a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

University of Waterloo

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.

A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion.

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.

The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion.

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.

Normal flux through ATP-citrate lyase or fatty acid synthase is not required for glucose-stimulated insulin secretion.

&#x76F8;&#x5BF9;&#x4E86;&#x89E3;&#x751A;&#x5C11;&#x4F53;&#x5185;&#x7684; alpha &#x4E9A;&#x57FA; heterotrimeric G &#x86CB;&#x767D; Gz (Galphaz) &#x7684;&#x804C;&#x80FD;&#x3002;Galphaz &#x6FC0;&#x6D3B;&#x6291;&#x5236;&#x8461;&#x8404;&#x7CD6;&#x523A;&#x6FC0;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x7624;&#x7EC6;&#x80DE;&#x682A;&#x7684;&#x53D1;&#x73B0;&#x4E0E;&#x6700;&#x8FD1;&#x51FA;&#x73B0;&#x7684;&#x4E00;&#x4E2A;&#x6F5C;&#x5728;&#x529F;&#x80FD;&#x7684;&#x7EBF;&#x7D22; &#xFF08;Kimple&#xFF0C;M.E.&#x3001; &#x5C3C;&#x514B;&#x677E;&#x3001; A.B.&#x51EF;&#x5229;&#xFF0C;&#x4F53;&#x80B2;&#x3001; &#x8D1D;&#x5229;&#x3001; C.L.&#x3001; &#x5E74;&#x8F7B;&#x3001; K.H.&#x3001; &#x5B57;&#x6BB5;&#x3001; T.A.&#x548C;&#x51EF;&#x897F;&#xFF0C;J.P.J.(2005 &#x5E74;) Biol.&#x5316;&#x5B66; 280&#xFF0C;31708 31713)&#x3002;&#x82E5;&#x8981;&#x6269;&#x5C55;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x5728;&#x4F53;&#x5185;&#xFF0C;&#x5229;&#x7528; Galphaz &#x6572;&#x9664;&#x9F20;&#x6A21;&#x578B;&#x4EE5;&#x786E;&#x5B9A;&#x662F;&#x5426; Galphaz &#x51FD;&#x6570;&#x4E2D;&#x6291;&#x5236;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x6BDB;&#x5F62;&#x6001;&#x7684;&#x80F0;&#x5C9B;&#x4E2D;&#x6216; Galphaz null &#x548C;&#x91CE;&#x751F;&#x578B;&#x5C0F;&#x9F20;&#x4E4B;&#x95F4;&#x7684;&#x80F0;&#x5C9B; DNA&#x3001; &#x86CB;&#x767D;&#x8D28;&#x3001; &#x6216;&#x80F0;&#x5C9B;&#x7D20;&#x5185;&#x5BB9;&#x53D1;&#x73B0;&#x6CA1;&#x6709;&#x5DEE;&#x522B;&#x3002;&#x4E5F;&#x6709;&#x7684; Galphaz null &#x5C0F;&#x9F20;&#x80F0;&#x5C9B;&#x7D20;&#x654F;&#x611F;&#x6027;&#x548C;&#x91CE;&#x751F;&#x578B;&#x63A7;&#x4EF6;&#xFF0C;&#x6765;&#x8861;&#x91CF;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x8010;&#x53D7;&#x6D4B;&#x8BD5;&#x4E4B;&#x95F4;&#x6CA1;&#x6709;&#x533A;&#x522B;&#x3002;Galphaz null &#x5C0F;&#x9F20;&#x505A;&#x7684;&#x90A3;&#x6837;&#xFF0C;&#x4F46;&#x662F;&#xFF0C;&#x663E;&#x793A;&#x589E;&#x52A0;&#x7684;&#x8840;&#x6D46;&#x80F0;&#x5C9B;&#x7D20;&#x6D53;&#x5EA6;&#x4E0E;&#x76F8;&#x5E94;&#x589E;&#x52A0;&#x8461;&#x8404;&#x7CD6;&#x6E05;&#x62C6;&#x540E;&#x8179;&#x8154;&#x548C;&#x53E3;&#x670D;&#x8461;&#x8404;&#x7CD6;&#x6311;&#x6218;&#x91CE;&#x751F;&#x578B;&#x5BF9;&#x7167;&#x7EC4;&#x8FDB;&#x884C;&#x6BD4;&#x8F83;&#x3002;Galphaz null &#x5C0F;&#x9F20;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x589E;&#x52A0;&#x7684;&#x8840;&#x6D46;&#x80F0;&#x5C9B;&#x7D20;&#x662F;&#x6700;&#x6709;&#x53EF;&#x80FD;&#x589E;&#x5F3A;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x7684;&#x76F4;&#x63A5;&#x7ED3;&#x679C;&#xFF0C;&#x56E0;&#x4E3A;&#x80F0;&#x5C9B;&#x5206;&#x79BB; Galphaz null &#x5C0F;&#x9F20;&#x8868;&#x73B0;&#x51FA;&#x6BD4;&#x91CE;&#x751F;&#x578B;&#x5C0F;&#x9F20;&#x660E;&#x663E;&#x9AD8;&#x4E8E;&#x8461;&#x8404;&#x7CD6;&#x523A;&#x6FC0;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x3002;&#x6700;&#x540E;&#xFF0C;Galphaz null &#x80F0;&#x5C9B;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x589E;&#x52A0;&#x7684;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x4F3C;&#x4E4E;&#x662F;&#x7531;&#x4E8E;&#x8865;&#x6291;&#x5236;&#x7684;&#x817A;&#x82F7;&#x9178;&#x73AF;&#x5316;&#x9176;&#xFF0C;&#x6551;&#x6D4E;&#x4F5C;&#x4E3A;&#x8425;&#x5730;&#x751F;&#x4EA7;&#x663E;&#x8457;&#x589E;&#x52A0;&#xFF0C;Galphaz null &#x80F0;&#x5C9B;&#x5916;&#x6E90;&#x523A;&#x6FC0;&#x7684;&#x7F3A;&#x5E2D;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E; Galphaz &#x53EF;&#x80FD;&#x65E8;&#x5728;&#x6539;&#x5584; 2 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x60A3;&#x8005; &beta; &#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x969C;&#x788D;&#x7684;&#x836F;&#x7269;&#x6CBB;&#x7597;&#x5B66;&#x7684;&#x6F5C;&#x5728;&#x65B0;&#x76EE;&#x6807;&#x3002;

