Mitochondrial function is impaired in the primary visual cortex in an experimental glaucoma model.

Glaucoma is a neurodegenerative disease that affects eye structures and brain areas related to the visual system. Oxidative stress plays a key role in the development and progression of the disease. The aims of the present study were to evaluate the mitochondrial function and its participation in the brain redox metabolism in an experimental glaucoma model. 3-month-old female Wistar rats were subjected to cauterization of two episcleral veins of the left eye to elevate the intraocular pressure. Seven days after surgery, animals were sacrificed, the brain was carefully removed and the primary visual cortex was dissected. Mitochondrial bioenergetics and ROS production, and the antioxidant enzyme defenses from both mitochondrial and cytosolic fractions were evaluated. When compared to control, glaucoma decreased mitochondrial ATP production (23%, p < 0.05), with an increase in superoxide and hydrogen peroxide production (30%, p < 0.01 and 28%, p < 0.05, respectively), whereas no changes were observed in membrane potential and oxygen consumption rate. In addition, the glaucoma group displayed a decrease in complex II activity (34%, p < 0.01). Moreover, NOX4 expression was increased in glaucoma compared to the control group (27%, p < 0.05). Regarding the activity of enzymes associated with the regulation of the redox status, glaucoma showed an increase in mitochondrial SOD activity (34%, p < 0.05), mostly due to an increase in Mn-SOD (50%, p < 0.05). A decrease in mitochondrial GST activity was observed (11%, p < 0.05). GR and TrxR activity were decreased in both mitochondrial (16%, p < 0.05 and 20%, p < 0.05 respectively) and cytosolic (21%, p < 0.01 and 50%, p < 0.01 respectively) fractions in the glaucoma group. Additionally, glaucoma showed an increase in cytoplasmatic GPx (50%, p < 0.01). In this scenario, redox imbalance took place resulting in damage to mitochondrial lipids (39%, p < 0.01) and proteins (70%, p < 0.05). These results suggest that glaucoma leads to mitochondrial function impairment in brain visual targets, that is accompanied by an alteration in both mitochondrial and cytoplasmatic enzymatic defenses. As a consequence of redox imbalance, oxidative damage to macromolecules takes place and can further affect vital cellular functions. Understanding the role of the mitochondria in the development and progression of the disease could bring up new neuroprotective therapies.

Glaucoma causes redox imbalance in the primary visual cortex by modulating NADPH oxidase-4, iNOS, and Nrf2 pathway in a rat experimental model.

The aim of this study was to elucidate the intracellular sources of oxidant species, the antioxidant response as well as the main signaling pathways involved in the regulation of the redox balance in the primary visual cortex of rats subjected to an experimental glaucoma model. 3-month female Wistar strain rats were operated under a microscope by cauterizing two of the episcleral veins in order to elevate the intraocular pressure (glaucoma group); the control group received a sham procedure. Seven days after surgery, the animals were sacrificed, the brains were carefully removed, and the primary visual cortex was dissected. NADPH oxidase (NOX) activity, as well as the inducible nitric oxide synthase (iNOS) expression, the enzymatic antioxidant defenses, the metabolism of glutathione, and the translocation of Nuclear factor-erythroid 2-related factor-2 (Nrf2) and Nuclear factor k-light-chain-enhancer of activated B cells (NF-κB) were assessed. Compared to control, glaucoma group displayed an increase in NOX activity (147%, p < 0.05), leading to a rise in the steady state concentration of oxidant species. Specifically, NOX4 expression was higher (90%, p < 0.05), suggesting that it could be a source of H2O2. In addition, iNOS expression was increased in glaucoma (47%, p < 0.05), as a source of NO in the brain, induced by NF-κB translocation to the nucleus (48%, p < 0.01). An increase in primary antioxidant enzymes superoxide dismutase (40%, p < 0.01) and glutathione peroxidase (55%, p < 0.05) was observed as an adaptive response to reactive oxygen species (ROS) production. However, an alteration in glutathione metabolism was shown in glaucoma due to a decrease in its recycling (40%, p < 0.05) as well as in its de novo synthesis (53%, p < 0.05), leading to a decreased in reduced/oxidized glutathione ratio (55%, p < 0.001). Moreover, a lower expression of Nfr2 was shown in glaucoma (40%, p < 0.05), suggesting that the cell signaling pathway that regulates the antioxidant capacity is compromised. In this context, redox imbalance takes place, resulting in oxidative damage to both lipids (70%, p < 0.001) and proteins (140%, p < 0.001). These results suggest that glaucoma damages not only eye structures but also brain visual targets such as the primary visual cortex. Redox imbalance takes place due to an enhancement in ROS and reactive nitrogen species production from different sources, such as NOX family and iNOS, respectively, in an onset where the antioxidant defenses are overwhelmed due to an impaired Nrf2 signaling, leading to oxidative damage to macromolecules.

