Thesis (Ph.D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed on Feb. 22, 2007). Includes bibliographical references (p. 124-134).

Thèse doctorat : Biochimie : Villeurbanne, INSA : 2005. / En annexe, 3 articles de revue rédigés en anglais auxquels l'auteur a participé, contenant un résumé et des notes bibliogr. Titre provenant de l'écran-titre. Bibliogr. p. 175-196. Index des figures.

L'apoptose des cellules bêta pancréatiques est une cause du diabète de type 2. La glycosylation par la voie des hexosamines modifie l'activité de protéines régulatrices. La déacétylase Sirt1 est connue pour son rôle dans la survie cellulaire et pour sa sensibilité face au métabolisme glucidique. Cette étude visait à montrer que l'activation directe de la voie des hexosamines stimule la mort de ces cellules par l'implication de Sirt-1. Le traitement des cellules Nit-1 avec la glucosamine a causé une augmentation temporelle significative de l'apoptose, jumelée à l'expression de facteurs pro-apoptotiques, la réduction des niveaux protéiques de Sirt-1 ainsi que sa glycosylation. De plus, l'attténuation génétique de Sirt1 a accentué la vulnérabilité des cellules à l'apoptose. Ces résultats montrent pour la première fois que Sirt1 joue un rôle important dans la survie de la cellule bêta et que sa glycosylation serait impliquée dans la perturbation de son rôle protecteur.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: OBJECTIVE – Oxidative stress increases flux through the hexosamine biosynthetic pathway (HBP) resulting in greater O-GlcNAcylation of target proteins. Since increased oxidative stress and HBP flux are associated with insulin resistance, we hypothesized that its activation leads to greater O-GlcNAcylation of BAD (pro-apoptotic) and increased myocardial apoptosis. RESEARCH DESIGN AND METHODS – To investigate our hypothesis, we employed two experimental models: 1) H9c2 cardiomyoblasts exposed to high glucose (33 mM glucose) ± HBP modulators ± antioxidant treatment vs. matched controls (5.5 mM glucose); and 2) a rat model of high fat diet-induced insulin resistance and hyperglycemia. We evaluated apoptosis in vitro by Hoechst nuclear staining, Annexin-V staining, caspase activity measurements and immunoblotting while in vivo apoptosis was assessed by immunoblotting. In vitro reactive oxygen species (ROS) levels were quantified by H2DCFDA staining (fluorescence microscopy, flow cytometry). We determined overall and BAD O-GlcNAcylation, both by immunoblotting and immunofluorescence microscopy. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and immunofluorescence microscopy (co-localization) to determine BAD-cl-2 dimerization. In vivo overall O-GlcNAcylation, BAD O-GlcNAcylation and BAD-Bcl-2 dimerization was determined by immunoprecipitation and immunoblotting. 4 RESULTS – High glucose treatment of cells significantly increased the degree of apoptosis as revealed by Hoechst nuclear staining (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V staining (43 ± 5%), caspase activity assay (26 ± 2%) and immunoblotting. In parallel, overall OGlcNAcylation (p<0.001 vs. 5.5 mM), BAD O-GlcNAcylation (p<0.05 vs. 5.5 mM) and ROS levels were increased (fluorescence microscopy – p<0.05 vs. 5.5 mM; flow cytometry – p<0.001 vs. 5.5 mM). HBP inhibition using DON and antioxidant treatment (α-OHCA) attenuated these effects while HBP activation by PUGNAc exacerbated it. Likewise, insulin resistant rat hearts exhibited significantly higher caspase-3 (p<0.05 vs. controls), overall