Galphaz &#x6D88;&#x6781;&#x8C03;&#x8282;&#x80F0;&#x5C9B;&#x7D20;&#x5206;&#x6CCC;&#x548C;&#x8461;&#x8404;&#x7CD6;&#x6E05;&#x5173;&#x3002;

Relatively little is known about the in vivo functions of the alpha subunit of the heterotrimeric G protein Gz (Galphaz). Clues to one potential function recently emerged with the finding that activation of Galphaz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708-31713). To extend this study in vivo, a Galphaz knock-out mouse model was utilized to determine whether Galphaz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Galphaz-null and wild-type mice. There was also no difference between the insulin sensitivity of Galphaz-null mice and wild-type controls, as measured by insulin tolerance tests. Galphaz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Galphaz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Galphaz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Galphaz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Galphaz-null islets in the absence of exogenous stimulation. These findings indicate that Galphaz may be a potential new target for therapeutics aimed at ameliorating beta-cell dysfunction in Type 2 diabetes.

Galphaz negatively regulates insulin secretion and glucose clearance.

Relativement peu de choses est connue sur les fonctions in vivo de la sous-unit&eacute; alpha de la prot&eacute;ine G h&eacute;t&eacute;rotrim&eacute;riques Gz (Galphaz). Indices pour une fonction potentielle a r&eacute;cemment &eacute;merg&egrave;rent avec la constatation que l&#39;activation de Galphaz inhibe la s&eacute;cr&eacute;tion d&#39;insuline stimul&eacute;e par le glucose dans une lign&eacute;e de cellules insulinome (Kimple, M. E., A. B., Nixon, Kelly, P., Bailey, C. L., jeune, K. H., champs, T. A. et Casey, J. (2005) J. Biol. Chem. 280, 31708-31713). Pour &eacute;tendre cette &eacute;tude in vivo, un mod&egrave;le de souris knock-out Galphaz a &eacute;t&eacute; utilis&eacute; pour d&eacute;terminer si la fonction Galphaz joue un r&ocirc;le dans l&#39;inhibition de la s&eacute;cr&eacute;tion d&#39;insuline. Aucune diff&eacute;rence n&#39;ont &eacute;t&eacute; d&eacute;couverts dans la morphologie g&eacute;n&eacute;rale des &icirc;lots pancr&eacute;atiques ou dans la teneur en ADN, prot&eacute;ine ou insuline &icirc;lot entre souris Galphaz null et sauvage. Il y n&#39;avait &eacute;galement aucune diff&eacute;rence entre la sensibilit&eacute; &agrave; l&#39;insuline des souris Galphaz null et les contr&ocirc;les de type sauvage, telle que mesur&eacute;e par les tests de tol&eacute;rance d&#39;insuline. Galphaz null souris a fait, cependant, affiche une augmentation de l&#39;insuline plasmatique et une augmentation correspondante dans la clairance du glucose apr&egrave;s le d&eacute;fi de glucose par voie orale et intrap&eacute;riton&eacute;ale comparativement aux t&eacute;moins de type sauvage. L&#39;insulin&eacute;mie accrue observ&eacute;e chez les souris Galphaz null est probablement une cons&eacute;quence directe de la s&eacute;cr&eacute;tion d&#39;insuline accrue, &eacute;tant donn&eacute; que les &icirc;lots pancr&eacute;atiques isolement de souris Galphaz null exhib&eacute; la s&eacute;cr&eacute;tion d&#39;insuline stimul&eacute;e par le glucose significativement plus &eacute;lev&eacute;e &agrave; celles des souris de type sauvage. Enfin, la s&eacute;cr&eacute;tion d&#39;insuline accrue observ&eacute;e dans les &icirc;lots Galphaz null semble &ecirc;tre due &agrave; la r&eacute;paration d&#39;une inhibition tonique de l&#39;ad&eacute;nylcyclase, comme la production d&#39;AMPc augmente significativement dans les &icirc;lots Galphaz null en l&#39;absence de stimulation exog&egrave;ne. Ces r&eacute;sultats indiquent que la Galphaz peut &ecirc;tre une nouvelle cible potentielle pour les th&eacute;rapies visant &agrave; am&eacute;liorer la dysfonction des cellules b&ecirc;ta dans le diab&egrave;te de Type 2.