Hyperinsulinemia is associated with increasing insulin secretion but not with decreasing insulin clearance in an age-related metabolic dysfunction mice model.

Human life expectancy is increasing faster lately and, consequently, the number of patients with age-related diseases such as type 2 diabetes (T2D) is rising every year. Cases of hyperinsulinemia have been extensively reported in elderly subjects and this alteration in blood insulin concentration is postulated to be a cause of insulin resistance, which in some cases triggers T2D onset. Thus, it is important to know the underlying mechanisms of age-dependent hyperinsulinemia to find new strategies to prevent T2D in elderly subjects. Two processes control blood insulin concentration: Insulin secretion by the endocrine portion of the pancreas and insulin clearance, which occurs mainly in the liver by the action of the insulin-degrading enzyme (IDE). Here, we demonstrated that 10-month-old mice (old) display increased body and fat pad weight, compared with 3-month-old mice (control), and these alterations were accompanied by glucose and insulin intolerance. We also confirm hyperinsulinemia in the old mice, which was related to increased insulin secretion but not to reduced insulin clearance. Although no changes in insulin clearance were observed, IDE activity was lower in the liver of old compared with the control mice. However, this decreased IDE activity was compensated by increased expression of IDE protein in the liver, thus explaining the similar insulin clearance observed in both groups. In conclusion, at the beginning of aging, 10-month-old mice do not display any alterations in insulin clearance. Therefore, hyperinsulinemia is initiated primarily due to a higher insulin secretion in the age-related metabolic dysfunction in mice.

Urban air pollution induces redox imbalance and epithelium hyperplasia in mice cornea.

The aim of the study was to evaluate the time course of the effects of urban air pollutants on the ocular surface, focusing on the morphological changes, the redox balance, and the inflammatory response of the cornea. 8-week-old mice were exposed to urban or filtered air (UA-group and FA-group, respectively) in exposure chambers for 1, 2, 4, and 12 weeks. After each time, the eyes were enucleated and the corneas were isolated for biochemical analysis. UA-group corneas exhibited a continuous increase in NADPH oxidase-4 levels throughout the exposure time, suggesting an increased production of reactive oxygen species (ROS). After 1 week, an early adaptive response to ROS was observed as an increase in antioxidant enzymes. After 4 weeks, the enzymatic antioxidants were decreased, meanwhile an increase of the glutathione was shown, as a later compensatory antioxidant response. However, redox imbalance took place, evidenced by the increased oxidized proteins, which persisted up to 12 weeks. At this time point, corneal epithelium hyperplasia was also observed. The inflammatory response was modulated by the increase in IL-10 levels after 1 week, which early regulates the release of TNF-α and IL-6. These results suggest that air pollution alters the ocular surface, supported by the observed cellular hyperplasia. The redox imbalance and the inflammatory response modulated by IL-10 play a key role in the response triggered by air pollutants on the cornea. Taking into account this time course study, the ocular surface should also be considered as a relevant target of urban air pollutants.

Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells.