O-GlcNAcylation (p<0.05 vs. controls) and BAD O-GlcNAcylation levels (p<0.05 vs. 5.5 mM). BAD-Bcl-2 dimer formation was increased in cells exposed to hyperglycemia [immunoprecipitation analysis and co-localization] and in insulin resistant hearts. CONCLUSIONS - Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress, resulting in increased HBP activation and increased BAD OGlcNAcylation. We also found greater BAD-Bcl-2 dimerization increasing myocardial apoptosis, suggesting that this pathway may play a crucial role in the onset of the diabetic cardiomyopathy. / AFRIKAANSE OPSOMMING: DOELWIT – Oksidatiewe stres verhoog fluks deur die heksosamien biosintetiese weg (HBW) wat in „n groter O-GlcNAsetilering van teiken proteïene resulteer. Weens die feit dat verhoogde oksidatiewe stres en HBW fluks verband hou met insulienweerstandigheid, hipotetiseer ons dat die aktivering hiervan tot groter O-GlcNAsetilering van BAD (pro-aptoptoties) en verhoogde miokardiale apoptose lei. NAVORSINGS ONTWERP EN METODES – Om die hipotese te ondersoek het ons twee modelle ontplooi: 1) H9c2 kardiomioblaste is blootgestel aan hoë glukose konsentrasie (33mM glucose) ± HBW moduleerders ± antioksidant behandeling vs. gepaarde kontrole (5.5mM glucose); en 2) „n hoë vet dieetgeïnduseerde insulienweerstandige rotmodel en hiperglukemie. Ons het apoptose in vitro deur middel van Hoescht nukleuskleuring geëvalueer, kasapase aktiwiteit bepalings en immunoblotting terwyl apoptose in vivo getoets is deur immunoblotting. Reaktiewe suurstofspesie (RSS) vlakke is deur middel van H2DCFDA verkleuring (fluoresensie mikroskopie, vloeisitometrie) bepaal. Algehele en BAD O-GlcNAsetilering is beide deur immunoblotting en immunofluoresensie mikroskopie bepaal. BAD-Bcl-2 dimeervorming bevorder apoptose, om BAD-cl-2 dimerisasie te bepaal is daar van immunopresipitering analise en immunofluoresensie mikroskopie (ko-lokalisasie) gebruik gemaak. In vivo is algehele OGlcNAsetiliering, BAD O-GlcNAsetiliering en BAD-Bcl-2 dimerisasie deur immunopresipitasie en immunoblotting bepaal. 6 RESULTE – Hoë glukose behandeling van selle het die graad van apotpose betekenisvol verhoog soos blootgelê deur Hoechst nukleuskleuring (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V kleuring (43 ± 5%), kaspase aktiviteit assay (26 ± 2%) en immunoblotting. In parallel, algehele OGlcNAsetilering (p<0.001 vs. 5.5 mM), BAD O-GlcNAsetilering (p<0.05 vs. 5.5 mM) en RSS vlakke is verhoog (fluoresensie mikroskopie– p<0.05 vs. 5.5 mM; vloeisitometrie– p<0.001 vs. 5.5 mM). HBW inhibering deur van DON en van antioksidant behandeling gebruik te maak (α- OHCA) het hierdie effekte verlaag terwyl HBW aktivering deur PUGNAc dit verhoog het. Netso, het insulienweerstandige rotharte betekenisvolle hoë kaspase -3 (p<0.05 vs. kontrole), algeheel O-GlcNAsetilering (p<0.05 vs. kontrole) en BAD O-GlcNAsetiliering vlakke (p<0.05 vs. 5.5 mM) getoon. BAD-Bcl-2 dimeervorming is verhoog in hiperglukemies blootgestelde selle [immunopresipitering analise en ko-lokalisering] en in insulienweerstandige harte. GEVOLGTREKKINGS – Ons studie het „n nuwe weg geïdenifiseer waar hiperglukemie in groter oksidatiewe stres resulteer wat weer HBW aktivering verhoog en BAD O-GlcNAsetilering verhoog het. Ons het verder bevind dat groter BAD-Bcl-2 dimerisasie miokardiale apoptose verhoog wat voorstel dat hierdie weg „n belangrike rol in diabetiese kardiomiopatie speel.

Indiana University-Purdue University Indianapolis (IUPUI) / Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.