Galphaz r&eacute;gule n&eacute;gativement la clairance de glucose et de la s&eacute;cr&eacute;tion d&#39;insuline.

&Uuml;ber die in-vivo-Funktionen von der alpha-Untereinheit des das Heterotrimeres G-Protein-Gz (Galphaz) ist relativ wenig bekannt. Hinweise auf eine m&ouml;gliche Funktion k&uuml;rzlich entstanden mit der Feststellung, dass die Aktivierung des Galphaz Glukose stimuliert Insulin-Sekretion in ein Insulinom-Zelllinie hemmt (Kimple, Kelly, M. E., Nixon, A. B. s. Bailey, C. L., Young, K. H., Felder, T. A. und Casey, s. J.j (2005). Biol Chem. 280, 31708-31713). Um in-vivo Studie zu erweitern, wurde ein Galphaz-Knock-Out Maus-Modell genutzt, um festzustellen, ob die Galphaz-Funktion spielt eine Rolle in der Hemmung der Insulinsekretion. Keine Unterschiede wurden in die grobe Morphologie von den pankreatischen kleinen Inseln oder im Ilet DNA, Protein oder Insulin Inhalt zwischen Galphaz-Null und Wildtyp M&auml;usen entdeckt. Auch gab es keinen Unterschied zwischen der Insulinempfindlichkeit des Galphaz-Null-M&auml;use und Wildtyp-Steuerelemente, wie durch Insulin-Toleranztests gemessen. Galphaz-Null-M&auml;use, jedoch erh&ouml;hte Insulin-Plasmakonzentrationen und einer entsprechenden Erh&ouml;hung in Glukose Freiraum nach intraperitoneal und oralen Glukose-Foulspiel im Vergleich zu Wildtyp Steuerelemente angezeigt. Das erh&ouml;hte Plasma-Insulin in Galphaz-Null-M&auml;use zu beobachten ist h&ouml;chstwahrscheinlich eine direkte Folge der verbesserten Insulin-Sekretion, da Pankreas Inselchen aus isoliert ausgestellt-Galphaz-Null-M&auml;use deutlich h&ouml;here Glukose stimuliert Insulin-Sekretion als jene von Wildtyp M&auml;usen. Schlie&szlig;lich erscheint die erh&ouml;hte Insulin-Sekretion in Galphaz-Null-Inselchen beobachtet werden durch das Relief eine tonische Hemmung der Fehlfunktion Guanylatcyclase, wie cAMP Produktion in Galphaz-Null-Inselchen in Ermangelung der exogene Stimulation deutlich erh&ouml;ht wurde. Diese Befunde zeigen, dass Galphaz m&ouml;glicherweise ein potenzielles neues Ziel f&uuml;r Therapeutika, die zur Betrachtung Beta-Zell-Dysfunktion bei Diabetes Typ 2.

Galphaz regelt negativ Insulin-Sekretion und Glukose-Abfertigung.

Relativamente poco si sa circa le funzioni in vivo della subunit&agrave; alfa della proteina eterotrimerica G Gz (Galphaz). Indizi di una funzione potenziale &egrave; emerso di recente con la constatazione che l&#39;attivazione di Galphaz inibisce la secrezione di insulina glucosio-stimolati in una linea cellulare di insulinoma (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., giovane, K. H., campi, T. A. e Casey, J. P. (2005) j Biol Chem. 280, 31708-31713). Per estendere questo studio in vivo, &egrave; stato utilizzato un modello di topo knock-out Galphaz per determinare se Galphaz funzione svolge un ruolo nell&#39;inibizione della secrezione dell&#39;insulina. Differenze non sono stati scoperti nella morfologia lorda delle isole pancreatiche o del contenuto di DNA, proteine o insulina isoletta tra topi Galphaz null e wild-type. Inoltre non c&#39;era alcuna differenza tra la sensibilit&agrave; dell&#39;insulina di topi Galphaz null e controlli wild-type, come misurato da insulina test di tolleranza. Null Galphaz topi ha fatto, tuttavia, visualizzare maggiori concentrazioni di insulina e un corrispondente aumento nella liquidazione di glucosio dopo la sfida di glucosio orale e intraperitoneale rispetto ai controlli wild-type. L&#39;insulina al plasma aumentato osservato nei topi Galphaz null &egrave; pi&ugrave; probabile un risultato diretto della secrezione dell&#39;insulina migliorata, poich&eacute; isole pancreatiche isolate da topi Galphaz null esposto secrezione di insulina glucosio stimolato significativamente pi&ugrave; elevata rispetto a quelli dei topi wild-type. Infine, la secrezione di insulina aumentata osservata in isolette Galphaz null sembra essere dovuto il rilievo di un&#39;inibizione tonica della adenil ciclasi, come produzione cAMP era significativamente aumentato in isolette Galphaz-null in assenza di stimolazione esogena. Questi risultati indicano che Galphaz pu&ograve; essere un potenziale obiettivo nuovo per terapie volte a migliorare la disfunzione delle cellule beta nel diabete di tipo 2.

