Skeletal muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibiting impaired glucose homeostasis

OBJECTIVE: To investigate whether changes in body energy balance induced by long-term high-fat feeding in adult rats could be associated with modifications in energetic behaviour and oxidative stress of skeletal muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial populations. DESIGN: Adult rats were fed low-fat or high-fat diet for 7 weeks. MEASUREMENTS: Body energy balance and composition analysis together with plasma insulin and glucose level determination in the whole animal. Oxidative capacity, basal and induced proton leaks as well as aconitase and superoxide dismutase activities in SS and IMF mitochondria from skeletal muscle. RESULTS: High-fat fed rats exhibit increased body lipid content, as well as hyperinsulinemia, hyperglycaemia and higher plasma non-esterified fatty acids. In addition, SS mitochondria display lower respiratory capacity and a different behaviour of SS and IMF mitochondria is found in the prevention from oxidative damage. CONCLUSIONS: A deleterious consequence of decreased oxidative capacity in SS mitochondria from rats fed high-fat diet would be a reduced utilization of energy substrates, especially fatty acids, which may lead to intracellular triglyceride accumulation, lipotoxicity and insulin resistance development. Our results thus reveal a possible role for SS mitochondria in the impairment of glucose homeostasis induced by high-fat diet.     

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso–redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso–redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO_2max. This study demonstrates that exercise results in a positive shift in the nitroso–redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.     

Insulin resistance is associated with impaired nitric oxide synthase activity in skeletal muscle of type 2 diabetic subjects

Type 2 diabetes is an insulin-resistant state characterized by hyperinsulinemia and accelerated atherosclerosis. In vitro and in vivo studies in rodents have suggested that nitric oxide generation plays an important role in glucose transport and insulin action. We determined nitric oxide synthase (NOS) activity in skeletal muscle of 10 type 2 diabetic (hemoglobin A(1C) = 6.8 +/- 0.1%) and 11 control subjects under basal conditions and during an 80 mU/m(2).min euglycemic insulin clamp performed with vastus lateralis muscle biopsies before and after 4 h of insulin. In diabetics, insulin-stimulated glucose disposal (Rd) was reduced by 50%, compared with controls (5.4 +/- 0.3 vs. 10.4 +/- 0.5 mg/kg.min, P < 0.01). Basal NOS activity was markedly reduced in the diabetic group (101 +/- 33 vs. 457 +/- 164 pmol/min.mg protein, P < 0.05). In response to insulin, NOS activity increased 2.5-fold in controls after 4 h (934 +/- 282 pmol/min.mg protein, P < 0.05 vs. basal), whereas insulin failed to stimulate NOS activity in diabetics (86 +/- 28 pmol/min.mg protein, P = NS from basal). Basal NOS protein content in muscle was similar in controls and diabetics and did not change following insulin. In controls, insulin-stimulated NOS activity correlated inversely with fasting plasma insulin concentration (r = -0.58, P = 0.05) and positively with Rd (r = 0.71, P = 0.03). In control and diabetic groups collectively, Rd correlated with insulin-stimulated NOS activity (r = 0.52, P = 0.02). We conclude that basal and insulin-stimulated muscle NOS activity is impaired in well-controlled type 2 diabetic subjects, and the defect in insulin-stimulated NOS activity correlates closely with the severity of insulin resistance. These results suggest that impaired NOS activity may play an important role in the insulin resistance in type 2 diabetic individuals.     

Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in Langendorff mouse heart

Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (-/-)], and its heterozygote [ecNOS (+/-)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (-/-), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g. beats/min, L-NAME increased glucose uptake from 0.1+/-0.1 to 3+/-0.4 microg/min x mg in ecNOS (+/-) mice and from 0.2+/-0.1 to 2.7+/-0.7 microg/min x mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (-/-) mice, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog or S-nitroso-N-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/-) mice, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.     

Estrogen stimulates neuronal nitric oxide synthase protein expression in human neutrophils

Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17beta-estradiol (10(-10) to 10(-8) mol/L) upregulated the expression of nNOS protein. The 17beta-estradiol receptor antagonists, tamoxifen (10(-8) mol/L) and ICI 182780 (10(-8) mol/L), inhibited the upregulation of nNOS protein induced by 17beta-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17beta-estradiol-incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17beta-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17beta-estradiol-incubated neutrophils derived from men to produce NO reduced their adhesive properties.     

