean in spite of an obesigenic environment is critical if we are to find new ways to reverse this trend. Levels of energy intake and physical activity both contribute to body weight management, but it is challenging for most to adopt major long-term changes in either factor. Physical activity outside of formal exercise, also referred to as activity of daily living, and in stricter form, spontaneous physical activity (SPA), may be an attractive modifiable variable for obesity prevention. In this review, we discuss individual variability in SPA and NEAT (nonexercise thermogenesis, or the energy expended by SPA) and its relationship to obesity resistance. The hypothalamic neuropeptide orexin (hypocretin) may play a key role in regulating SPA and NEAT. We discuss how elevated orexin signaling capacity, in the context of a brain network modulating SPA, may play a major role in defining individual variability in SPA and NEAT. Greater activation of this SPA network leads to a lower propensity for fat mass gain and therefore may be an attractive target for obesity prevention and therapy.

Brain orexin promotes obesity resistance.

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection.

Obesity has increased in prevalence worldwide, attributed in part to the influences of an obesity-promoting environment and genetic factors. While obesity and overweight increasingly seem to be the norm, there remain individuals who resist obesity. We present here an overview of data supporting the idea that hypothalamic neuropeptide orexin A (OXA; hypocretin 1) may be a key component of brain mechanisms underlying obesity resistance. Prior work with models of obesity and obesity resistance in rodents has shown that increased orexin and/or orexin sensitivity is correlated with elevated spontaneous physical activity (SPA), and that orexin-induced SPA contributes to obesity resistance via increased non-exercise activity thermogenesis (NEAT). However, central hypothalamic orexin signaling mechanisms that regulate SPA remain undefined. Our ongoing studies and work of others support the hypothesis that one such mechanism may be upregulation of a hypoxia-inducible factor 1 alpha (HIF-1α)-dependent pathway, suggesting that orexin may promote obesity resistance both by increasing SPA and by influencing the metabolic state of orexin-responsive hypothalamic neurons. We discuss potential mechanisms based on both animal and in vitro pharmacological studies, in the context of elucidating potential molecular targets for obesity prevention and therapy.

Orexin: pathways to obesity resistance?

We have previously shown that mitochondrial uncoupling protein-2 (UCP-2) is increased in a swine model of hibernating myocardium (HM). Although UCP-2 reduces oxidant stress, it can promote inefficiency of the electron transport chain. In this study, we tested whether UCP-2 remains increased in revascularized HM (RHM) after coronary artery bypass grafting (CABG).

Expression of uncoupling protein-2 remains increased within hibernating myocardium despite successful coronary artery bypass grafting at 4 wk post-revascularization.

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.

Sleep disorders, obesity, and aging: the role of orexin.

Hibernating myocardium is characterized by viable yet dysfunctional myocardium secondary to chronic ischemia, with studies demonstrating incomplete early recovery after coronary artery bypass graft (CABG). We tested whether mitochondrial fusion proteins, an indicator of mitochondrial biogenesis, are increased in hibernating myocardium post-CABG.

Mitochondrial fusion proteins in revascularized hibernating hearts.

Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

Role of orexin A signaling in dietary palmitic acid-activated microglial cells.

The peroxisome proliferator-activated receptor (PPAR)-γ drug pioglitazone (PIO) has been shown to protect tissue against oxidant stress. In a swine model of chronic myocardial ischemia, we tested whether PIO increases PGC1-α signaling and the expression of mitochondrial antioxidant peptides. Eighteen pigs underwent a thoracotomy with placement of a fixed constrictor around the LAD artery. At 8 weeks, diet was supplemented with either PIO (3 mg/kg) or placebo for 4 weeks. Regional myocardial function and blood flow were determined at the time of the terminal study. PGC1-α expression was quantified from nuclear membranes by gels and respiration, oxidant stress markers and proteomics by iTRAQ were determined from isolated mitochondria. In the chronically ischemic LAD region, wall thickening from the PIO and control groups was 42 ± 6 and 45 ± 5 %, respectively (NS) with no intergroup differences in basal blood flow (0.72 ± 0.04 versus 0.74 ± 0.04 ml/min g, respectively; NS). In the PIO group, the expression of nuclear bound PGC1-α was higher (11.3 ± 2.6 versus 4.4 ± 1.4 AU; P < 0.05) and the content of mitochondrial antioxidant peptides including superoxide dismutase 2, aldose reductase, glutathione S-transferase and thioredoxin reductase were greater than controls. Although isolated mitochondria from the PIO group showed lower state 3 respiration (102 ± 13 versus 161 ± 22 nmol/min mg; P < 0.05), no differences in oxidant stress were noted by protein carbonyl (1.7 ± 0.7 versus 1.1 ± 0.1 nmol/mg). Chronic pioglitazone does not reduce regional myocardial blood flow or function in a swine model of chronic myocardial ischemia, but may have an important role in increasing expression of antioxidant proteins through PGC1-α signaling.