Galphaz regola negativamente la liquidazione di secrezione e glucosio insulina.

&#x6BD4;&#x8F03;&#x7684;&#x5C11;&#x3057;&#x306E; Gz (Galphaz) &#x306E;&#x30D8;&#x30C6;&#x30ED;&#x4E09;&#x91CF;&#x4F53; G &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30A2;&#x30EB;&#x30D5;&#x30A1; &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x751F;&#x4F53;&#x5185;&#x6A5F;&#x80FD;&#x306B;&#x3064;&#x3044;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x6A5F;&#x80FD;&#x306E; 1 &#x3064;&#x306E;&#x624B;&#x304C;&#x304B;&#x308A;&#x3092;&#x6700;&#x8FD1;&#x6D6E;&#x4E0A;&#x3057;&#x3066;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9;&#x523A;&#x6FC0;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x3059;&#x308B;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30CE;&#x30FC;&#x30DE;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; Galphaz &#x306E;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x3068; &#xFF08;Kimple&#x3001;m. e.&#x3001;&#x30CB;&#x30AF;&#x30BD;&#x30F3;&#x3001;A. B.&#x3001;&#x30B1;&#x30EA;&#x30FC;&#x3001;P.&#x3001;&#x30D9;&#x30A4;&#x30EA;&#x30FC;&#x3001;C. L.&#x3001;&#x30E4;&#x30F3;&#x30B0;&#x3001;K. H.&#x3001;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3001;T. A. &#x304A;&#x3088;&#x3073; J. J. P. (2005) &#x751F;&#x7269;&#x5316;&#x5B66; 280&#x3001;&#x30B1;&#x30FC;&#x30B7;&#x30FC; 31708 31713)&#x3002;&#x3053;&#x306E;&#x7814;&#x7A76;&#x306F; in vivo &#x3067;&#x62E1;&#x5F35;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;Galphaz &#x95A2;&#x6570;&#x306F;&#x3001;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x306E;&#x6291;&#x5236;&#x306B;&#x306F;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B; Galphaz &#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9; &#x30E2;&#x30C7;&#x30EB;&#x304C;&#x5229;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x81B5;&#x5CF6;&#x306E;&#x7DCF;&#x5F62;&#x614B;&#x307E;&#x305F;&#x306F; Galphaz null &#x304A;&#x3088;&#x3073;&#x91CE;&#x751F;&#x578B;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x9593;&#x306E;&#x81B5;&#x5CF6;&#x306E; DNA&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3001;&#x307E;&#x305F;&#x306F;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x306E;&#x5185;&#x5BB9;&#x306E;&#x76F8;&#x9055;&#x70B9;&#x306F;&#x691C;&#x51FA;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x307E;&#x305F;&#x3001;Galphaz null &#x30DE;&#x30A6;&#x30B9;&#x306E;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x611F;&#x53D7;&#x6027;&#x3068;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x8CA0;&#x8377;&#x8A66;&#x9A13;&#x306B;&#x3088;&#x3063;&#x3066;&#x6E2C;&#x5B9A;&#x3055;&#x308C;&#x305F;&#x3068;&#x3057;&#x3066;&#x306E;&#x91CE;&#x751F;&#x578B;&#x306E;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x3068;&#x306E;&#x9055;&#x3044;&#x306F;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;Galphaz null &#x30DE;&#x30A6;&#x30B9;&#x305F;&#x3060;&#x3057;&#x3001;&#x5897;&#x52A0;&#x8840;&#x6F3F;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x6FC3;&#x5EA6;&#x3068;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x5897;&#x52A0;&#x306F;&#x3001;&#x30D6;&#x30C9;&#x30A6;&#x7CD6;&#x30AF;&#x30EA;&#x30A2;&#x30E9;&#x30F3;&#x30B9;&#x306E;&#x8179;&#x8154;&#x5185;&#x304A;&#x3088;&#x3073;&#x7D4C;&#x53E3;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9; &#x30C1;&#x30E3;&#x30EC;&#x30F3;&#x30B8;&#x91CE;&#x751F;&#x578B;&#x306E;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x3001;&#x6B21;&#x306E;&#x8868;&#x793A;&#x3001;&#x3067;&#x3057;&#x305F;&#x3002;Galphaz null &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x5897;&#x52A0;&#x8840;&#x6F3F;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x306F;&#x6700;&#x3082;&#x53EF;&#x80FD;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x5F37;&#x5316;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x306E;&#x76F4;&#x63A5;&#x306E;&#x7D50;&#x679C;&#x3067;&#x3059;&#x3001;&#x81B5;&#x5CF6;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3055;&#x308C;&#x305F;&#x306E;&#x3067; Galphaz null &#x30DE;&#x30A6;&#x30B9;&#x91CE;&#x751F;&#x578B;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x305D;&#x308C;&#x3089;&#x3088;&#x308A;&#x6709;&#x610F;&#x306B;&#x9AD8;&#x3044;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9;&#x523A;&#x6FC0;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x3092;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;cAMP &#x306E;&#x7523;&#x751F;&#x306F; Galphaz null &#x5CF6;&#x306E;&#x5916;&#x6765;&#x523A;&#x6FC0;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x6709;&#x610F;&#x306B;&#x5897;&#x52A0;&#x3057;&#x305F;&#x3001;Galphaz null &#x5CF6;&#x3067;&#x89B3;&#x6E2C;&#x3055;&#x308C;&#x305F;&#x5897;&#x52A0;&#x3059;&#x308B;&#x30A4;&#x30F3;&#x30B7;&#x30E5;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x30A2;&#x30C7;&#x30CB;&#x30EB;&#x9178;&#x30B7;&#x30AF;&#x30E9;&#x30FC;&#x30BC;&#x306E;&#x30C8;&#x30CB;&#x30C3;&#x30AF;&#x6291;&#x5236;&#x3092;&#x8EFD;&#x6E1B;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;Galphaz &beta; &#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x4E0D;&#x5168; 2 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x306E;&#x6539;&#x5584;&#x3092;&#x76EE;&#x6307;&#x3057;&#x305F;&#x6CBB;&#x7642;&#x306E;&#x65B0;&#x3057;&#x3044;&#x6A19;&#x7684;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