Exhaled nitric oxide is not reduced in infants with cystic fibrosis.

Fractional exhaled nitric oxide (F(eNO)) has been reported to be reduced in cystic fibrosis (CF) patients. However, data from young children are conflicting and it is not clear whether this is a primary feature of the disease or a secondary response. The present study compared F(eNO) between CF and healthy infants using a validated single-breath technique. A total of 23 healthy infants (11 females; mean age 40.1 weeks) and 18 infants with CF (nine females; 64.9 weeks) underwent tests of lung function and F(eNO). Bronchoalveolar lavage (BAL) was collected from all CF infants 2-5 days after lung function testing. There was no significant difference in F(eNO) between the CF and healthy infants (geometric mean: 23.1 parts per billion (ppb) and 17.0 ppb, respectively). There was an inverse relationship between age and F(eNO) in the CF patients, but not in the healthy group. Within the CF group, there was no association between F(eNO) and any marker of airway inflammation measured in the BAL. Exhaled nitric oxide is not reduced in cystic fibrosis infants, but does decrease with age. The current data indicate that F(eNO) is not a good marker of airway inflammation in cystic fibrosis.     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

Skeletal muscle inflammation and nitric oxide in patients with COPD.

In chronic obstructive pulmonary disease (COPD) the presence of systemic inflammation has been associated with peripheral muscle abnormalities and weight loss. To study whether inflammatory factors are important in these processes, the present study compared the skeletal muscle levels of nitrite, nitrate, nitrotyrosine, neuronal, endothelial and inducible nitric oxide synthases (nNOS, eNOS, and iNOS, respectively), and inflammatory markers (tumour necrosis factor (TNF)-alpha, CD154 and CD163) in 15 patients (forced expiratory volume in one second 43+/-11%) and 14 controls. All these markers were also compared between patients with normal and low body weight. Nitrite (12.5+/-2.6 versus 17.0+/-3.4 micromol.mg(-1) protein), nitrate (20.7+/-2.4 versus 24.4+/-4.5 micromol.mg(-1) protein) and eNOS (31.9+/-4.6 versus 43.6+/-7.5 ng.mg(-1) protein) were lower in COPD patients than in controls. Nitrotyrosine (25.6+/-5.4 versus 6.6+/-3.3 ng.mg(-1) protein), iNOS expression (32+/-9.5 versus 7.16+/-2.7 ng.mg(-1) protein), TNF-alpha (257+/-160 versus 48.3+/-4.4 pg.mg(-1) protein) and CD163 (6.4+/-2.1 versus 0.8+/-0.4 ng.mg(-1) protein) were higher in COPD patients than in controls. CD154 levels were 15.7+/-7.0 ng.mg(-1) protein in COPD patients and undetectable in controls. Similar levels of all these markers were observed in COPD patients with normal and low body weight. In conclusion, these findings suggest the presence of an inflammatory process in the muscle tissue of chronic obstructive pulmonary disease patients, and argue in favour of its participation in the pathogenesis of skeletal muscle abnormalities.     

Endothelial nitric oxide synthase protein is reduced in the renal medulla of two-kidney, one-clip hypertensive rats

OBJECTIVE : We studied endothelial nitric oxide synthase (eNOS) expression in the kidneys of two-kidney, one-clip renal hypertensive rats (2K1C) before and after removal of the clip (unclipping, UC). We hypothesised that the haemodynamic changes induced by 2K1C and UC would change eNOS expression in the two kidneys. METHODS : Six weeks after inducing 2K1C, mean arterial pressure (MAP) was measured in conscious rats and hypertension reversed by UC. Left and right kidney eNOS protein in cortex and outer medulla was semi-quantified using immunoblotting. Groups were; normotensive (n = 10), 2K1C (n = 10), 3 h (n = 10), 48 h (n = 7) and 4 weeks (n = 7) after UC. The effect of 7 days of aldosterone or angiotensin II (Ang II) infusion on medullary eNOS protein was tested as well as the effect of L-NAME (nitric oxide (NO) synthase inhibitor) on medullary blood flow (MBF) in anaesthetized 2K1C. RESULTS : UC reduced MAP from 178 +/- 5 to 134 +/- 3 mmHg after 3 h and normalized MAP at 48 h and 4 weeks. The medulla from 2K1C kidneys contained about 33% less eNOS protein compared with normotensive kidneys (P < 0.05). This difference was still evident at 3 h (P < 0.05), but completely reversed at 48 h and 4 weeks after UC. Similar levels of eNOS expression were seen in the left and right kidney at all time points. Cortical eNOS was increased in kidneys from 2K1C. Neither Ang II nor aldosterone affected eNOS expression in the medulla. MBF was under similar influence of NO in 2K1C compared with normotensive kidneys. CONCLUSIONS : 2K1C is associated with reduced levels of eNOS protein in the renal medulla of both clipped and contralateral kidney. eNOS expression in right and left kidney was not changed despite expected large changes in haemodynamics of the two kidneys. The reduced level of eNOS may be associated with a reduction in MBF and thus be of patho-physiological importance in renovascular hypertension.     