The aim of this study was to evaluate the time course of oxidative stress markers and inflammatory mediators in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP) for 1, 3, and 24 h. Reactive oxygen species (ROS) production, lipid and protein oxidation, Nrf2 pathway activation, enzymatic antioxidants, glutathione (GSH) levels and synthesis, as well as cytokine release and cell proliferation were analyzed. Cells exposed to DEP showed an increase in ROS at all time points. The induction of NADPH oxidase-4 appeared later than mitochondrial superoxide anion production, when the cell also underwent a proinflammatory response mediated by IL-6. DEP exposure triggered the activation of Nrf2 in IOBA-NHC, as a strategy for increasing cellular antioxidant capacity. Antioxidant enzyme activities were significantly increased at early stages except for glutathione reductase (GR) that showed a significant decrease after a 3-h-incubation. GSH levels were found increased after 1 and 3 h of incubation with DEP, despite the increase in its consumption by the antioxidant enzymes as it works as a cofactor. GSH recycling and the de novo synthesis were responsible for the maintenance of its content at these time points, respectively. After 24 h, the decrease in GR and glutamate cysteine ligase as wells as the enhanced activity of glutathione peroxidase and glutathione S-transferase produced a depletion in the GSH pool. Lipid-peroxidation was found increased in cells exposed to DEP after 1-h-incubation, whereas protein oxidation was found increased in cells exposed to DEP after a 3-h-incubation that persisted after a longer exposure. Furthermore, DEP lead IOBA-NHC cells to hyperplasia after 1 and 3 h of incubation, but a decrease in cell proliferation was found after longer exposure. ROS production seems to be an earlier event triggered by DEP on IOBA-NHC, comparing to the proinflammatory response mediated by IL-6. Despite the fact that under short periods of exposure to DEP lipids and then proteins are targets of oxidative damage, the viability of the cells is not affected at early stages, since cell hyperplasia was detected as compensatory mechanism. Although after 24 h Nrf2 pathway is still enhanced, the epithelial cell capacity to maintain redox balance is exceeded. The antioxidant enzymes activation and the depleted GSH pool are not capable of counteracting the increased ROS production, leading to oxidative damage.

Leucine supplementation does not affect protein turnover and impairs the beneficial effects of endurance training on glucose homeostasis in healthy mice.

Endurance exercise training as well as leucine supplementation modulates glucose homeostasis and protein turnover in mammals. Here, we analyze whether leucine supplementation alters the effects of endurance exercise on these parameters in healthy mice. Mice were distributed into sedentary (C) and exercise (T) groups. The exercise group performed a 12-week swimming protocol. Half of the C and T mice, designated as the CL and TL groups, were supplemented with leucine (1.5 % dissolved in the drinking water) throughout the experiment. As well known, endurance exercise training reduced body weight and the retroperitoneal fat pad, increased soleus mass, increased VO2max, decreased muscle proteolysis, and ameliorated peripheral insulin sensitivity. Leucine supplementation had no effect on any of these parameters and worsened glucose tolerance in both CL and TL mice. In the soleus muscle of the T group, AS-160(Thr-642) (AKT substrate of 160 kDa) and AMPK(Thr-172) (AMP-Activated Protein Kinase) phosphorylation was increased by exercise in both basal and insulin-stimulated conditions, but it was reduced in TL mice with insulin stimulation compared with the T group. Akt phosphorylation was not affected by exercise but was lower in the CL group compared with the other groups. Leucine supplementation increased mTOR phosphorylation at basal conditions, whereas exercise reduced it in the presence of insulin, despite no alterations in protein synthesis. In trained groups, the total FoxO3a protein content and the mRNA for the specific isoforms E2 and E3 ligases were reduced. In conclusion, leucine supplementation did not potentiate the effects of endurance training on protein turnover, and it also reduced its positive effects on glucose homeostasis.

Experimental glaucoma triggers a pro-oxidative and pro-inflammatory state in the rat cornea.

BACKGROUND: Increasing evidence suggests that glaucoma affects the ocular surface. We aimed to investigate the cellular mechanisms underlying the glaucoma-associated corneal alterations in an animal model. METHODS: Wistar rats underwent the cauterization of two episcleral veins of the left eye to elevate the intraocular pressure (ipsilateral, G-IL). Control animals received a sham procedure (C-IL). Contralateral eyes did not receive any procedure (G-CL or C-CL). Enzymes related to the redox status, oxidative damage to macromolecules, and inflammatory markers were assessed in corneal lysates. RESULTS: Compared to C-IL, NOX4, NOX2, and iNOS expression was increased in G-IL (68%, p < 0.01; 247%, p < 0.01; and 200%, p < 0.001, respectively). We found an increase in SOD activity in G-IL (60%, p < 0.05). The GSH/GSSG ratio decreased in G-IL (80%, p < 0.05), with a decrease in GR activity (40%, p < 0.05). G-IL displayed oxidative (90%, p < 0.01) and nitrosative (40%, p < 0.05) protein damage, and enhanced lipid peroxidation (100%, p < 0.01). G-IL group showed an increased in CD45, CD68 and F4/80 expression (50%, p < 0.05; 190%, p < 0.001 and 110%, p < 0.05, respectively). G-CL displayed a higher expression of Nrf2 (60%, p < 0.001) and increased activity of SOD, CAT, and GPx (60%, p < 0.05; 90%, p < 0.01; and 50%, p < 0.05, respectively). CONCLUSIONS: Glaucoma induces a redox imbalance in the ipsilateral cornea with an adaptive response of the contralateral one. GENERAL SIGNIFICANCE: Our study provides a possible mechanism involving oxidative stress and inflammation that explains the corneal alterations observed in glaucoma. We demonstrate that these changes extend not only to the ipsilateral but also to the contralateral cornea.