Galphaz &#x306F;&#x3001;&#x5426;&#x5B9A;&#x7684;&#x306B;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x5206;&#x6CCC;&#x3068;&#x30D6;&#x30C9;&#x30A6;&#x7CD6;&#x30AF;&#x30EA;&#x30A2;&#x30E9;&#x30F3;&#x30B9;&#x3092;&#x8ABF;&#x6574;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC0C1;&#xB300;&#xC801;&#xC73C;&#xB85C; &#xC791;&#xC740; heterotrimeric G &#xB2E8;&#xBC31;&#xC9C8; Gz (Galphaz)&#xC758; &#xC54C;&#xD30C; &#xC18C; &#xB2E8;&#xC704;&#xC758; in vivo &#xAE30;&#xB2A5;&#xC5D0; &#xB300; &#xD55C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. &#xD55C; &#xC7A0;&#xC7AC;&#xC801; &#xD568;&#xC218;&#xC5D0; &#xB300; &#xD55C; &#xB2E8;&#xC11C; &#xCC3E;&#xB294; Galphaz&#xC758; &#xD65C;&#xC131;&#xD654; insulinoma &#xC140; &#xB77C;&#xC778;&#xC5D0;&#xC11C; &#xD3EC;&#xB3C4; &#xB2F9; &#xC790;&#xADF9; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44; &#xC5B5;&#xC81C;&#xC640; &#xCD5C;&#xADFC; &#xB4F1;&#xC7A5; (Kimple, M. E., &#xB2C9;&#xC2A8;, A. B., &#xCF08;&#xB9AC;, &#xD53C;, &#xBCA0;&#xC77C;&#xB9AC;, C. L., &#xC80A;&#xC740;, K. H., &#xD544;&#xB4DC;, T. A.&#xC640; &#xCF00;&#xC774;&#xC2DC;, &#xC81C;&#xC774; &#xD53C; &#xC81C;&#xC774; (2005) Biol. &#xD654;&#xD559; 280, 31708-31713). In vivo&#xC774; &#xC5F0;&#xAD6C;&#xB97C; &#xD655;&#xC7A5; &#xD558;&#xB824;&#xBA74; Galphaz &#xB178;&#xD06C; &#xC544;&#xC6C3; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378; Galphaz &#xD568;&#xC218;&#xC5D0;&#xC11C; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44;&#xC758; &#xC5B5;&#xC81C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4; &#xC5EC;&#xBD80; &#xD655;&#xC778; &#xC774;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. &#xCDCC; &#xC7A5; &#xB3C5;&#xB3C4;&#xC758; &#xC2EC;&#xD55C; &#xD615;&#xD0DC; &#xB610;&#xB294; Galphaz null &#xBC0F; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xB9C8;&#xC6B0;&#xC2A4; &#xC0AC;&#xC774; &#xC12C; DNA, &#xB2E8;&#xBC31;&#xC9C8;, &#xB610;&#xB294; &#xC778;&#xC290;&#xB9B0; &#xB0B4;&#xC6A9; &#xCC28;&#xC774;&#xAC00; &#xBC1C;&#xACAC; &#xB410;&#xB2E4;. &#xB610;&#xD55C; Galphaz null &#xC950;&#xC758; &#xC778;&#xC290;&#xB9B0; &#xAC10;&#xB3C4; &#xBC0F; &#xC778;&#xC290;&#xB9B0; &#xB0B4;&#xC131; &#xD14C;&#xC2A4;&#xD2B8;&#xC5D0; &#xC758;&#xD574; &#xCE21;&#xC815; &#xB41C;&#xB85C; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xCEE8;&#xD2B8;&#xB864; &#xC0AC;&#xC774; &#xCC28;&#xC774;&#xAC00; &#xC788;&#xC5C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; Galphaz null &#xC950;,, intraperitoneal &#xBC0F; &#xACBD;&#xAD6C; &#xD3EC;&#xB3C4; &#xB2F9; &#xB3C4;&#xC804; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xCEE8;&#xD2B8;&#xB864;&#xC5D0; &#xBE44;&#xD574; &#xB2E4;&#xC74C;&#xACFC; &#xAC19;&#xC740; &#xD3EC;&#xB3C4; &#xB2F9; &#xC815;&#xB9AC;&#xC5D0; &#xC99D;&#xAC00; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xC778;&#xC290;&#xB9B0; &#xB18D;&#xB3C4; &#xBC0F; &#xC99D;&#xAC00; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. Galphaz null &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xC99D;&#xAC00; &#xD50C;&#xB77C;&#xC988;&#xB9C8; &#xC778;&#xC290;&#xB9B0;&#xC740; &#xB300;&#xBD80;&#xBD84; &#xD5A5;&#xC0C1; &#xB41C; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44;&#xC758; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xACB0;&#xACFC;, Galphaz null &#xC950; &#xC804;&#xC2DC; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xB9C8;&#xC6B0;&#xC2A4;&#xC758; &#xAC83; &#xBCF4;&#xB2E4; &#xD6E8;&#xC52C; &#xB354; &#xB192;&#xC740; &#xD3EC;&#xB3C4; &#xB2F9; &#xC790;&#xADF9; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44; &#xCDCC; &#xC7A5; &#xB3C5;&#xB3C4;&#xC5D0;&#xC11C; &#xBD84;&#xB9AC; &#xB41C; &#xC774;&#xD6C4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, Galphaz null &#xB3C5;&#xB3C4;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xC99D;&#xAC00; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44;&#xB294; adenylyl cyclase &#xD1A0; &#xB2C9; &#xC5B5;&#xC81C; &#xC644;&#xD654; &#xD588;&#xAE30; &#xB54C;&#xBB38;&#xC77C; exogenous &#xC790;&#xADF9;&#xC758; &#xBD80;&#xC7AC;&#xC5D0;&#xC11C; Galphaz null &#xB3C5;&#xB3C4; &#xCEA0;&#xD504; &#xC0DD;&#xC0B0; &#xC99D;&#xAC00; &#xD06C;&#xAC8C;&#xB294; &#xB098;&#xD0C0;&#xB0A9;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; Galphaz &#xD0C0;&#xC785;-2 &#xB2F9;&#xB1E8;&#xBCD1;&#xC5D0;&#xC11C; &#xBCA0;&#xD0C0; &#xC138;&#xD3EC; &#xAE30;&#xB2A5; &#xC7A5;&#xC560; &#xC7A5;&#xD669;&#xC744; &#xACA8;&#xB0E5; &#xD55C; &#xCE58;&#xB8CC; &#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC0C8; &#xB300;&#xC0C1; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