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Aims/hypothesis

Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect.

Methods

Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays.

Results

nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes.

Conclusions/interpretation

Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.     

Brain neuronal nitric oxide synthase neuron-mediated sympathoinhibition is enhanced in hypertensive Dahl rats

OBJECTIVE: To elucidate the role of central neurons containing neuronal nitric oxide synthase (nNOS neurons) in the sympathetic nervous system in hypertensive Dahl salt-sensitive (DS) rats. DESIGN AND METHODS: Dahl rats were fed either a regular-salt (0.4% NaCl) or high-salt (8% NaCl) diet for 4 weeks. The effect of intracerebroventricular administration of S-methyl-L-thiocitrulline, a selective nNOS inhibitor, on renal sympathetic nerve activity was examined in chronically instrumented conscious DS rats. The activity and protein amount of brain nNOS was evaluated by enzyme assay and western blot analysis. The distribution and number of nNOS neurons in the brainstem were examined immunohistochemically in hypertensive and normotensive DS rats. RESULTS: S-methyl-L-thiocitrulline induced a larger increase in tonic renal sympathetic nerve activity generated before baroreflex-mediated inhibition in hypertensive DS rats than normotensive DS rats. Hypertensive DS rats showed increased nNOS activity in the brainstem, but not in the diencephalon or cerebellum. High nNOS activity was confirmed by an increase in the amount of nNOS protein. nNOS Neurons were localized in several nuclei throughout the brainstem; the dorsolateral periaqueductal gray, pedunculopontine tegmental nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, lateral parabrachial nucleus, rostral ventrolateral medulla, nucleus tractus solitarius and raphe magnus. The number of nNOS neurons in these nuclei, except for the two raphes, was significantly greater in hypertensive than in normotensive DS rats. CONCLUSIONS: These findings suggest that central nNOS-mediated sympathoinhibition may be enhanced in salt-sensitive hypertensive Dahl rats. The upregulated nNOS-mediated inhibition may occur in the central sympathetic control system generated before baroreflex-mediated inhibition.     

Exhaled nitric oxide is reduced in infants with rhinorrhea

In infants, the effect of colds and other respiratory tract infections (RTI) on exhaled nitric oxide (FE(NO)) is not clear. In this study, we measured FE(NO) in 24 infants (14 boys) who presented with rhinorrhea, with or without cough but not wheeze. Twelve of these infants had a history of recurrent wheeze. Levels were compared with a group of 23 healthy infants (13 boys). Further, 8 infants (5 with a history of recurrent wheeze) with rhinorrhea were tested after symptoms had resolved. Infants with rhinorrhea had significantly lower FE(NO) than the healthy infants (11.9 vs. 23.8 ppb, respectively, P < 0.0007). Levels increased from 7.5 ppb to 34.1 ppb in the 8 infants tested with and without symptoms (P = 0.0002). Infants with rhinorrhea have reduced FE(NO), irrespective of their respiratory history.     

