The Vascular Function of Resistance Arteries Depends on NADPH Oxidase 4 and Is Exacerbated by Perivascular Adipose Tissue.

The NADPH oxidase NOX4 that releases H2O2 can mediate vasoprotective mechanisms under pathophysiological conditions in conductive arteries. However, the role of NOX4 in resistance arteries and in perivascular adipose tissue is not well understood. We hypothesized that NOX4 is of functional importance in resistance arteries and perivascular adipose tissue under dyslipidemia conditions. We detected elevated NOX4 expression in murine and human vessels under dyslipidemia. Diminishing Nox4 under these conditions led to endothelial dysfunction in resistance arteries. The mesenteric arteries of Nox4-/-/Ldlr-/- mice revealed decreased eNos mRNA expression. Inhibition of eNOS in those vessels did not affect vascular function, while in Ldlr-/- mice endothelial function was significantly altered. Anticontractile properties of perivascular adipose tissue at resistance arteries were diminished in Nox4-/-/Ldlr-/- compared with Ldlr-/- mice. In addition, the presence of perivascular adipose tissue further worsened endothelial dysfunction in mesenteric arteries under dyslipidemia conditions. Perivascular adipose tissue from mesenteric arteries revealed a higher expression of markers of white adipocytes compared to markers of beige/brown adipocytes. Among those white adipocyte markers, leptin was significantly less expressed in perivascular adipose tissue from Nox4-/-/Ldlr-/- mice compared with Ldlr-/- mice. Furthermore, in human perivascular adipose tissue with a profound pattern of white adipocyte marker genes, we detected a correlation of NOX4 and LEP expression. In addition, incubating arterial vessels with leptin induced nitrite release, indicating increased eNOS activity. In humans, a higher expression of leptin in perivascular adipose tissue correlated with eNOS expression in the corresponding left internal mammary artery. In conclusion, vascular function of resistance arteries was dependent on Nox4-derived H2O2, especially under dyslipidemia conditions. Perivascular adipose tissue of the mesenteric arteries with white adipose tissue characteristics further aggravated endothelial function through reduced leptin-eNOS signaling.

Impact of Dietary Sodium Reduction on the Development of Obesity and Type 2 Diabetes in db/db Mice.

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice.

Convergence of sphingolipid desaturation across over 500 million years of plant evolution.

For plants, acclimation to low temperatures is fundamental to survival. This process involves the modification of lipids to maintain membrane fluidity. We previously identified a new cold-induced putative desaturase in Physcomitrium (Physcomitrella) patens. Lipid profiles of null mutants of this gene lack sphingolipids containing monounsaturated C24 fatty acids, classifying the new protein as sphingolipid fatty acid denaturase (PpSFD). PpSFD mutants showed a cold-sensitive phenotype as well as higher susceptibility to the oomycete Pythium, assigning functions in stress tolerance for PpSFD. Ectopic expression of PpSFD in the Atads2.1 (acyl coenzyme A desaturase-like 2) Arabidopsis thaliana mutant functionally complemented its cold-sensitive phenotype. While these two enzymes catalyse a similar reaction, their evolutionary origin is clearly different since AtADS2 is a methyl-end desaturase whereas PpSFD is a cytochrome b5 fusion desaturase. Altogether, we suggest that adjustment of membrane fluidity evolved independently in mosses and seed plants, which diverged more than 500 million years ago.

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

AIMS: Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. METHODS AND RESULTS: Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice. CONCLUSIONS: Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Elevated Steroid Hormone Production in the db/db Mouse Model of Obesity and Type 2 Diabetes.

