Provocative PERG testing serves as a noninvasive test in the living organism to identify early damage to the visual system, which may reflect corresponding damage in the brain that is not otherwise detectable by noninvasive means. This provides the basis for developing an earlier diagnostic test to identify patients at risk for developing chronic CNS and visual system damage after TBI at an earlier stage when treatments may be more effective in preventing these sequelae. In addition, treatment with the neuroprotective agent P7C3-S243 after TBI protects from visual system dysfunction after TBI.

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Mitochondrial reactive oxygen species have been implicated in both diabetic complications and the progression of the underlying diabetic state. However, it is not clear whether mitochondria of diabetic origin are intrinsically altered to generate excess reactive oxygen species independent of the surrounding diabetic milieu. Mitochondria were isolated from gastrocnemius, heart, and liver of 2-wk and 2-month streptozotocin diabetic rats and controls. We rigidly quantified mitochondrial superoxide, respiration and ATP production, respiratory coupling, the expression of several proteins with antioxidant properties, and the redox state of glutathione. Both fluorescent assessment and electron paramagnetic spectroscopy revealed that superoxide production was unchanged or reduced in the 2-month diabetic mitochondria compared with controls. Kinetic analysis of the proton leak showed that diabetic heart and muscle mitochondria were actually more coupled compared with control despite an approximate 2- to 4-fold increase in uncoupling protein-3 content. Adenine nucleotide translocator type 1 expression was reduced by approximately 50% in diabetic muscle mitochondria. Catalase was significantly up-regulated in muscle and heart tissue and in heart mitochondria, whereas glutathione peroxidase expression was increased in liver mitochondria of diabetic rats. We conclude that gastrocnemius, heart, and liver mitochondria of streptozotocin diabetic rats are not irrevocably altered toward excess superoxide production either by complex I or complex III. Moreover, gastrocnemius and heart mitochondria demonstrate increased, not decreased, respiratory coupling. Mitochondria of insulin-deficient diabetic rats do show signs of adaptation to antecedent oxidative stress manifested as tissue-specific enzyme and uncoupling protein expression but remain remarkably robust with respect to superoxide production.

Interpreting mitochondrial function as affected by comparative physiologic conditions is confounding because individual functional parameters are interdependent. Here, we studied muscle mitochondrial function in insulin-deficient diabetes using a novel, highly sensitive, and specific method to quantify ATP production simultaneously with reactive oxygen species (ROS) at clamped levels of inner mitochondrial membrane potential (ΔΨ), enabling more detailed study. We used a 2-deoxyglucose (2DOG) energy clamp to set ΔΨ at fixed levels and to quantify ATP production as 2DOG conversion to 2DOG-phosphate measured by one-dimensional 1H and two-dimensional 1H/13C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional 31P nuclear magnetic resonance and allowed high-throughput study of small mitochondrial isolates. Over conditions ranging from state 4 to state 3 respiration, ATP production was lower and ROS per unit of ATP generated was greater in mitochondria isolated from diabetic muscle. Moreover, ROS began to increase at a lower threshold for inner membrane potential in diabetic mitochondria. Further, ATP production in diabetic mitochondria is limited not only by respiration but also by limited capacity to use ΔΨ for ATP synthesis. In summary, we describe novel methodology for measuring ATP and provide new mechanistic insight into the dysregulation of ATP production and ROS in mitochondria of insulin-deficient rodents.

Adrenergic responsiveness to acute hypoglycemia is impaired after prior episodes of hypoglycemia. Although circulating epinephrine responses are blunted, associated alterations in adrenal sympathetic nerve activity (SNA) have not been reported. We examined adrenal nerve traffic in normal conscious rats exposed to acute insulin-induced hypoglycemia compared with insulin with (clamped) euglycemia. We also examined adrenal SNA and catecholamine responses to insulin-induced hypoglycemia in normal conscious rats after two antecedent episodes of hypoglycemia (days ؊2 and ؊1) compared with prior episodes of sham treatment. Acute insulin-induced hypoglycemia increased adrenal sympathetic nerve traffic compared with insulin administration with clamped euglycemia (165 ؎ 12 vs. 118 ؎ 21 spikes/s [P < 0.05]; or to 138 ؎ 8 vs. 114 ؎ 10% of baseline [P < 0.05]). In additional experiments, 2 days of antecedent hypoglycemia (days -2 and -1) compared with sham treatment significantly enhanced baseline adrenal SNA measured immediately before subsequent acute hypoglycemia on day 0 (180 ؎ 11 vs. 130 ؎ 12 spikes/s, respectively; P < 0.005) and during subsequent acute hypoglycemia (229 ؎ 17 vs. 171 ؎ 16 spikes/s; P < 0.05). However, antecedent hypoglycemia resulted in a nonsignificant reduction in hypoglycemic responsiveness of adrenal SNA when expressed as percent increase over baseline (127 ؎ 5% vs. 140 ؎ 14% of baseline). Antecedent hypoglycemia, compared with sham treatment, resulted in diminished epinephrine responsiveness to subsequent hypoglycemia. Norepinephrine responses to hypoglycemia were not significantly altered by antecedent hypoglycemia. In summary, prior hypoglycemia in normal rats increased adrenal sympathetic tone, but impaired epinephrine responsiveness to acute hypoglycemia. Hence, these data raise the intriguing possibility that increased sympathetic tone resulting from antecedent hypoglycemia downregulates subsequent epinephrine responsiveness to hypoglycemia. Alternatively, it is possible that the decrease in epinephrine responsiveness after antecedent hypoglycemia could be the result of reduced adrenal sympathetic nerve responsiveness. Diabetes 50:1119 -1125, 2001 I mpaired hypoglycemic counterregulation and hypoglycemia unawareness are well-recognized complications of long-standing diabetes (1,2). Hypoglycemia unawareness has also been observed as a reversible consequence of transient prior hypoglycemic events (3-5) and has been observed even in individuals without diabetes (6). However, the pathophysiology of hypoglycemic counterregulation and hypoglycemia unawareness is still unclear. Although much has been learned of catecholamine and other hormonal responses to hypoglycemia, the role of sympathetic neural activity per se to adrenal and other tissues has received only limited study.Peripheral sympathetic nerve activity (SNA) originating in hypothalamic centers may be important in the counterregulatory response to hypoglycemia. Hoffman et al. (7) found that skeletal muscle SNA in type 1 diabetic and nondia...

