High-plasma soluble prorenin receptor is associated with vascular damage in male, but not female, mice fed a high-fat diet.

Plasma soluble prorenin receptor (sPRR) displays sexual dimorphism and is higher in women with type 2 diabetes mellitus (T2DM). However, the contribution of plasma sPRR to the development of vascular complications in T2DM remains unclear. We investigated if plasma sPRR contributes to sex differences in the activation of the systemic renin-angiotensin-aldosterone system (RAAS) and vascular damage in a model of high-fat diet (HFD)-induced T2DM. Male and female C57BL/6J mice were fed either a normal fat diet (NFD) or an HFD for 28 wk to assess changes in blood pressure, cardiometabolic phenotype, plasma prorenin/renin, sPRR, and ANG II. After completing dietary protocols, tissues were collected from males to assess vascular reactivity and aortic reactive oxygen species (ROS). A cohort of male mice was used to determine the direct contribution of increased systemic sPRR by infusion. To investigate the role of ovarian hormones, ovariectomy (OVX) was performed at 32 wk in females fed either an NFD or HFD. Significant sex differences were found after 28 wk of HFD, where only males developed T2DM and increased plasma prorenin/renin, sPRR, and ANG II. T2DM in males was accompanied by nondipping hypertension, carotid artery stiffening, and aortic ROS. sPRR infusion in males induced vascular thickening instead of material stiffening caused by HFD-induced T2DM. While intact females were less prone to T2DM, OVX increased plasma prorenin/renin, sPRR, and systolic blood pressure. These data suggest that sPRR is a novel indicator of systemic RAAS activation and reflects the onset of vascular complications during T2DM regulated by sex.NEW & NOTEWORTHY High-fat diet (HFD) for 28 wk leads to type 2 diabetes mellitus (T2DM) phenotype, concomitant with increased plasma soluble prorenin receptor (sPRR), nondipping blood pressure, and vascular stiffness in male mice. HFD-fed female mice exhibiting a preserved cardiometabolic phenotype until ovariectomy revealed increased plasma sPRR and blood pressure. Plasma sPRR may indicate the status of systemic renin-angiotensin-aldosterone system (RAAS) activation and the onset of vascular complications during T2DM in a sex-dependent manner.

Peroxynitrite decomposition catalyst enhances respiratory function in isolated brain mitochondria.

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

Effects of prolonged type 2 diabetes on mitochondrial function in cerebral blood vessels.

One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS.NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.

Impaired Mitochondrial Respiration in Large Cerebral Arteries of Rats with Type 2 Diabetes.

Mitochondrial dysfunction has been suggested as a potential underlying cause of pathological conditions associated with type 2 diabetes (T2DM). We have previously shown that mitochondrial respiration and mitochondrial protein levels were similar in the large cerebral arteries of insulin-resistant Zucker obese rats and their lean controls. In this study, we extend our investigations into the mitochondrial dynamics of the cerebral vasculature of 14-week-old Zucker diabetic fatty obese (ZDFO) rats with early T2DM. Body weight and blood glucose levels were significantly higher in the ZDFO group, and basal mitochondrial respiration and proton leak were significantly decreased in the large cerebral arteries of the ZDFO rats compared with the lean controls (ZDFL). The expression of the mitochondrial proteins total manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC) were significantly lower in the cerebral microvessels, and acetylated MnSOD levels were significantly reduced in the large arteries of the ZDFO group. Additionally, superoxide production was significantly increased in the microvessels of the ZDFO group. Despite evidence of increased oxidative stress in ZDFO, exogenous SOD was not able to restore mitochondrial respiration in the ZDFO rats. Our results show, for the first time, that mitochondrial respiration and protein levels are compromised during the early stages of T2DM.

The mitochondrial function of the cerebral vasculature in insulin-resistant Zucker obese rats.

Little is known about mitochondrial functioning in the cerebral vasculature during insulin resistance (IR). We examined mitochondrial respiration in isolated cerebral arteries of male Zucker obese (ZO) rats and phenotypically normal Zucker lean (ZL) rats using the Seahorse XFe24 analyzer. We investigated mitochondrial morphology in cerebral blood vessels as well as mitochondrial and nonmitochondrial protein expression levels in cerebral arteries and microvessels. We also measured reactive oxygen species (ROS) levels in cerebral microvessels. Under basal conditions, the mitochondrial respiration components (nonmitochondrial respiration, basal respiration, ATP production, proton leak, and spare respiratory capacity) showed similar levels among the ZL and ZO groups with the exception of maximal respiration, which was higher in the ZO group. We examined the role of nitric oxide by measuring mitochondrial respiration following inhibition of nitric oxide synthase with N(ω)-nitro-l-arginine methyl ester (l-NAME) and mitochondrial activation after administration of diazoxide (DZ). Both ZL and ZO groups showed similar responses to these stimuli with minor variations.l-NAME significantly increased the proton leak, and DZ decreased nonmitochondrial respiration in the ZL group. Other components were not affected. Mitochondrial morphology and distribution within vascular smooth muscle and endothelium as well as mitochondrial protein levels were similar in the arteries and microvessels of both groups. Endothelial nitric oxide synthase (eNOS) and ROS levels were increased in cerebral microvessels of the ZO. Our study suggests that mitochondrial function is not significantly altered in the cerebral vasculature of young ZO rats, but increased ROS production might be due to increased eNOS in the cerebral microcirculation during IR.

Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide.

The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.

Diversity of mitochondria-dependent dilator mechanisms in vascular smooth muscle of cerebral arteries from normal and insulin-resistant rats.

Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by generation of mitochondrial reactive oxygen species (ROS), which sequentially promotes frequency of calcium sparks and activation of large conductance calcium-activated potassium channels (BKCa) in vascular smooth muscle (VSM). We previously demonstrated that cerebrovascular insulin resistance accompanies aging and obesity. It is unclear whether mitochondrial depolarization without the ROS generation enhances calcium sparks and vasodilation in phenotypically normal [Sprague Dawley (SD); Zucker lean (ZL)] and insulin-resistant [Zucker obese (ZO)] rats. We compared the mechanisms underlying the vasodilation to ROS-dependent (diazoxide) and ROS-independent [BMS-191095 (BMS)] mitoKATP channel activators in normal and ZO rats. Arterial diameter studies from SD, ZL, and ZO rats showed that BMS as well as diazoxide induced vasodilation in endothelium-denuded cerebral arteries. In normal rats, BMS-induced vasodilation was mediated by mitochondrial depolarization and calcium sparks generation in VSM and was reduced by inhibition of BKCa channels. However, unlike diazoxide-induced vasodilation, scavenging of ROS had no effect on BMS-induced vasodilation. Electron spin resonance spectroscopy confirmed that diazoxide but not BMS promoted vascular ROS generation. BMS- as well as diazoxide-induced vasodilation, mitochondrial depolarization, and calcium spark generation were diminished in cerebral arteries from ZO rats. Thus pharmacological depolarization of VSM mitochondria by BMS promotes ROS-independent vasodilation via generation of calcium sparks and activation of BKCa channels. Diminished generation of calcium sparks and reduced vasodilation in ZO arteries in response to BMS and diazoxide provide new insights into mechanisms of cerebrovascular dysfunction in insulin resistance.

Acute severe hypoglycemia alters mouse brain microvascular proteome.

Hypoglycemia increases the risk related to stroke and neurodegenerative diseases, however, the underlying mechanisms are unclear. For the first time, we studied the effect of a single episode (acute) of severe (ASH) and mild (AMH) hypoglycemia on mouse brain microvascular proteome. After four-hour fasting, insulin was administered (i.p) to lower mean blood glucose in mice and induce ∼30 minutes of ASH (∼30 mg/dL) or AMH (∼75 mg/dL), whereas a similar volume of saline was given to control mice (∼130 mg/dL). Blood glucose was allowed to recover over 60 minutes either spontaneously or by 20% dextrose administration (i.p). Twenty-four hours later, the brain microvessels (BMVs) were isolated, and tandem mass tag (TMT)-based quantitative proteomics was performed using liquid chromatography-mass spectrometry (LC/MS). When compared to control, ASH significantly downregulated 13 proteins (p ≤ 0.05) whereas 23 proteins showed a strong trend toward decrease (p ≤ 0.10). When compared to AMH, ASH significantly induced the expression of 35 proteins with 13 proteins showing an increasing trend. AMH downregulated only 3 proteins. ASH-induced downregulated proteins are involved in actin cytoskeleton maintenance needed for cell shape and migration which are critical for blood-brain barrier maintenance and angiogenesis. In contrast, ASH-induced upregulated proteins are RNA-binding proteins involved in RNA splicing, transport, and stability. Thus, ASH alters BMV proteomics to impair cytoskeletal integrity and RNA processing which are critical for cerebrovascular function.

Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function.