Galphaz&#xB294; &#xBD80;&#xC815;&#xC801;&#xC778; &#xC778;&#xC290;&#xB9B0; &#xBD84; &#xBE44;&#xC640; &#xD608;&#xB2F9; &#xD074;&#xB9AC;&#xC5B4;&#xB7F0;&#xC2A4; &#xC870;&#xC808;.

Relativamente poco se sabe acerca de las funciones en vivo de la subunidad alfa de la prote&iacute;na heterotrim&eacute;ricas G Gz (Galphaz). Pistas para una funci&oacute;n potencial surgieron recientemente con el hallazgo de que la activaci&oacute;n de Galphaz inhibe la secreci&oacute;n de insulina estimulada por glucosa en una l&iacute;nea celular de insulinoma (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., campos, T. A. y Casey, J. P. J. (2005) Biol Chem. 280, 31708-31713). Para extender este estudio in vivo, un modelo con ratones knock-out Galphaz fue utilizado para determinar si la funci&oacute;n de Galphaz desempe&ntilde;a un papel en la inhibici&oacute;n de la secreci&oacute;n de insulina. Diferencias fueron descubiertas en la morfolog&iacute;a gruesa de los islotes pancre&aacute;ticos o en el contenido de ADN, prote&iacute;nas o insulina islote entre ratones Galphaz nulo y salvaje. Tambi&eacute;n hubo diferencias entre la sensibilidad a la insulina de ratones Galphaz nulo y los controles de tipo salvaje, seg&uacute;n lo medido por pruebas de tolerancia de insulina. Ratones Galphaz nulo, sin embargo, mostr&oacute; las concentraciones plasm&aacute;ticas de aumento de la insulina y un aumento correspondiente en la remoci&oacute;n de glucosa despu&eacute;s de desaf&iacute;o de glucosa intraperitoneal y oral en comparaci&oacute;n con controles de tipo salvaje. La insulina del plasma creciente observada en ratones Galphaz nulo probablemente es consecuencia directa de la secreci&oacute;n de insulina mayor, ya que los islotes pancre&aacute;ticos aislaron de ratones Galphaz nulo exhibieron la secreci&oacute;n de insulina estimulada por glucosa significativamente mayor que los de los ratones de tipo salvaje. Finalmente, la secreci&oacute;n de insulina aumento observada en islotes de Galphaz-null parece ser debido a la relevaci&oacute;n de una inhibici&oacute;n t&oacute;nica de cyclase del adenylyl, como campo producci&oacute;n aument&oacute; significativamente en islotes de Galphaz-null en la ausencia de estimulaci&oacute;n ex&oacute;gena. Estos resultados indican que el Galphaz puede ser un potencial nuevo objetivo para terapias tendientes a mejorar la disfunci&oacute;n de la c&eacute;lula beta en la diabetes tipo 2.