Obesity and type 2 diabetes have become a major public health problem worldwide. Steroid hormone dysfunction appears to be linked to development of obesity and type 2 diabetes and correction of steroid abnormalities may offer new approaches to therapy. We therefore analyzed plasma steroids in 15-16 week old obese and diabetic db/db mice using liquid chromatography-tandem mass spectrometry. Lean db/+ served as controls. Db/db mice developed obesity, hyperglycemia, hyperleptinemia, and hyperlipidemia. Hepatic triglyceride storage was increased and adiponectin and pancreatic insulin were lowered. Aldosterone, corticosterone, 11-deoxycorticosterone, and progesterone were respectively increased by 3.6-, 2.9-, 3.4, and 1.7-fold in db/db mice compared to controls. Ratios of aldosterone-to-progesterone and corticosterone-to-progesterone were respectively 2.0- and 1.5-fold higher in db/db mice. Genes associated with steroidogenesis were quantified in the adrenal glands and gonadal adipose tissues. In adrenals, Cyp11b2, Cyp11b1, Cyp21a1, Hsd3b1, Cyp11a1, and StAR were all significantly increased in db/db mice compared with db/+ controls. In adipose tissue, no Cyp11b2 or Cyp11b1 transcripts were detected and no differences in Cyp21a1, Hsd3b1, Cyp11a1, or StAR expression were found between db/+ and db/db mice. In conclusion, the present study showed an elevated steroid hormone production and adrenal steroidogenesis in the db/db model of obesity and type 2 diabetes.

Aldosterone Synthase Inhibition Improves Glucose Tolerance in Zucker Diabetic Fatty (ZDF) Rats.

Plasma aldosterone is elevated in type 2 diabetes and obesity in experimental and clinical studies and can act to inhibit both glucose-stimulated insulin secretion by the ß-cell and insulin signaling. Currently mineralocorticoid receptor antagonism is the best characterized treatment to ameliorate aldosterone-mediated effects. A second alternative is inhibition of aldosterone synthase, an approach with protective effects on end-organ damage in heart or kidney in animal models. The effect of aldosterone synthase inhibition on metabolic parameters in type 2 diabetes is not known. Therefore, male Zucker diabetic fatty (ZDF) rats were treated for 11 weeks with the aldosterone synthase inhibitor FAD286, beginning at 7 weeks of age. Results were compared with the mineralocorticoid receptor antagonist eplerenone. Plasma aldosterone was abolished by FAD286 and elevated more than 9-fold by eplerenone. The area under the curve calculated from an oral glucose tolerance test (OGTT) was lower and overall insulin response during OGTT was increased by FAD286. In contrast, eplerenone elevated blood glucose levels and blunted insulin secretion during the OGTT. Fasting glucose was lowered and fasting insulin was increased by FAD286 in the prediabetic state. Glycated hemoglobin was lowered by FAD286, whereas eplerenone showed no effect. We conclude that aldosterone synthase inhibition, in contrast to mineralocorticoid receptor antagonism, has the potential for beneficial effects on metabolic parameters in type 2 diabetes.

Knockout of GH3 genes in the moss Physcomitrella patens leads to increased IAA levels at elevated temperature and in darkness.

Two proteins of the GRETCHEN HAGEN3 (GH3) family of acyl acid amido synthetases from the moss Physcomitrella patens conjugate indole-3-acetic acid (IAA) to a series of amino acids. The possible function of altered auxin levels in the moss in response to two different growth perturbations, elevated temperatures and darkness, was analyzed using a) the recently described double knockout lines in both P. patens GH3 genes (GH3-doKO) and b) a previously characterized line harboring an auxin-inducible soybean GH3 promoter::reporter fused to ß-glucuronidase (G1-GUS). The GUS activity as marker of the auxin response increased at higher temperatures and after cultivation in the darkness for a period of up to four weeks. Generally, the double knockout plants grew more slowly than the wild type (WT). The altered growth conditions influenced the phenotypes of the double knockout lines differently from that of WT moss. Higher temperatures negatively affected GH3-doKO plants compared to WT which was shown by stronger loss of chlorophyll. On the other hand, a positive effect was found on the concentrations of free IAA which increased at 28 °C in the GH3-doKO lines compared to WT plants. A different factor, namely darkness vs. a light/dark cycle caused the adverse phenotype concerning chlorophyll concentrations. Mutant moss plants showed higher chlorophyll concentrations than WT and these correlated with higher free IAA in the plant population that was classified as green. Our data show that growth perturbations result in higher free IAA levels in the GH3-doKO mutants, but in one case - growth in darkness - the mutants could cope better with the condition, whereas at elevated temperatures the mutants were more sensitive than WT. Thus, GH3 function in P. patens WT could lie in the regulation of IAA concentrations under unfavorable environmental conditions.

Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.