(1) Background: Apolipoprotein E (ApoE) is a critical plasma apolipoprotein for lipid transport and nonlipid-related functions. Humans possess three isoforms of ApoE (2, 3, and 4). ApoE2, which exhibits beneficial effects on cardiac health, has not been adequately studied. (2) Methods: We investigated the cardiac phenotypes of the humanized ApoE knock-in (hApoE KI) rats and compared to wild-type (WT) and ApoE knock-out (ApoE KO) rats using echocardiography, ultrasound, blood pressure measurements, histology strategies, cell culture, Seahorse XF, cardiomyocyte contractility and intracellular Ca2+ tests, and Western blotting; (3) Results: hApoE2 rats exhibited enhanced heart contractile function without signs of detrimental remodeling. Isolated adult hApoE2 cardiomyocytes had faster and stronger sarcomere contractility because of more mitochondrial energy generation and stimulation-induced fast and elevated intracellular Ca2+ transient. The abundant energy is a result of elevated mitochondrial function via fatty acid ß-oxidation. The fast and elevated Ca2+ transient is associated with decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) and increased expression of cardiac ryanodine receptor 2 (RyR2) conducting a potent Ca2+ release from SR.; (4) Conclusions: Our studies validated the association of polymorphic ApoEs with cardiac health in the rat model, and revealed the possible mechanisms of the protective effect of ApoE2 against heart diseases.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Amyloid [Formula: see text] (1-42) peptide impairs mitochondrial respiration in primary human brain microvascular endothelial cells: impact of dysglycemia and pre-senescence.

Diabetes increases the risk of Alzheimer's disease (AD). We investigated the impact of glucose concentrations on the ß-amyloid (Aß)-induced alteration of mitochondrial/cellular energetics in primary human brain microvascular endothelial cells (HBMECs). HBMECs were grown and passaged in media containing 15 mmol/l glucose (normal) based on which the glucose levels in the media were designated as high (25 mmol/L) or low (5 mmol/L). HBMECs were treated with Aß (1-42) (5 µmol/l) or a scrambled peptide for 24 h and mitochondrial respiratory parameters were measured using Seahorse Mito Stress Test. Aß (1-42) decreased the mitochondrial ATP production at normal glucose levels and decreased spare respiratory capacity at high glucose levels. Aß (1-42) diminished all mitochondrial respiratory parameters markedly at low glucose levels that were not completely recovered by restoring normal glucose levels in the media. The addition of mannitol (10 mmol/l) to low and normal glucose-containing media altered the Aß (1-42)-induced bioenergetic defects. Even at normal glucose levels, pre-senescent HMBECs (passage 15) displayed greater Aß (1-42)-induced mitochondrial respiratory impairments than young cells (passages 7-9). Thus, hypoglycemia, osmolarity changes, and senescence are stronger instigators of Aß (1-42)-induced mitochondrial respiration and energetics in HBMECs and contributors to diabetes-related increased AD risk than hyperglycemia.

Aging related impairment of brain microvascular bioenergetics involves oxidative phosphorylation and glycolytic pathways.

Mitochondrial and glycolytic energy pathways regulate the vascular functions. Aging impairs the cerebrovascular function and increases the risk of stroke and cognitive dysfunction. The goal of our study is to characterize the impact of aging on brain microvascular energetics. We measured the oxygen consumption and extracellular acidification rates of freshly isolated brain microvessels (BMVs) from young (2-4 months) and aged (20-22 months) C57Bl/6 male mice. Cellular ATP production in BMVs was predominantly dependent on oxidative phosphorylation (OXPHOS) with glucose as the preferred energy substrate. Aged BMVs exhibit lower ATP production rate with diminished OXPHOS and glycolytic rate accompanied by increased utilization of glutamine. Impairments of glycolysis displayed by aged BMVs included reduced compensatory glycolysis whereas impairments of mitochondrial respiration involved reduction of spare respiratory capacity and proton leak. Aged BMVs showed reduced levels of key glycolysis proteins including glucose transporter 1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 but normal lactate dehydrogenase activity. Mitochondrial protein levels were mostly unchanged whereas citrate synthase activity was reduced, and glutamate dehydrogenase was increased in aged BMVs. Thus, for the first time, we identified the dominant role of mitochondria in bioenergetics of BMVs and the alterations of the energy pathways that make the aged BMVs vulnerable to injury.

Glycolytic and Oxidative Phosphorylation Defects Precede the Development of Senescence in Primary Human Brain Microvascular Endothelial Cells.

Alterations of mitochondrial and glycolytic energy pathways related to aging could contribute to cerebrovascular dysfunction. We studied the impact of aging on energetics of primary human brain microvascular endothelial cells (HBMECs) by comparing the young (passages 7-9), pre-senescent (passages 13-15), and senescent (passages 20-21) cells. Pre-senescent HBMECs displayed decreased telomere length and undetectable telomerase activity although markers of senescence were unaffected. Bioenergetics in HBMECs were determined by measuring the oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Cellular ATP production in young HBMECs was predominantly dependent on glycolysis with glutamine as the preferred fuel for mitochondrial oxidative phosphorylation (OXPHOS). In contrast, pre-senescent HBMECs displayed equal contribution to ATP production rate from glycolysis and OXPHOS with equal utilization of glutamine, glucose, and fatty acids as mitofuels. Compared to young, pre-senescent HBMECs showed a lower overall ATP production rate that was characterized by diminished contribution from glycolysis. Impairments of glycolysis displayed by pre-senescent cells included reduced basal glycolysis, compensatory glycolysis, and non-glycolytic acidification. Furthermore, impairments of mitochondrial respiration in pre-senescent cells involved the reduction of maximal respiration and spare respiratory capacity but intact basal and ATP production-related OCR. Proton leak and non-mitochondrial respiration, however, were unchanged in the pre-senescent HBMECs. HBMECS at passages 20-21 displayed expression of senescence markers and continued similar defects in glycolysis and worsened OXPHOS. Thus, for the first time, we characterized the bioenergetics of pre-senescent HBMECs comprehensively to identify the alterations of the energy pathways that could contribute to aging.