Regulation of bone mass and bone turnover by neuronal nitric oxide synthase

Nitric oxide (NO) is produced by NO synthase (NOS) and plays an important role in the regulation of bone cell function. The endothelial NOS isoform is essential for normal osteoblast function, whereas the inducible NOS isoform acts as a mediator of cytokine effects in bone. The role of the neuronal isoform of NOS (nNOS) in bone has been studied little thus far. Therefore, we investigated the role of nNOS in bone metabolism by studying mice with targeted inactivation of the nNOS gene. Bone mineral density (BMD) was significantly higher in nNOS knockout (KO) mice compared with wild-type controls, particularly the trabecular BMD (P < 0.01). The difference in BMD between nNOS KO and control mice was confirmed by histomorphometric analysis, which showed a 67% increase in trabecular bone volume in nNOS KO mice when compared with controls (P < 0.001). This was accompanied by reduced bone remodeling, with a significant reduction in osteoblast numbers and bone formation surfaces and a reduction in osteoclast numbers and bone resorption surfaces. Osteoblasts from nNOS KO mice, however, showed increased levels of alkaline phosphatase and no defects in proliferation or bone nodule formation in vitro, whereas osteoclastogenesis was increased in nNOS KO bone marrow cultures. These studies indicate that nNOS plays a hitherto unrecognized but important physiological role as a stimulator of bone turnover. The low level of nNOS expression in bone and the in vitro behavior of nNOS KO bone cells indicate that these actions are indirect and possibly mediated by a neurogenic relay.     

Expression of nitric oxide synthase isoforms in the human placenta is not altered by labor

Nitric oxide has various biological activities including smooth muscle relaxation, anti-inflammatory activity, anti-coagulatory activity. As the human placenta is known to express nitric oxide synthases, this study investigated the possible effect of labor on the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in human placental tissues at term. Both eNOS and iNOS mRNA expression in placental tissues in labor were significantly higher than those in the amnion, chorion laeve, decidua vera and myometrium. The eNOS mRNA and protein expressions in placental tissues in labor (n = 12) were 1.6023 +/- 0.1652 (eNOS/GAPDH, mean +/- SEM) and 12.8 +/- 1.3 arbitrary units (AU), respectively, which were similar to those not in labor (n = 10), 1.5806 +/- 0.2042 (eNOS/GAPDH) and 11.4 +/- 1.8 AU. The iNOS mRNA and protein expressions in the placental tissues in labor were 1.2831 +/- 0.2436 (iNOS/GAPDH) and 10.7 +/- 2.1 AU respectively, similar to those not in labor, 1.9254 +/- 0.8004 (iNOS/GAPDH) and 13.3 +/- 1.8 AU. The guanosine 3',5'-cyclic monophosphate (cGMP) concentration in the placental tissues in labor was 23.6 +/- 1.4 fmol/g wet tissue, similar to that not in labor, 26.1 +/- 2.0 fmol/g wet tissue. These findings suggest that nitric oxide production in the human placenta is maintained during labor.     

Inhibition of neuronal nitric oxide synthase activity does not alter vasopressin secretion in septic rats

Background/Purpose

During the early phase of sepsis, hypotension is accompanied by increase of plasma vasopressin hormone (AVP) levels, which decline during the late phase. This hypotension is due in part to increase of nitric oxide (NO) synthesis by nitric oxide synthase (NOS) enzyme. Neuronal isoform of this enzyme (nNOS) is present in vasopressinergics neurons of hypothalamus, but its role in vasopressin secretion during sepsis is unknown.

Methods

We evaluated the role of nNOS in NO production and vasopressin secretion during sepsis. Wistar rats received 7-nitroindazole (50 mg/kg, i.p.), an inhibitor of nNOS activity, or vehicle and were submitted to septic stimulus by cecal ligation and puncture (CLP). At the time points 0, 4, 6, 18 and 24 h after sepsis induction the animals were decapitated and neurohypophysis and hypothalamus were removed for analysis of vasopressin content and NOS activity, respectively. Hematocrit, serum sodium, osmolality, proteins and plasmatic AVP were quantified.

Results

Mortality was not affected by 7-nitroindazole (7-NI). Sodium and plasma proteins levels decreased after CLP and the treatment anticipated the protein loss, and delayed serum sodium decrease. Septic animals treated with 7-NI showed decrease of osmolality 4 h after CLP. Nitric oxide synthase activity in hypothalamus increased at 4 and 24 h after CLP and was reduced with 7-NI. Neurohypophysis content of AVP diminished after CLP and 7-NI did not alter this parameter. Plasma AVP levels increased at 6 h and decreased 18 and 24 h after CLP. Treatment with 7-NI did not alter plasma vasopressin levels.

Conclusion

We concluded that nNOS does not have a substantial role in vasopressin secretion during experimental sepsis.