title: Insulin-induced recurrent hypoglycemia exacerbates diabetic brain mitochondrial dysfunction and oxidative imbalance.
abstract: Intensive insulin therapy can prevent or slow the progression of long-term diabetes complications but, at the same time, it increases the risk for episodes of severe hypoglycemia. In our study, we used a protocol intended to mimic the levels of blood glucose that occur in type 1 diabetic patients under an intensive insulin therapy. Streptozotocin (STZ)-induced diabetic rats were treated subcutaneously with twice-daily insulin injections for 2weeks to induce hypoglycemic episodes. Brain cortical and hippocampal mitochondria were isolated and mitochondrial bioenergetics (respiratory chain and phosphorylation system) and oxidative status parameters (malondialdehyde (MDA) levels, mitochondrial aconitase activity and enzymatic and non-enzymatic antioxidant defenses) were analyzed. The protein levels of synaptophysin, a marker of synaptic integrity, and caspase 9 activity were also evaluated in cortical and hippocampal homogenates. Brain cortical mitochondria isolated from hyper- and recurrent hypoglycemic animals presented higher levels of MDA and α-tocopherol together with an increased glutathione disulfide reductase activity, lower manganese superoxide dismutase (MnSOD) activity and glutathione-to-glutathione disulfide (GSH/GSSG) ratio. No significant alterations were found in cortical mitochondrial respiratory chain and oxidative phosphorylation system. Hippocampal mitochondria from both experimental groups presented an impaired oxidative phosphorylation system characterized by a decreased mitochondrial energization potential and ATP levels and higher repolarization lag phase. In addition, higher MDA levels and decreased GSH/GSSG, α-tocopherol levels, and aconitase, glutathione peroxidase and MnSOD activities were observed in both groups of animals. Hippocampal mitochondria from recurrent hypoglycemic animals also showed an impairment of the respiratory chain characterized by a lower state 3 of respiration, respiratory control ratio and ADP/O index, and a higher state 4 of respiration. Additionally, a non-statistically significant decrease in synaptophysin protein levels was observed in cortical homogenates from recurrent hypoglycemic rats as well as in hippocampal homogenates from hyperglycemic and recurrent hypoglycemic rats. An increase in caspase 9 activity was also observed in hippocampal homogenates from hyperglycemic and recurrent hypoglycemic animals. Our results show that mitochondrial dysfunction induced by long-term hyperglycemic effects is exacerbated by recurrent hypoglycemia, which may compromise the function and integrity of brain cells.
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Cardoso
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Renato X
authors.initials: RX
authors.lastname: Santos
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Sónia C
authors.initials: SC
authors.lastname: Correia
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Carvalho
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Santos
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Laboratory of Neurochemistry, Coimbra University Hospital, Portugal; Neurology Department, Faculty of Medicine, University of Coimbra, Portugal.
authors.forename: Inês
authors.initials: I
authors.lastname: Baldeiras
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Portugal.
authors.forename: Catarina R
authors.initials: CR
authors.lastname: Oliveira
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Institute of Physiology, Faculty of Medicine, University of Coimbra, 3000-354 Coimbra, Portugal. Electronic address: venta@ci.uc.pt.
authors.forename: Paula I
authors.initials: PI
authors.lastname: Moreira
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D007003:Hypoglycemia / Q000139:chemically induced / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000008:administration & dosage / Q000009:adverse effects*; D007328:Insulin / Q000008:administration & dosage / Q000009:adverse effects*; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D011897:Random Allocation; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000378:metabolism; D016708:Synaptophysin / Q000378:metabolism; D024502:alpha-Tocopherol / Q000378:metabolism
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D016708:Synaptophysin; C569016:Syp protein, rat; D008315:Malondialdehyde; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; C506435:Casp9 protein, rat; D053453:Caspase 9; D000154:Aconitate Hydratase; D005978:Glutathione; D024502:alpha-Tocopherol; D019803:Glutathione Disulfide