Culture at low glucose up-regulates mitochondrial function in pancreatic β cells with accompanying effects on viability

Abstract

We tested whether exposure of β cells at reduced glucose leads to mitochondrial adaptions and whether such adaptions modulate effects of hypoxia. Rat islets, human islets and INS-1 832/13 cells were pre-cultured short term at half standard glucose concentrations (5.5 mM for rat islets and cells, 2.75 mM for human islets) without overtly negative effects on subsequently measured function (insulin secretion and cellular insulin contents) or on viability. Culture at half standard glucose upregulated complex I and tended to upregulate complex II in islets and INS-1 cells alike. An increased release of lactate dehydrogenase that followed exposure to hypoxia was attenuated in rat islets which had been pre-cultured at half standard glucose. In INS-1 cells exposure to half standard glucose attenuated hypoxia-induced effects on several viability parameters (MTT, cell number and incremental apoptotic DNA). Thus culture at reduced glucose of pancreatic islets and clonal β cells leads to mitochondrial adaptions which possibly lessen the negative impact of hypoxia on β cell viability. These findings appear relevant in the search for optimization of pre-transplant conditions in a clinical setting.

Keywords: INS-1; hypoxia; insulin secretion; low glucose; mitochondrial complexes; oxygen consumption; rat and human islets; viability.

Figure 1.

General study design. Standard glucose concentrations in culture media were for rat islets and INS-1 832/13 cells 11 mM (half standard: 5.5 mM) and for human islets 5.5 mM (half standard: 2.75 mM). The media for rat and human islets were renewed to contain standard glucose concentrations before exposure to hypoxia. For INS-1 832/13 cells the media were renewed without changing the glucose concentrations before exposure to hypoxia. Oxygen levels during hypoxia were 0.8 or 2.8% for rat islets and 0.8% for human islets while INS-1 832/13 cells were exposed to 0.3–0.5% oxygen. The media employed in the re-oxygenation period always contained standard glucose concentrations for both islets and cells. (Not indicated are selected experiments in which measurements were made also immediately after hypoxia).

Figure 2.

Culture at half standard glucose increases levels of mitochondrial complex proteins. Levels of protein subunits of complexes I and II were measured by Western blotting. (A) shows protein levels after culture at half standard (1/2 Std) vs. standard (Std) glucose (G) (for rat islets and cells 1/2 Std = 5.5 mM G and for human islets 1/2 Std = 2.75 mM G). Data are mean ± SEM of 8 (rat islets), 3 (human islets) and 9 (INS-1 832/13 cells) experiments, **p < 0.002 and *p < 0.03 vs. protein levels after culture at standard glucose. (B) Representative Western blots are shown of levels of complex I and complex II at standard (Std) and half standard (1/2 Std) glucose (G). Band images from human islet experiments were from separate parts of the same gel.

Figure 3.

Effects of hypoxia followed by re-oxygenation on insulin secretion after pre-culture at different glucose (G) concentrations. Levels of insulin release after culture at half standard glucose are indicated by red bars and standard glucose by blue bars. (A) Insulin release from rat islets at 3.3 and 16.7 mM G, ^#p < 0.02 for the effect of hypoxia, n = 4. (B) Insulin release from human islets at 1.6 and 16.7 mM G, n = 4 (one or 3 experiments per donor, 4 donors). (C) Insulin release from INS-1 832/13 cells at 3.3, 11 and 27 mM glucose, *p < 0.05 for the effect of culture at half standard G, ^#p < 0.05 for the effect of hypoxia, n = 5. Effects of hypoxia at standard G were previously reported. All data are mean ± SEM.

Figure 4.

Effects of hypoxia on viability after pre-culture at different glucose (G) concentrations. Blue bars denote pre-culture at standard (std) glucose (for rat islets and cells: 11 mM, for human islets: 5.5 mM), red bars at half standard glucose. (A) Rat islets were exposed to 0.8% oxygen during hypoxia. There was no significant effects due to glucose concentrations on MTT signals, n = 11. (B) Human islets, n = 2 (2 experiments per donor, 2 donors). (C and D) INS-1 832/13 cells. Cell number is depicted in (C) and MTT signals in (D), after 18 h of hypoxia. Attenuating effects on hypoxia-reoxygenation by half vs. standard glucose were significant *p < 0.03 (C) and **p < 0.002 (D), n = 4-5.

Figure 5.

Half standard glucose attenuates hypoxia-induced release of lactate dehydrogenase (LDH). Hypoxia (2.8% oxygen for 5.5 h followed by re-oxygenation) increased the release of LDH from rat islets cultured continuously at 11 mM glucose (blue patterned bar). The release was attenuated in islets pre-cultured at 5.5 mM glucose (red patterned bar, *p < 0.03 for difference). Mean ± SEM of 8 experiments.

Figure 6.

Effects of hypoxia on oxygen consumption in INS-1 832/13 cells after culture at 5.5 or 11 mM glucose (G). Blue bars represent culture at standard G (11 mM), red bars represent culture at half standard G (5.5 mM). Patterned bars indicate exposure to 8 h of hypoxia followed by re-oxygenation. (A) Respiration at basal conditions, followed by conditions of ATP synthase inhibition by oligomycin and finally a non-coupled state achieved by FCCP. (B) Uncoupled (oligomycin) to FCCP-induced (FCCP) respiration ratio. ^#p < 0.05 or less for effects of hypoxia-reoxygenation. Data are mean ± SEM of 5 separate experiments, each consisting of 2-4 parallel measurements. Effects with culture at 11 mM glucose throughout were previously reported.