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 mM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF-and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics.

Antecedent hypoglycemia is well known to impair sympathetic responses to subsequent hypoglycemia. However, it is less clear whether this occurs through altered sympathetic neural traffic or through decreased adrenal catecholamine release per se. It is also not clear whether antecedent hypoglycemia impairs sympathetic responsiveness to subsequent nonhypoglycemic sympathetic stimuli. We exposed rats to two episodes of insulin-induced hypoglycemia or sham hypoglycemia (n = 15 per group) on d -2 and -1 before exposure to transient (10 min) hypotension on d 0. Adrenal sympathetic nerve activity (SNA) was directly recorded in the conscious state and plasma catecholamine concentrations were assessed. We also examined the effect of antecedent hypoglycemia on phosphorylated and nonphosphorylated tyrosine hydroxylase (TH) protein expression as well as the expression of phenylethanolamine N-methyltransferase. Adrenal SNA was not significantly altered by antecedent hypoglycemia either at baseline of d 0 (before hypotension) or in response to hypotension. In contrast, plasma epinephrine (EPI) responsiveness was impaired by more than 50% (P = 0.025) in rats exposed to antecedent vs. sham hypoglycemia. Antecedent hypoglycemia had no effect on norepinephrine responsiveness to hypotension. In studies of adrenal tissue from separate rats, antecedent hypoglycemia decreased adrenal EPI content but did not significantly alter the expression of TH, phosphorylated TH, or phenylethanolamine N-methyltransferase. In summary, antecedent hypoglycemia impaired EPI responsiveness to subsequent hypotension despite no reduction in adrenal SNA and in association with reduced adrenal EPI content. Thus, antecedent hypoglycemia impaired responsiveness to a subsequent nonhypoglycemic sympathetic stimulus, an effect mediated at the level of the adrenal medullae.

Mitochondrial-targeted analogs of coenzyme Q (CoQ) are under development to reduce oxidative damage induced by a variety of disease states. However, there is a need to understand the bioenergetic effects of these agents and whether or not these effects are related to redox properties, including their known pro-oxidant effects. We examined the bioenergetic effects of two mitochondrial-targeted CoQ analogs in their quinol forms, mitoquinol (MitoQ) and plastoquinonyl-decyl-triphenylphosphonium (SkQ1), in bovine aortic endothelial cells. We used an extracellular oxygen and proton flux analyzer to assess mitochondrial action at the intact-cell level. Both agents, in dose-dependent fashion, reduced the oxygen consumption rate (OCR) directed at ATP turnover (OCR ATP ) (IC 50 values of 189 Ϯ 13 nM for MitoQ and 181 Ϯ 7 for SKQ1; difference not significant) while not affecting or mildly increasing basal oxygen consumption. Both compounds increased extracellular acidification in the basal state consistent with enhanced glycolysis. Both compounds enhanced mitochondrial superoxide production assessed by using mitochondrial-targeted dihydroethidium, and both increased H 2 O 2 production from mitochondria of cells treated before isolation of the organelles. The manganese superoxide dismutase mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin did not alter or actually enhanced the actions of the targeted CoQ analogs to reduce OCR ATP . In contrast, N-acetylcysteine mitigated this effect of MitoQ and SkQ1. In summary, our data demonstrate the important bioenergetic effects of targeted CoQ analogs. Moreover, these effects are mediated, at least in part, through superoxide production but depend on conversion to H 2 O 2 . These bioenergetic and redox actions need to be considered as these compounds are developed for therapeutic purposes.