Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice.

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 µg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4-6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.

The variation of macro- and micro-minerals of tissues in diabetic and non-diabetic rats.

This study determined the levels of Ca, Mg, Fe, Zn, Cu, and Na in various tissues samples (liver, brain, kidney, intestines, muscle and hair) of diabetic and non-diabetic rats by flame atomic absorption spectroscopy, in order to assess the role of element levels during T2DM. The ratios of Ca/Mg, Zn/Cu, Ca/Zn, and Mg/Zn in diabetic and non-diabetic rat tissues were also calculated. The determined element levels were further subjected to a student-t test statistical analysis and multiple-linear-regression in order to evaluate similarities, differences, and an inter-element association in tissues of diabetic and non-diabetic rats. The results of the study showed high variability in element levels and Ca/Mg Zn/Cu Mg/Zn Ca/Zn ratios in the tissues of diabetic and non-diabetic rats, but are tissue- and element-dependent, suggesting differences in the accumulation of the elements in tissues of diabetics and non-diabetics. The obtained significant differences in the levels of elements and Ca/Mg Zn/Cu Mg/Zn Ca/Zn ratios in several tissues of diabetic and non-diabetic rats in this study suggest that the investigated elements play considerable roles in the T2DM disease process. Strong inter-element associations (R2≥0.9) were observed for some elements in tissues of diabetic and non-diabetics rats. However, poor inter-elemental associations were obtained for some elements in the tissues of diabetic and non-diabetic rats.

Inducing Muscle Heat Shock Protein 70 Improves Insulin Sensitivity and Muscular Performance in Aged Mice.

Heat shock proteins (HSPs) are molecular chaperones with roles in longevity and muscular preservation. We aimed to show elevating HSP70 improves indices of health span. Aged C57/BL6 mice acclimated to a western diet were randomized into: geranylgeranylacetone (GGA)-treated (100 mg/kg/d), biweekly heat therapy (HT), or control. The GGA and HT are well-known pharmacological and environmental inducers of HSP70, respectively. Assessments before and after 8 weeks of treatment included glycemic endpoints, body composition, and muscular endurance, power, and perfusion. An HT mice had more than threefold, and GGA mice had a twofold greater HSP70 compared with control. Despite comparable body compositions, both treatment groups had significantly better insulin sensitivity and insulin signaling capacity. Compared with baseline, HT mice ran 23% longer than at study start, which was significantly more than GGA or control. Hanging ability (muscular endurance) also tended to be best preserved in HT mice. Muscle power, contractile force, capillary perfusion, and innervation were not different. Heat treatment has a clear benefit on muscular endurance, whereas HT and GGA both improved insulin sensitivity. Different effects may relate to muscle HSP70 levels. An HSP induction could be a promising approach for improving health span in the aged mice.

Estrogen therapy, independent of timing, improves cardiac structure and function in oophorectomized mRen2.Lewis rats.

OBJECTIVE: mRen2.Lewis rats exhibit exacerbated increases in blood pressure, left ventricular (LV) remodeling, and diastolic impairment after the loss of estrogens. In this same model, depletion of estrogens has marked effects on the cardiac biopterin profile concomitant with suppressed nitric oxide release. With respect to the establishment of overt systolic hypertension after oophorectomy (OVX), we assessed the effects of timing long-term 17ß-estradiol (E2) therapy on myocardial function, myocardial structure, and the cardiac nitric oxide system. METHODS: OVX (n = 24) or sham operation (Sham; n = 13) was performed in 4-week-old female mRen2.Lewis rats. After randomization, OVX rats received E2 immediately (OVX + E2-early; n = 7), E2 at 11 weeks of age (OVX + E2-late; n = 8), or no E2 at all (OVX; n = 9). RESULTS: E2-early was associated with lower body weight, less hypertension-related cardiac remodeling, and decreased LV filling pressure compared with OVX rats without E2 supplementation. E2-late similarly attenuated the adverse effects of ovarian hormone loss on tissue Doppler-derived LV filling pressures and perivascular fibrosis, and significantly improved myocardial relaxation or mitral annular velocity (e'). Early and late exposures to E2 decreased dihydrobiopterin, but only E2-late yielded significant increases in cardiac nitrite concentrations. CONCLUSIONS: Although there are some similarities between E2-early and E2-late treatments in relation to preservation of diastolic function and cardiac structure after OVX, the lusitropic potential of E2 is most consistent with late supplementation. The cardioprotective effects of E2-late are independent of blood pressure and may have occurred through regulation of cardiac biopterins and nitric oxide production.

Plasma nitrate and nitrite are increased by a high-nitrate supplement but not by high-nitrate foods in older adults.