Urinary angiotensinogen increases in the absence of overt renal injury in high fat diet-induced type 2 diabetic mice.

AIM OF THE STUDY: During type 2 diabetes (T2D) and hypertension there is stimulation of renal proximal tubule angiotensinogen (AGT), but whether urinary excretion of AGT (uAGT) is an indicator of glomerular damage or intrarenal RAS activation is unclear. We tested the hypothesis that elevations in uAGT can be detected in the absence of albuminuria in a mouse model of T2D. METHODS: Male C57BL/6 mice (N = 10) were fed a high fat (HFD; 45% Kcal from fat) for 28 weeks, and the metabolic phenotype including body weight, blood pressures, glucose, insulin, ippGTT, HOMA-IR, and cholesterol was examined. In addition, kidney Ang II content and reactive oxygen species (ROS) was measured along with urinary albumin, creatinine, Ang II, and AGT. RESULTS: All parameters consistent with T2D were present in mice after 12-14 weeks on the HFD. Systolic BP increased after 18 weeks in HFD but not NFD mice. Intrarenal ROS and Ang II concentrations were also increased in HFD mice. Remarkably, these changes paralleled the augmentation uAGT excretion (3.66 ±â€¯0.50 vs. 0.92 ±â€¯0.13 ng/mg by week 29; P < 0.01), which occurred in the absence of overt albuminuria. CONCLUSIONS: In HFD-induced T2D mice, increases in uAGT occur in the absence of overt renal injury, indicating that this biomarker accurately detects early intrarenal RAS activation.

G Protein-Coupled Estrogen Receptor Protects From Angiotensin II-Induced Increases in Pulse Pressure and Oxidative Stress.

Our previous work showed that the G protein-coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II-dependent hypertension by inhibiting oxidative stress. The goal of the current study was to assess the impact of GPER deletion on sex differences in Ang II-induced hypertension and oxidative stress. Male and female wildtype and GPER knockout mice were implanted with radiotelemetry probes for measurement of baseline blood pressure before infusion of Ang II (700 ng/kg/min) for 2 weeks. Mean arterial pressure was increased to the same extent in all groups, but female wildtype mice were protected from Ang II-induced increases in pulse pressure, aortic wall thickness, and Nox4 mRNA. In vitro studies using vascular smooth muscle cells found that pre-treatment with the GPER agonist G-1 inhibited Ang II-induced ROS and NADP/NADPH. Ang II increased while G-1 decreased Nox4 mRNA and protein. The effects of Ang II were blocked by losartan and Nox4 siRNA, while the effects of G-1 were inhibited by adenylyl cyclase inhibition and mimicked by phosphodiesterase inhibition. We conclude that during conditions of elevated Ang II, GPER via the cAMP pathway suppresses Nox4 transcription to limit ROS production and prevent arterial stiffening. Taken together with our previous work, this study provides insight into how acute estrogen signaling via GPER provides cardiovascular protection during Ang II hypertension and potentially other diseases characterized by increased oxidative stress.

Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice.

Cerebral blood flow (CBF) is uniquely regulated by the anatomical design of the cerebral vasculature as well as through neurovascular coupling. The process of directing the CBF to meet the energy demands of neuronal activity is referred to as neurovascular coupling. Microvasculature in the brain constitutes the critical component of the neurovascular coupling. Mitochondria provide the majority of ATP to meet the high-energy demand of the brain. Impairment of mitochondrial function plays a central role in several age-related diseases such as hypertension, ischemic brain injury, Alzheimer's disease, and Parkinson disease. Interestingly, microvessels and small arteries of the brain have been the focus of the studies implicating the vascular mechanisms in several age-related neurological diseases. However, the role of microvascular mitochondrial dysfunction in age-related diseases remains unexplored. To date, high-throughput assay for measuring mitochondrial respiration in microvessels is lacking. The current study presents a novel method to measure mitochondrial respiratory parameters in freshly isolated microvessels from mouse brain ex vivo using Seahorse XFe24 Analyzer. We validated the method by demonstrating impairments of mitochondrial respiration in cerebral microvessels isolated from old mice compared to the young mice. Thus, application of mitochondrial respiration studies in microvessels will help identify novel vascular mechanisms underlying a variety of age-related neurological diseases.