Galphaz regula negativamente la separaci&oacute;n de glucosa y la secreci&oacute;n de insulina.

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism.

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of beta-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in beta-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K(+) (K(ATP)) channels and activate voltage-gated Ca(2+) channels, leading to stimulation of insulin granule exocytosis. Whereas this K(ATP) channel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet beta-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and alpha-ketoglutarate, in control of GSIS.

Metabolic cycling in control of glucose-stimulated insulin secretion.

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

The mitochondrial 2-oxoglutarate carrier is part of a metabolic pathway that mediates glucose- and glutamine-stimulated insulin secretion.

Gemini surfactants have demonstrated significant potential for use in constructing non-viral transfection vectors for the delivery of genes into cells to induce protein expression. Previously, two asymmetric gemini surfactants containing pyrenyl groups in one of the alkyl tails of the surfactants were synthesized as fluorescence probes for use in mechanistic studies of the transfection process. Here we present the results of a thermodynamic investigation of the binding interaction(s) between the pyrenyl-modified surfactants and DNA. The thermodynamics of the interactions have been examined using isothermal titration calorimetry, light scattering, zeta potential, and circular dichroism measurements. Distinct differences are observed between the interaction of 12-s-12 vs. the pyrene modified py-s-12 surfactants with DNA; an intercalated binding is found for the py-s-12 surfactants that disrupts the typical interactions observed between DNA and gemini surfactants.

Thermodynamic investigation of the binding of dissymmetric pyrenyl-gemini surfactants to DNA.

The metabolic pathways that are involved in regulating insulin secretion from pancreatic β-cells are still incompletely understood. One potential regulator of the metabolic phenotype of β-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1β. ARNT/HIF-1β levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in β-cells that lack ARNT/HIF-1β. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1β inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1β knockdown. We demonstrate that β-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of β-cells treated with siRNAs against the ARNT/HIF-1β gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1β knockdown. Together, our data suggest an important role for ARNT/HIF-1β in anaplerosis, and it may play a critical role in maintaining normal secretion competence of β-cells.

Aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor-1{beta} plays a critical role in maintaining glucose-stimulated anaplerosis and insulin release from pancreatic {beta}-cells.

The ability of the pancreatic β-cells to adapt the rate of insulin release in accordance to changes in circulating glucose levels is essential for glucose homeostasis. Two spatial barriers imposed by the plasma membrane and inner mitochondrial membrane need to be overcome in order to achieve stringent coupling between the different steps in the stimulus-secretion cascade.   The first spatial barrier is overcome by the presence of a glucose transporter (GLUT) in the plasma membrane, whereas a low affinity hexokinase IV (glucokinase, GK) in the cytosol conveys glucose availability into a metabolic flux that triggers and accelerates insulin release. The mitochondrial inner membrane comprises a second spatial barrier that compartmentalizes glucose metabolism into glycolysis (cytosol) and tricarboxylate (TCA) cycle (mitochondrial matrix). The exchange of metabolites between cytosol and mitochondrial matrix is mediated via a set of mitochondrial carriers, including the aspartate-glutamate carrier (aralar1), α- ketoglutarate carrier (OGC), ATP/ADP carrier (AAC), glutamate carrier (GC1), dicarboxylate carrier (DIC) and citrate/isocitrate carrier (CIC). The scope of this review is to provide an overview of the role these carriers play in stimulus-secretion coupling and discuss the importance of these findings in the context of the exquisite glucose responsive state of the pancreatic β-cell.

Overcoming the spatial barriers of the stimulus secretion cascade in pancreatic β-cells.

Defining the key metabolic pathways that are important for fuel-regulated insulin secretion is critical to providing a complete picture of how nutrients regulate insulin secretion. We have performed a detailed metabolomics study of the clonal β-cell line 832/13 using a gas chromatography-mass spectrometer (GC-MS) to investigate potential coupling factors that link metabolic pathways to insulin secretion. Mid-polar and polar metabolites, extracted from the 832/13 β-cells, were derivatized and then run on a GC/MS to identify and quantify metabolite concentrations. Three hundred fifty-five out of 527 chromatographic peaks could be identified as metabolites by our metabolomic platform. These identified metabolites allowed us to perform a systematic analysis of key pathways involved in glucose-stimulated insulin secretion (GSIS). Of these metabolites, 41 were consistently identified as biomarker for GSIS by orthogonal partial least-squares (OPLS). Most of the identified metabolites are from common metabolic pathways including glycolytic, sorbitol-aldose reductase pathway, pentose phosphate pathway, and the TCA cycle suggesting these pathways play an important role in GSIS. Lipids and related products were also shown to contribute to the clustering of high glucose sample groups. Amino acids lysine, tyrosine, alanine and serine were upregulated by glucose whereas aspartic acid was downregulated by glucose suggesting these amino acids might play a key role in GSIS. In summary, a coordinated signaling cascade elicited by glucose metabolism in pancreatic β-cells is revealed by our metabolomics platform providing a new conceptual framework for future research and/or drug discovery.

Metabolomic analysis of pancreatic β-cell insulin release in response to glucose.

Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.

Mitochondrial glutathione transport is a key determinant of neuronal susceptibility to oxidative and nitrosative stress.

Our understanding of adult human β-cells is advancing, but we know little about the function and plasticity of β-cells from infants. We therefore characterized islets and single islet cells from human infants after isolation and culture. Although islet morphology in pancreas biopsies was similar to that in adults, infant islets after isolation and 24-48 hours of culture had less insulin staining, content, and secretion. The cultured infant islets expressed pancreatic and duodenal homeobox 1 and several (Glut1, Cav1.3, Kir6.2) but not all (syntaxin 1A and synaptosomal-associated protein 25) markers of functional islets, suggesting a loss of secretory phenotype in culture. The activity of key ion channels was maintained in isolated infant β-cells, whereas exocytosis was much lower than in adults. We examined whether a functional exocytotic phenotype could be reestablished under conditions thought to promote β-cell differentiation. After a 24- to 28-day expansion and maturation protocol, we found preservation of endocrine markers and hormone expression, an increased proportion of insulin-positive cells, elevated expression of syntaxin 1A and synaptosomal-associated protein 25, and restoration of exocytosis to levels comparable with that in adult β-cells. Thus, human infant islets are prone to loss of their exocytotic phenotype in culture but amenable to experimental approaches aimed at promoting expansion and functional maturation. Control of exocytotic protein expression may be an important mechanism underlying the plasticity of the secretory machinery, an increased understanding of which may lead to improved regenerative approaches to treat diabetes.

Functional plasticity of the human infant β-cell exocytotic phenotype.

Biphasic glucose-stimulated insulin secretion involves a rapid first phase followed by a prolonged second phase of insulin secretion. The biochemical pathways that control these 2 phases of insulin secretion are poorly defined. In this study, we used a gas chromatography mass spectroscopy-based metabolomics approach to perform a global analysis of cellular metabolism during biphasic insulin secretion. A time course metabolomic analysis of the clonal β-cell line 832/13 cells showed that glycolytic, tricarboxylic acid, pentose phosphate pathway, and several amino acids were strongly correlated to biphasic insulin secretion. Interestingly, first-phase insulin secretion was negatively associated with L-valine, trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, DL-3-aminoisobutyric acid, L-glutamine, sarcosine, L-lysine, and thymine and positively with L-glutamic acid, flavin adenine dinucleotide, caprylic acid, uridine 5'-monophosphate, phosphoglycerate, myristic acid, capric acid, oleic acid, linoleic acid, and palmitoleic acid. Tricarboxylic acid cycle intermediates pyruvate, α-ketoglutarate, and succinate were positively associated with second-phase insulin secretion. Other metabolites such as myo-inositol, cholesterol, DL-3-aminobutyric acid, and L-norleucine were negatively associated metabolites with the second-phase of insulin secretion. These studies provide a detailed analysis of key metabolites that are either negatively or positively associated with biphasic insulin secretion. The insights provided by these data set create a framework for planning future studies in the assessment of the metabolic regulation of biphasic insulin secretion.

Assessment of the metabolic pathways associated with glucose-stimulated biphasic insulin secretion.

It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

Article	Year
A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion. The Journal of biological chemistry\| PubMed ID: 16912049	2006
The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion. The Journal of biological chemistry\| PubMed ID: 17001083	2006
Normal flux through ATP-citrate lyase or fatty acid synthase is not required for glucose-stimulated insulin secretion. The Journal of biological chemistry\| PubMed ID: 17823126	2007
Galphaz negatively regulates insulin secretion and glucose clearance. The Journal of biological chemistry\| PubMed ID: 18096703	2008
Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism. The Journal of biological chemistry\| PubMed ID: 18381287	2008
Metabolic cycling in control of glucose-stimulated insulin secretion. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 18728221	2008
The mitochondrial 2-oxoglutarate carrier is part of a metabolic pathway that mediates glucose- and glutamine-stimulated insulin secretion. The Journal of biological chemistry\| PubMed ID: 20356834	2010
Thermodynamic investigation of the binding of dissymmetric pyrenyl-gemini surfactants to DNA. Physical chemistry chemical physics : PCCP\| PubMed ID: 20428564	2010
Aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor-1{beta} plays a critical role in maintaining glucose-stimulated anaplerosis and insulin release from pancreatic {beta}... The Journal of biological chemistry\| PubMed ID: 21059654	2011
Overcoming the spatial barriers of the stimulus secretion cascade in pancreatic β-cells. Islets\| PubMed ID: 22143007	2012
Metabolomic analysis of pancreatic β-cell insulin release in response to glucose. Islets\| PubMed ID: 22847496	2012
Mitochondrial glutathione transport is a key determinant of neuronal susceptibility to oxidative and nitrosative stress. The Journal of biological chemistry\| PubMed ID: 23283974	2013
Functional plasticity of the human infant β-cell exocytotic phenotype. Endocrinology\| PubMed ID: 23449893	2013
Assessment of the metabolic pathways associated with glucose-stimulated biphasic insulin secretion. Endocrinology\| PubMed ID: 24564396	2014
Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion. The Journal of biological chemistry\| PubMed ID: 24675076	2014

Jamie W. Joseph

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