Hyperglycemia-mediated onset of myocardial insulin resistance – unraveling molecular mechanisms and identifying therapeutic targets

Joseph, Danzil Eugene (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Background - Although acute hyperglycemic episodes are linked to lower glucose uptake, underlying mechanisms driving this process remain unclear. We hypothesized that acute hyperglycemia triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP) and protein kinase C (PKC) activation. These mechanisms attenuate cellular function, and may indeed decrease insulin-mediated cardiac glucose uptake. The role of the pentose phosphate pathway (PPP) under high glucose/diabetic conditions is a subject of contention. Activation of the PPP enzyme transketolase (TK) (by benfotiamine/BFT or thiamine) reduces flux via the other four NOGPs, and is associated with beneficial outcomes. Our aim was therefore to evaluate the effects of acute hyperglycemia on insulin-mediated glucose uptake in a cardiac-derived cell line. Specifically, we aimed to elucidate the role of ROS and NOGP induction under these conditions. Methodology - H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24 hr vs. 5.5 mM glucose controls ± modulating agents during last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250 μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100 μM DPI), c) NOGP inhibitors – 100 μM aminoguanidine (AGE), 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). We also employed BFT (50 and 100 μM) in vitro, while the effects of in vivo thiamine administration were assessed in hearts of an obese/diabetic rat model of pre-diabetes and diabetes, the OLETF strain. We evaluated insulin sensitivity by glucose uptake assay (flow cytometry), GLUT4 translocation (transfection of HA-GLUT4-GFP construct) and protein kinase B (Akt) activity assay. ROS levels (mitochondrial, intracellular) were measured by flow cytometry analysis of specific fluorescent probes. Markers of each NOGP were also assessed. Results - Acute hyperglycemia elevated ROS, activated NOGPs and blunted glucose uptake. However, TK activity (marker of PPP) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed BFT (known to shunt flux away from NOGPs and into the PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under acute hyperglycemic conditions. In vivo thiamine administration reduced markers of the other NOGP, while it attenuated (mainly in the pre-diabetic phase) the metabolic dysfunction observed in the OLETF rats. Conclusions - This study demonstrates that acute hyperglycemia elicits a series of maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.

AFRIKAANSE OPSOMMING: Agtergrond – Alhoewel akute hiperglisemie voorvalle gekoppel is aan verlaagde glukose opname, is die onderliggende meganismes wat die proses dryf steeds onduidelik. Ons hipotetiseer dat akute hiperglisemie aanleiding gee tot die produksie van reaktiewe suurstofspesies (RSS) en toename in nie-oksidatiewe glukose weg (NOGW) aktivering, i.e. stimulering van gevorderde glukasie eindprodukte (GGE), poliolweg (PW), heksosamien biosintetiese weg (HBW) en proteïenkinase C (PKC) aktivering. Hierdie meganismes verminder sellulêre funksie, en mag inderdaad insulien-bemiddelde kardiêre glukose opname verlaag. Die rol van die pentosefosfaatweg (PFW) onder hoë glukose/diabetiese kondisies is ‘n onderwerp van stryd. Aktivering van die PFW ensiem transketolase (TK) (deur benfotiamien/BFT of tiamien) verminder fluks deur die ander vier NOGWë, en is geassosieer met voordelige uitkomste. Ons doel was dus om die effekte van akute hiperglisemie op insulien-bemiddelde glukose opname te evalueer in ‘n kardiaal-afkomstige sellyn. Meer bepaald het ons gepoog om die rol van RSS en NOGW induksie onder hierdie kondisies te verstaan. Metode – H9c2 kardiomioblaste is aan 25 mM glukose vir 24 h blootgestel vs. 5.5 mM glukose kontroles ± moduleeragente tydens die laaste uur van glukose blootstelling: a) anti-oksidant #1 vir mitochondriese RSS (250 μM 4-OHCA), b) anti-oksidant #2 vir NADPH oksidase-gegenereerde RSS (100 μM DPI), c) NOGW inhibeerders – 100 μM aminoguanidien (GGE), 5 μM cheleritrien (PKC); 40 μM DON (HBW); en 10 μM zopolrestaat (PW). Ons het ook BFT (50 en 100 μM) in vitro aangewend, terwyl die effek van in vivo tiamien aanwending geassesseer is in die harte van ‘n vetsugtige/diabetiese rotmodel van pre-diabetes en diabetes, die OLETF lyn. Ons het insuliensensitiwiteit deur ‘n glukose opname toets (vloeisitometrie), GLUT4 translokasie (transfeksie van HA-GLUT4-GFP konstruk) en proteïenkinase B (Akt) aktiwiteitstoets, geëvalueer. RSS vlakke (mitochondries, intrasellulêr) is gemeet deur vloeisitometriese analise van spesifieke fluoresserende peilers. Merkers van elke NOGW is ook geassesseer. Resultate - Akute hiperglisemie het RSS verhoog, NOGWë geaktiveer en glukose opname versag. TK aktiwiteit (merker van PFW) het egter nie verander nie. Onderskeidelike 4-OHCA en DPI behandeling het RSS produksie versag, NOGW aktivering verminder en glukose opname genormaliseer. NOGW onderdrukking studies het PKCβII geïdentifiseer as ‘n sleutel deelnemer in verlaging van insulien-bemiddelde glukose opname. Die aanwending van BFT (bekend vir die wegvoer van fluks vanaf NOGWë na die PFW), het RSS skepping en NOGW aktivering verlaag, en glukose opname herwin onder akute hiperglisemiese kondisies. In vivo tiamien toediening het merkers van die ander NOGW verlaag, terwyl dit die metaboliese disfunksie waargeneem in die OLETF rotte (hoofsaaklik in die pre-diabetiese fase) verminder het. Gevolgtrekking – Hierdie studie demonstreer dat akute hiperglisemie ‘n reeks van wanaangepaste voorvalle ontlok wat gesamentlik funksioneer om glukose opname te verlaag, en dat anti-oksidant behandeling en/of vermindering van NOGW aktivering (PKC, poliolweg), die aanvang van insulien weerstand mag beperk.

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