Abstract
Purpose :
Müller glial cells (MGCs) provide critical metabolic support to the retina, and MGC dysfunction contributes to the pathogenesis of diabetic retinopathy (DR). Our previous studies in retinal endothelial cells and in diabetic mice have shown that interleukin-6 (IL-6) trans-signaling (TS) plays a role in metabolic changes associated with DR. The purpose of this study was to assess the contribution of hyperglycemia to IL-6 trans-signaling mediated metabolic dysregulation in MGCs.
Methods :
Primary mouse MGCs were exposed to normal glucose (5.5 mM, NG) or high glucose (30 mM, HG) overnight with or without IL-6 TS activation using IL-6 (50 ng/mL) and sIL-6R (150 ng/mL). Mitochondrial metabolic function was assessed by measuring oxygen consumption rate (OCR) using a Seahorse bioenergetic analyzer. Mitochondrial superoxide and membrane potential were examined by live-cell staining for MitoSOX and JC-1 dye, respectively. Apoptosis was determined using RT-PCR, western blot analysis, and TUNEL assay. Fluorescent images were captured using a confocal microscope and quantified with ImageJ software. Statistical significance (p<0.05) was determined by 2-way ANOVA with Tukey’s multiple comparisons.
Results :
Under normal glucose, TS increased spare respiratory capacity (74.77 pmol/min vs. 48.95 pmol/min) and maximal respiration (131.37 pmol/min vs. 101.31 pmol/min) relative to untreated (UT), and TS+HG significantly reduced this effect. Regardless of glucose concentration, IL-6 TS activation increased mitochondrial superoxide generation (TS: 1.32-fold; TS+HG: 1.58-fold) and increased pro-apoptotic voltage-dependent anion channel 1 (VDAC1) levels (TS: 1.31-fold; TS+HG: 1.42-fold). Anti-apoptotic regulator Bcl2 was also decreased by TS under both glucose conditions (TS: 0.52-fold; TS+HG: 0.71-fold), however anti-apoptotic Mcl1 showed a glucose-dependent effect (0.62-fold vs. UT+HG). Mitochondrial depolarization (5.36-fold vs. UT+HG) and cleaved caspase-3 levels (1.81-fold vs. UT+HG) were also significantly upregulated in the TS+HG group, and apoptosis induction followed a similar trend as confirmed by TUNEL assay (3.07-fold vs. UT+HG).
Conclusions :
In this study, we demonstrate that IL-6 trans-signaling independently affects MGC mitochondrial function, whereas many of the pro-apoptotic functions of trans-signaling occur only under hyperglycemic conditions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.