The role of GABA in islet function.

Gamma aminobutyric acid (GABA) is a non-proteinogenic amino acid and neurotransmitter that is produced in the islet at levels as high as in the brain. GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), of which the 65 kDa isoform (GAD65) is a major autoantigen in type 1 diabetes. Originally described to be released via synaptic-like microvesicles or from insulin secretory vesicles, beta cells are now understood to release substantial quantities of GABA directly from the cytosol via volume-regulated anion channels (VRAC). Once released, GABA influences the activity of multiple islet cell types through ionotropic GABAA receptors and metabotropic GABAB receptors. GABA also interfaces with cellular metabolism and ATP production via the GABA shunt pathway. Beta cells become depleted of GABA in type 1 diabetes (in remaining beta cells) and type 2 diabetes, suggesting that loss or reduction of islet GABA correlates with diabetes pathogenesis and may contribute to dysfunction of alpha, beta, and delta cells in diabetic individuals. While the function of GABA in the nervous system is well-understood, the description of the islet GABA system is clouded by differing reports describing multiple secretion pathways and effector functions. This review will discuss and attempt to unify the major experimental results from over 40 years of literature characterizing the role of GABA in the islet.

ARHGAP21 Acts as an Inhibitor of the Glucose-Stimulated Insulin Secretion Process.

ARHGAP21 is a RhoGAP protein implicated in the modulation of insulin secretion and energy metabolism. ARHGAP21 transient-inhibition increase glucose-stimulated insulin secretion (GSIS) in neonatal islets; however, ARHGAP21 heterozygote mice have a reduced insulin secretion. These discrepancies are not totally understood, and it might be related to functional maturation of beta cells and peripheral sensitivity. Here, we investigated the real ARHGAP21 role in the insulin secretion process using an adult mouse model of acute ARHGAP21 inhibition, induced by antisense. After ARHGAP21 knockdown induction by antisense injection in 60-day old male mice, we investigated glucose and insulin tolerance test, glucose-induced insulin secretion, glucose-induced intracellular calcium dynamics, and gene expression. Our results showed that ARHGAP21 acts negatively in the GSIS of adult islet. This effect seems to be due to the modulation of important points of insulin secretion process, such as the energy metabolism (PGC1α), Ca2+ signalization (SYTVII), granule-extrusion (SNAP25), and cell-cell interaction (CX36). Therefore, based on these finds, ARHGAP21 may be an important target in Diabetes Mellitus (DM) treatment.

Interleukin-6 increases the expression and activity of insulin-degrading enzyme.

Impairment of the insulin-degrading enzyme (IDE) is associated with obesity and type 2 diabetes mellitus (T2DM). Here, we used 4-mo-old male C57BL/6 interleukin-6 (IL-6) knockout mice (KO) to investigate the role of this cytokine on IDE expression and activity. IL-6 KO mice displayed lower insulin clearance in the liver and skeletal muscle, compared with wild type (WT), due to reduced IDE expression and activity. We also observed that after 3-h incubation, IL-6, 50 and 100 ng ml-1, increased the expression of IDE in HEPG2 and C2C12 cells, respectively. In addition, during acute exercise, the inhibition of IL-6 prevented an increase in insulin clearance and IDE expression and activity, mainly in the skeletal muscle. Finally, IL-6 and IDE concentrations were significantly increased in plasma from humans, after an acute exercise, compared to pre-exercise values. Although the increase in plasma IDE activity was only marginal, a positive correlation between IL-6 and IDE activity, and between IL-6 and IDE protein expression, was observed. Our outcomes indicate a novel function of IL-6 on the insulin metabolism expanding the possibilities for new potential therapeutic strategies, focused on insulin degradation, for the treatment and/or prevention of diseases related to hyperinsulinemia, such as obesity and T2DM.