Time dynamics of elevated glucose and beta-hydroxybutyrate on beta cell mitochondrial metabolism

Abstract

Chronic hyperglycemia impairs mitochondrial function of beta cells. Changes in mitochondrial function preceding a negative glucose effect have not been fully characterized, nor interactions with ketones. To compare effects on beta cell mitochondrial function by short and longer exposures to elevated glucose and interactions with ketones oxygen consumption rate (OCR) was measured in intact clonal beta cells by an OROBOROS and in rat islets by a Seahorse instrument. Proteins (subunits) of mitochondrial complexes (C) were measured by immunoblotting. ATP and ROS were measured in islets. In INS-1 832/13 cells, overnight exposure to 27 vs. 11 mm glucose increased OCR and uncoupled mitochondrial respiration. These effects vanished when prolonging the exposure time of elevated glucose. C1 was decreased after two days of culture with high glucose. Interactions with racemic 5 and 20 mm beta-hydroxybutyrate (BHB) were not detected. In islets, culture overnight at 27 vs.11 mm glucose enhanced basal OCR. No decrease in glucose-induced OCR was seen after prolonging 27 mm glucose for two days. Interactions with 5 mm BHB were not detected. Prolonged exposure to 27 mm glucose enhanced basal ECAR (extracellular acidification rate) and an ECAR response to acute elevation of glucose. C1 and 3 and 4 were decreased after two days of 27 vs. 11 mm glucose. ATP levels were decreased at this time-point and extracellular ROS increased. High glucose time-dependently affects mitochondrial function in clonal beta cells and islets. C1 was uniformly decreased. Interactions with BHB were not detected.

Keywords: Beta-hydroxybutyrate; INS-1 832/13 cells; elevated glucose; mitochondrial metabolism; oxygen consumption rate; rat pancreatic islets.

Graphical abstract

Figure 1.

Effects of glucose on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in rat islets. The figure shows results from measurements performed after overnight (22 h) (A, B, E and F) or two days (46 h) (C, D, G and H) of culture at 11- or 27- mM glucose. Islets of four rats were – separately for each rat – placed in parallel wells and processed in the same experiment. Measurements over time of OCR are depicted in A-B and of ECAR in E-F after overnight culture in 11- and 27-mM glucose, and for OCR in C-D and for ECAR in G-H after culture for two days. Integrated responses for basal (3.3 mm glucose) and glucose-stimulated (16.7 mm glucose) OCR are shown in C and for ECAR in D following culture overnight in 11- and 27-mM glucose, and in I and J after culture for two days. FCCP-stimulated (maximum) values of OCR are shown in E and K. Residual total OCR after oligomycin is calculated as lowest recorded value after oligomycin in % of FCCP-stimulated OCR (highest recorded value) and is shown in F (for overnight culture) and L (for two days culture). All data are mean ± SEM, n = 4. *p < .05 for differences as indicated in the figure.

Figure 2.

High glucose culture decreases intra-islet ATP and increases ROS in islet culture media. Electron paramagnetic resonance (EPR) was used for the detection of ROS. Results are expressed as arbitrary units (A.U.). Data on ATP and ROS are expressed as mean ± SEM of eight and four separate experiments respectively. *p < 0.05 for differences vs. levels at 11 mm glucose.