Islet Biology/Insulin Secretion
Both type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are caused by islet dysfunction and eventual β-cell failure. In T2DM pathogenesis, β-cell dysfunction takes years to progress, providing an opportunity for intervention prior to the development of frank hyperglycemia. However, current biomarkers of diabetes, such as C-peptide and HbA1c, only provide a measure of β-cell function late in the disease process. New biomarkers that are evident earlier in the disease process are needed to
... e preemptive preventative strategies. Our previous studies have shown that the β-cell failure underlying T1DM can be monitored by measuring demethylated β-cell insulin (deMeth-β) DNA that is released into the circulation; e.g., we have reported serum deMeth-β DNA as a biomarker of β-cell loss in murine T1D models and in T1D patients. In this study, we adapted our assay for the rhesus macaque. We utilized our well-characterized non-human primate (NHP) model of diet-induced obesity (DIO) to determine deMeth-β DNA's utility as an early T2DM biomarker. Three adult male NHPs (10-13 yrs old) were fed a high-fat, high-fructose diet (HFFD; 35% of calories from fat) and demonstrated signifi cant weight gain and the gradual onset of insulin resistance. Pre-diabetes was determined by an increase in insulin secretion during a glucose tolerance test (GTT), indicative of early β-cell dysfunction. Serum analysis from these NHPs found that, compared to levels prior to the start of the HFFD, deMeth-β DNA levels more than doubled with DIO. This increase in deMeth-β DNA coincided with increased insulin secretion during the GTT. These data suggest that an increase in circulating deMeth-β DNA levels may serve as a biomarker of early β-cell dysfunction in pre-T2DM. Beta cells are highly susceptible to reactive oxygen species (ROS) which are elevated by pro-infl ammatory cytokines (PICs) along with other diabetogenic stimuli. NADPH oxidase-1 (NOX1) is an enzyme that generates ROS. NOX1 is expressed in beta cells and is upregulated following exposure to PICs. We have also shown that NOX1 is upregulated by 12-lipoxygenase (12-LO) activity POSTERS ISLET BIOLOGY-APOPTOSIS in beta cells. 12-HETE (a product of 12-LO activity) upregulates NOX1 13±3fold (p<0.01) and a selective inhibitor of 12-LO blocks PIC-stimulated NOX1 80±5% (p<0.01) in human islets and INS1 beta cells. Inhibitors of NOX activity reduce cellular ROS and the expression of NOX1. This observation suggests a feed-forward regulation of NOX1 in beta cells involving ROS elevation. Antioxidants (GEE, BHA) inhibited PIC-induced NOX1 26±11%, 29±15% (p<0.05) and pro-oxidants (H 2 0 2 , pyocynin) directly elevated NOX1 expression 9.3±2.1, 2.8±0.3 fold (p<0.05) in beta cells. The redox-responsive kinase, Src-kinase, regulated NOX1. An inhibitor of Src-kinase, PP2 blocked NOX1 expression induced by PICs or H 2 0 2 in beta cells (52±8.5% and 48±5.4% resp, p<0.001). This was not seen with a structurally related but inactive compound PP3. Functionally, inhibition of NOX1 with a selective pyrazolopyridine dione, or inhibition of Src-kinase with PP2, preserved function and survival in beta cells exposed to PICs. Induction of apoptosis by PICs, measured by caspase-3 activation and Yo-Pro-1 fl uorescence microscopy was blocked by PP2, 95±2.5% (p<0.01). Uncoupling of glucose stimulated insulin secretion by PICs was preserved by PP2 and NOX1 inhibition. Collectively, these data indicate NOX1 upregulation is an important component in PIC-induced beta cell dysfunction. A feed-forward regulation of NOX1 and ROS production could rapidly result in beta cell pathology. Targeted inhibition of NOX1 feedback could open a new strategy to preserve and protect beta cell mass in the presence of elevated PICs.