Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

Abstract

ObjectiveBeta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.Methods and DesignRat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.ResultsInsulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.Conclusions1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia

“…The increase in maximum ETS capacity in intact INS-1832/13 cells indicates adaptation to hypoxia by increasing levels and activities of mitochondrial complexes. These observations differ from the global depression of respiration in intact rat islets, previously reported [ 18 ]. However, adaptation (up-regulation of mitochondrial complexes) could be demonstrated in rat islets by employing here a lesser degree of hypoxia than in previous experiments ( Fig 6 ) in a setting in which glucose-induced insulin secretion was not affected.…”

“…Rat islets were exposed for 5.5 hours to hypoxia (rather than 18 hours as for INS-1 cells). Due to previous observations indicating increased susceptibility to damage by hypoxia [ 18 ], the oxygen concentration was kept at 2.8% rather than at < 1%. Human islets were also exposed to 5.5 hours of hypoxia.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…In the generic cell, the acceleration of glycolysis in response to a lowered ⌬G p is generally sufficient to maintain ATP levels for the duration of the experiment. In the case of the ␤-cell, with its restriction on anaerobic glycolysis due to the low levels of lactic dehydrogenase and/or the monocarboxylate transporter, such compensation cannot occur, with the result that ATP depletion may limit the respiration achieved in the presence of the protonophore (184,306,531). To overcome this limitation, a separate experiment, involving the addition of oligomycin together with FCCP, allows maximal respiration to be monitored prior to ATP depletion.…”

Encapsulated Islet Transplantation: Where Do We Stand?

“…Unfortunately, these oxygen-generating systems cannot produce enough oxygen required in a clinical setting. Other approaches to enhance oxygenation at the subcuta-neous site include delivering oxygen through an external source [159], making the islet cells hypoxic resistant [160][161][162], and inducing neoangiogenesis around the transplanted device. Several groups have demonstrated the benefit of inducing neovascularization using fibroblast growth factor (FGF) [163][164][165] and hemoglobin cross-linking [166] with improved transplant outcomes in rodents.…”