Pioglitazone increases PGC1-α signaling within chronically ischemic myocardium.

There is conflicting clinical evidence whether administration of coenzyme Q10 (CoQ10) improves function following coronary artery bypass graft surgery (CABG). Using a swine model of hibernating myocardium, we tested whether daily CoQ10 would improve contractile function by MRI at 4-week post-CABG. Twelve pigs underwent a thoracotomy and had a constrictor placed on the left anterior descending (LAD). At 12 weeks, they underwent off-pump bypass and received daily dietary supplements of either CoQ10 (10 mg/kg/day) or placebo. At 4-week post-CABG, circumferential strain measurements in the hibernating LAD region from placebo and CoQ10 groups were not different and increased to a similar extent with dobutamine (-14.7 ± 0.6 versus -14.8 ± 0.1, respectively (NS)). Post-sacrifice, oxidant stress markers were obtained in the mitochondrial isolates and protein carbonyl in the placebo, and CoQ10 groups were 6.14 ± 0.36 and 5.05 ± 0.32 nmol/mg, respectively (NS). In summary, CoQ10 did not improve contractile reserve or reduce oxidant stress at 4-week post-CABG.

Cardiac Strain in a Swine Model of Regional Hibernating Myocardium: Effects of CoQ10 on Contractile Reserve Following Bypass Surgery.

Palmitic acid (PA), an abundant dietary saturated fatty acid, contributes to obesity and hypothalamic dysregulation in part through increase in oxidative stress, insulin resistance, and neuroinflammation. Increased production of reactive oxygen species (ROS) as a result of PA exposure contributes to the onset of neuronal apoptosis. Additionally, high fat diets lead to changes in hypothalamic gene expression profiles including suppression of the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) and upregulation of the pro-apoptotic protein B cell lymphoma 2 associated X protein (Bax). Orexin A (OXA), a hypothalamic peptide important in obesity resistance, also contributes to neuroprotection. Prior studies have demonstrated that OXA attenuates oxidative stress induced cell death. We hypothesized that OXA would be neuroprotective against PA induced cell death. To test this, we treated an immortalized hypothalamic cell line (designated mHypoA-1/2) with OXA and PA. We demonstrate that OXA attenuates PA-induced hypothalamic cell death via reduced caspase-3/7 apoptosis, stabilization of Bcl-2 gene expression, and reduced Bax/Bcl-2 gene expression ratio. We also found that OXA inhibits ROS production after PA exposure. Finally, we show that PA exposure in mHypoA-1/2 cells significantly reduces basal respiration, maximum respiration, ATP production, and reserve capacity. However, OXA treatment reverses PA-induced changes in intracellular metabolism, increasing basal respiration, maximum respiration, ATP production, and reserve capacity. Collectively, these results support that OXA protects against PA-induced hypothalamic dysregulation, and may represent one mechanism through which OXA can ameliorate effects of obesogenic diet on brain health.

Article	Year
Simvastatin stimulates production of the antiapoptotic protein Bcl-2 via endothelin-1 and NFATc3 in SH-SY5Y cells. Molecular neurobiology\| PubMed ID: 20369390	2010
Altered Expression of Mitochondrial Electron Transport Proteins and Improved Myocardial Energetic State During Late Ischemic Preconditioning. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 22389388	2012
Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model. Neuroscience letters\| PubMed ID: 22796468	2012
Brain orexin promotes obesity resistance. Annals of the New York Academy of Sciences\| PubMed ID: 22803681	2012
Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection. Journal of molecular and cellular cardiology\| PubMed ID: 23562790	2013
Orexin: pathways to obesity resistance? Reviews in endocrine & metabolic disorders\| PubMed ID: 24005942	2013
Expression of uncoupling protein-2 remains increased within hibernating myocardium despite successful coronary artery bypass grafting at 4 wk post-revascularization. The Journal of surgical research\| PubMed ID: 25199570	2015
Sleep disorders, obesity, and aging: the role of orexin. Ageing research reviews\| PubMed ID: 25462194	2015
Mitochondrial fusion proteins in revascularized hibernating hearts. The Journal of surgical research\| PubMed ID: 25655995	2015
Role of orexin A signaling in dietary palmitic acid-activated microglial cells. Neuroscience letters\| PubMed ID: 26306651	2015
Pioglitazone increases PGC1-α signaling within chronically ischemic myocardium. Basic research in cardiology\| PubMed ID: 27138931	2016
Cardiac Strain in a Swine Model of Regional Hibernating Myocardium: Effects of CoQ10 on Contractile Reserve Following Bypass Surgery. Journal of cardiovascular translational research\| PubMed ID: 27184805	2016
Orexin A attenuates palmitic acid-induced hypothalamic cell death. Molecular and cellular neurosciences\| PubMed ID: 27449757	2016

Tammy Butterick

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