Little is known about the effect of dietary nitrate on the nitrate/nitrite/nitric oxide cycle in older adults. We examined the effect of a 3-day control diet vs high-nitrate diet, with and without a high-nitrate supplement (beetroot juice), on plasma nitrate and nitrite kinetics and blood pressure using a randomized 4-period crossover controlled design. We hypothesized that the high-nitrate diet would show higher levels of plasma nitrate/nitrite and lower blood pressure compared with the control diet, which would be potentiated by the supplement. Participants were 8 normotensive older men and women (5 female, 3 male, 72.5 ± 4.7 years old) with no overt disease or medications that affect nitric oxide metabolism. Plasma nitrate and nitrite levels and blood pressure were measured before and hourly for 3 hours after each meal. The mean daily changes in plasma nitrate and nitrite were significantly different from baseline for both control diet + supplement (P < .001 and P = .017 for nitrate and nitrite, respectively) and high-nitrate diet + supplement (P = .001 and P = .002), but not for control diet (P = .713 and P = .741) or high-nitrate diet (P = .852 and P = .500). Blood pressure decreased from the morning baseline measure to the three 2-hour postmeal follow-up time points for all treatments, but there was no main effect for treatment. In healthy older adults, a high-nitrate supplement consumed at breakfast elevated plasma nitrate and nitrite levels throughout the day. This observation may have practical utility for the timing of intake of a nitrate supplement with physical activity for older adults with vascular dysfunction.

Tetrahydrobiopterin restores diastolic function and attenuates superoxide production in ovariectomized mRen2.Lewis rats.

After oophorectomy, mRen2.Lewis rats exhibit diastolic dysfunction associated with elevated superoxide, increased cardiac neuronal nitric oxide synthase (nNOS) expression, and diminished myocardial tetrahydrobiopterin (BH4) content, effects that are attenuated with selective nNOS inhibition. BH4 is an essential cofactor of nNOS catalytic activity leading to nitric oxide production. Therefore, we assessed the effect of 4 wk BH4 supplementation on diastolic function and left ventricular (LV) remodeling in oophorectomized mRen2.Lewis rats compared with sham-operated controls. Female mRen2.Lewis rats underwent either bilateral ovariectomy (OVX) (n = 19) or sham operation (n = 13) at 4 wk of age. Beginning at 11 wk of age, OVX rats were randomized to receive either BH4 (10 mg/kg · d) or saline, whereas the sham rats received saline via sc mini-pumps. Loss of ovarian hormones reduced cardiac BH4 when compared with control hearts; this was associated with impaired myocardial relaxation, augmented filling pressures, increased collagen deposition, and thickened LV walls. Additionally, superoxide production increased and nitric oxide decreased in hearts from OVX compared with sham rats. Chronic BH4 supplementation after OVX improved diastolic function and attenuated LV remodeling while restoring myocardial nitric oxide release and preventing reactive oxygen species generation. These data indicate that BH4 supplementation protects against the adverse effects of ovarian hormonal loss on diastolic function and cardiac structure in mRen2.Lewis rats by restoring myocardial NO release and mitigating myocardial O2⁻ generation. Whether BH4 supplementation is a therapeutic option for the management of diastolic dysfunction in postmenopausal women will require direct testing in humans.

Neuronal nitric oxide synthase inhibition improves diastolic function and reduces oxidative stress in ovariectomized mRen2.Lewis rats.

OBJECTIVE: The loss of estrogen in mRen2.Lewis rats leads to an exacerbation of diastolic dysfunction. Because specific neuronal nitric oxide synthase (nNOS) inhibition reverses renal damage in the same model, we assessed the effects of inhibiting neuronal nitric oxide on diastolic function, left ventricular remodeling, and the components of the cardiac nitric oxide system in ovariectomized (OVX) and sham-operated mRen2.Lewis rats treated with N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO; 0.5 mg/kg per day for 28 d) or vehicle (saline). METHODS: Female mRen2.Lewis rats underwent either bilateral oophorectomy (OVX; n = 15) or sham operation (or surgical procedure) (sham; n = 19) at 4 weeks of age. Beginning at 11 weeks of age, the rats were randomized to receive either L-VNIO or vehicle. RESULTS: The surgical loss of ovarian hormones, particularly estrogen, led to exacerbated hypertension, impaired myocardial relaxation, diminished diastolic compliance, increased perivascular fibrosis, and increased relative wall thickness. The cardiac tetrahydrobiopterin-to-dihydrobiopterin levels were lower among OVX rats compared with sham-operated rats, and this altered cardiac biopterin profile was associated with enhanced myocardial superoxide production and decreased nitric oxide release. L-VNIO decreased myocardial reactive oxygen species production, increased nitrite concentrations, attenuated cardiac remodeling, and improved diastolic function. CONCLUSIONS: Impaired relaxation, diastolic stiffness, and cardiac remodeling were found among OVX mRen2.Lewis rats. A possible mechanism for this unfavorable cardiac phenotype may have resulted from a deficiency in available tetrahydrobiopterin and subsequent increase in nNOS-derived superoxide and reduction in nitric oxide synthase metabolites within the heart. Selective nNOS inhibition with L-VNIO attenuated cardiac superoxide production and limited remodeling, leading to improved diastolic function in OVX mRen2.Lewis rats.

Acute effect of a high nitrate diet on brain perfusion in older adults.

