Assessment of Proinflammatory Mediators of Beta-Cell Circadian Clock Dysfunction in Diabetes

Abstract

Both type 1 and type 2 diabetes are characterized by a decline in β-cell function and mass partly attributed to exposure of β-cells to proinflammatory cytokines. The molecular mechanisms underlying this process are not well understood, however several lines of evidence have linked the circadian clock with regulation of the deleterious proinflammatory effects in diabetes. Therefore, in this study, we assessed the effect of known proinflammatory mediators of β-cell failure in diabetes (IL-1β, TNFα, IL-6, and IFNγ) on the β-cell circadian clock. Through real-time bioluminescence tracking of islets from Per2:luciferase (a key circadian clock gene reporter) knock-in mice, our data revealed that IL-1β (2 ng/ml) diminished β-cell circadian rhythmicity via impairment of the amplitude and the phase of oscillations (p<0.vs. vehicle). This was in contrast to TNFα, IL-6, and IFNγ, which showed either mild suppression or no effect on the β-cell clock. We next assessed the ability of proinflammatory cytokines to modulate Bmal1 and Clock (two main transcription factors in the circadian pathway) gene and protein expression. Interestingly, studies using INS-1 832/13 cells and cadaveric human islets revealed that IL-1β addition (0.2-5 ng/ml) for 24 h significantly reduced Bmal1 (but not Clock) at both the mRNA (∼40-50%, p<0.05) and protein (10-65%, p<0.05) level in a dose dependent manner. In contrast, other cytokines (IL-6, TNFα, and IFNγ) only modestly diminished Bmal1 expression after 24 h. In assessing a molecular link between IL-1β and the β-cell clock, Bmal1 promoter activity was determined using a BMAL1:luciferase expressing INS-1 832/13 stable-transfected cell line. Importantly, IL-1β (0.01-5 ng/ml) showed a dose dependent decrease in Bmal1 promoter activation (up to 90%). This body of work provides the first novel connection between diabetogenic inflammatory cytokines, specifically IL-1β, as the dominant mediator of β-cell clock dysfunction in diabetes. Disclosure N. Javeed: None. K. Rakshit: None. A. Matveyenko: None.