AIMS: Poor blood flow and hypoxia/ischemia contribute to many disease states and may also be a factor in the decline of physical and cognitive function in aging. Nitrite has been discovered to be a vasodilator that is preferentially harnessed in hypoxia. Thus, both infused and inhaled nitrite are being studied as therapeutic agents for a variety of diseases. In addition, nitrite derived from nitrate in the diet has been shown to decrease blood pressure and improve exercise performance. Thus, dietary nitrate may also be important when increased blood flow in hypoxic or ischemic areas is indicated. These conditions could include age-associated dementia and cognitive decline. The goal of this study was to determine if dietary nitrate would increase cerebral blood flow in older adults. METHODS AND RESULTS: In this investigation we administered a high vs. low nitrate diet to older adults (74.7±6.9 years) and measured cerebral perfusion using arterial spin labeling magnetic resonance imaging. We found that the high nitrate diet did not alter global cerebral perfusion, but did lead to increased regional cerebral perfusion in frontal lobe white matter, especially between the dorsolateral prefrontal cortex and anterior cingulate cortex. CONCLUSION: These results suggest that dietary nitrate may be useful in improving regional brain perfusion in older adults in critical brain areas known to be involved in executive functioning.

Hyperthermia-induced Hsp90·eNOS preserves mitochondrial respiration in hyperglycemic endothelial cells by down-regulating Glut-1 and up-regulating G6PD activity.

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mM) or hyperglycemia (30 mM). With hyperglycemia treatment, O(2) consumption rate was reduced (from V(O(2)max) = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10(6) cells), whereas in heat-shocked cells, O(2) consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10(6) cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.

Effects of a single sickling event on the mechanical fragility of sickle cell trait erythrocytes.

Hemolysis contributes to the pathology associated with sickle cell disease. However, the mechanism of hemolysis or relative contribution of sickling due to hemoglobin (Hb) polymerization vs. oxidative damage remains unknown. Earlier studies aimed at deciphering the relative importance of these two mechanisms have been complicated by the fact that sickle red cells (SS) have already been affected by multiple rounds of sickling and oxidative damage before they are collected. In our study, we examine the mechanical fragility of sickle cell trait cells, which do not sickle in vivo, but can be made to do so in vitro. Thus, our novel approach explores the effects of sickle Hb polymerization on cells that have never been sickled before. We find that the mechanical fragility of these cells increases dramatically after a single sickling event, suggesting that a substantial amount of hemolysis in vivo probably occurs in polymer-containing cells.

Plasma nitrite response in older women to a physical function test.

BACKGROUND AND AIMS: Nitric oxide (NO) may play a critical role in facilitating the delivery of blood to active skeletal muscle, ultimately impacting functional health in older adults. Plasma nitrite is a useful marker of vascular NO bioavailability. The aim of the current investigation was to examine the effect of a widely used physical function test on plasma nitrite concentrations in older adults. METHODS: Venous blood was drawn before, immediately following, and 10 minutes following the completion of a 400-m walk test. Blood samples were added to heparin and frozen for subsequent analysis of nitrite levels using chemiluminescence. RESULTS: Twenty six (79±4 yrs) women participated in this study. Plasma nitrite levels decreased approximately 22% from baseline following a 400-m walk. Percent change in plasma nitrite was related to walking speed (r=-0.550, p=0.004). CONCLUSIONS: These data suggest an alteration in plasma nitrite concentration following a functional test which may impact functional health.

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome c oxidase in low oxygen concentrations.

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42 degrees C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV.

Activation of Hsp90-eNOS and increased NO generation attenuate respiration of hypoxia-treated endothelial cells.

Hypoxia induces various adoptive signaling in cells that can cause several physiological changes. In the present work, we have observed that exposure of bovine aortic endothelial cells (BAECs) to extreme hypoxia (1-5% O(2)) attenuates cellular respiration by a mechanism involving heat shock protein 90 (Hsp90) and endothelial nitric oxide (NO) synthase (eNOS), so that the cells are conditioned to consume less oxygen and survive in prolonged hypoxic conditions. BAECs, exposed to 1% O(2), showed a reduced respiration compared with 21% O(2)-maintained cells. Western blot analysis showed an increase in the association of Hsp90-eNOS and enhanced NO generation on hypoxia exposure, whereas there was no significant accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). The addition of inhibitors of Hsp90, phosphatidylinositol 3-kinase, and NOS significantly alleviated this hypoxia-induced attenuation of respiration. Thus we conclude that hypoxia-induced excess NO and its derivatives such as ONOO(-) cause inhibition of the electron transport chain and attenuate O(2) demand, leading to cell survival at extreme hypoxia. More importantly, such an attenuation is found to be independent of HIF-1alpha, which is otherwise thought to be the key regulator of respiration in hypoxia-exposed cells, through a nonphosphorylative glycolytic pathway. The present mechanistic insight will be helpful to understand the difference in the magnitude of endothelial dysfunction.

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin.

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angeli's salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference.

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.