Diminished Neurogenic Femoral Artery Vasoconstrictor Response in a Zucker Obese Rat Model: Differential Regulation of NOS and COX Derivatives

Objective

Peripheral arterial disease is one of the macrovascular complications of type 2 diabetes mellitus. This study addresses femoral artery regulation in a prediabetic model of obese Zucker rats (OZR) by examining cross-talk between endothelial and neural factors.

Methods and Results

Arterial preparations from lean (LZR) and OZR were subjected to electrical field stimulation (EFS) on basal tone. Nitric oxide synthase (NOS) and cyclooxygenase (COX) isoform expression patterns were determined by immunohistochemical labelling and Western blotting. Results indicate significantly reduced noradrenergic contractions in preparations from OZR compared with those of LZR. Functional inhibition of endothelial NOS (eNOS) indicated a predominant role of this isoform in LZR and its modified activity in OZR. Neural (nNOS) and inducible NOS (iNOS) were activated and their expression was higher in femoral arteries from OZR. Neurotransmission modulated by large-conductance Ca²⁺-activated (BK_Ca) or voltage-dependent (K_V) K⁺ channels did not seem compromised in the obese animals. Endothelial COX-1 and COX-2 were expressed in LZR and an additional adventitial location of COX-2 was also observed in OZR, explaining the higher COX-2 protein levels detected in this group. Prostanoids derived from both isoforms helped maintain vasoconstriction in LZR while in OZR only COX-2 was active. Superoxide anion inhibition reduced contractions in endothelium-intact arteries from OZR.

Conclusions

Endothelial dysfunction led to reduced neurogenic vasoconstriction in femoral arteries from OZR. In a setting of obesity, NO-dependent nNOS and iNOS dilation activity could be an alternative mechanism to offset COX-2- and reactive oxygen species-mediated vasoconstriction, along with impaired endothelial NO relaxation.     

Upregulation of SK3 and IK1 Channels Contributes to the Enhanced Endothelial Calcium Signaling and the Preserved Coronary Relaxation in Obese Zucker Rats

Background and Aims

Endothelial small- and intermediate-conductance K_Ca channels, SK3 and IK1, are key mediators in the endothelium-derived hyperpolarization and relaxation of vascular smooth muscle and also in the modulation of endothelial Ca²⁺ signaling and nitric oxide (NO) release. Obesity is associated with endothelial dysfunction and impaired relaxation, although how obesity influences endothelial SK3/IK1 function is unclear. Therefore we assessed whether the role of these channels in the coronary circulation is altered in obese animals.

Methods and Results

In coronary arteries mounted in microvascular myographs, selective blockade of SK3/IK1 channels unmasked an increased contribution of these channels to the ACh- and to the exogenous NO- induced relaxations in arteries of Obese Zucker Rats (OZR) compared to Lean Zucker Rats (LZR). Relaxant responses induced by the SK3/IK1 channel activator NS309 were enhanced in OZR and NO- endothelium-dependent in LZR, whereas an additional endothelium-independent relaxant component was found in OZR. Fura2-AM fluorescence revealed a larger ACh-induced intracellular Ca²⁺ mobilization in the endothelium of coronary arteries from OZR, which was inhibited by blockade of SK3/IK1 channels in both LZR and OZR. Western blot analysis showed an increased expression of SK3/IK1 channels in coronary arteries of OZR and immunohistochemistry suggested that it takes place predominantly in the endothelial layer.

Conclusions

Obesity may induce activation of adaptive vascular mechanisms to preserve the dilator function in coronary arteries. Increased function and expression of SK3/IK1 channels by influencing endothelial Ca²⁺ dynamics might contribute to the unaltered endothelium-dependent coronary relaxation in the early stages of obesity.     

Effects of Aerobic Exercise Training on Cardiac Renin-Angiotensin System in an Obese Zucker Rat Strain

Objective

Obesity and renin angiotensin system (RAS) hyperactivity are profoundly involved in cardiovascular diseases, however aerobic exercise training (EXT) can prevent obesity and cardiac RAS activation. The study hypothesis was to investigate whether obesity and its association with EXT alter the systemic and cardiac RAS components in an obese Zucker rat strain.

Methods

The rats were divided into the following groups: Lean Zucker rats (LZR); lean Zucker rats plus EXT (LZR+EXT); obese Zucker rats (OZR) and obese Zucker rats plus EXT (OZR+EXT). EXT consisted of 10 weeks of 60-min swimming sessions, 5 days/week. At the end of the training protocol heart rate (HR), systolic blood pressure (SBP), cardiac hypertrophy (CH) and function, local and systemic components of RAS were evaluated. Also, systemic glucose, triglycerides, total cholesterol and its LDL and HDL fractions were measured.

Results

The resting HR decreased (∼12%) for both LZR+EXT and OZR+EXT. However, only the LZR+EXT reached significance (p<0.05), while a tendency was found for OZR versus OZR+EXT (p = 0.07). In addition, exercise reduced (57%) triglycerides and (61%) LDL in the OZR+EXT. The systemic angiotensin I-converting enzyme (ACE) activity did not differ regardless of obesity and EXT, however, the OZR and OZR+EXT showed (66%) and (42%), respectively, less angiotensin II (Ang II) plasma concentration when compared with LZR. Furthermore, the results showed that EXT in the OZR prevented increase in CH, cardiac ACE activity, Ang II and AT2 receptor caused by obesity. In addition, exercise augmented cardiac ACE2 in both training groups.

Conclusion

Despite the unchanged ACE and lower systemic Ang II levels in obesity, the cardiac RAS was increased in OZR and EXT in obese Zucker rats reduced some of the cardiac RAS components and prevented obesity-related CH. These results show that EXT prevented the heart RAS hyperactivity and cardiac maladaptive morphological alterations in obese Zucker rats.     

COX-2 is involved in vascular oxidative stress and endothelial dysfunction of renal interlobar arteries from obese Zucker rats

Obesity is related to vascular dysfunction through inflammation and oxidative stress and it has been identified as a risk factor for chronic renal disease. In the present study, we assessed the specific relationships among reactive oxygen species (ROS), cyclooxygenase 2 (COX-2), and endothelial dysfunction in renal interlobar arteries from a genetic model of obesity/insulin resistance, the obese Zucker rats (OZR). Relaxations to acetylcholine (ACh) were significantly reduced in renal arteries from OZR compared to their counterpart, the lean Zucker rat (LZR), suggesting endothelial dysfunction. Blockade of COX with indomethacin and with the selective blocker of COX-2 restored the relaxations to ACh in obese rats. Selective blockade of the TXA₂/PGH₂ (TP) receptor enhanced ACh relaxations only in OZR, while inhibition of the prostacyclin (PGI₂) receptor (IP) enhanced basal tone and inhibited ACh vasodilator responses only in LZR. Basal production of superoxide was increased in arteries of OZR and involved NADPH and xanthine oxidase activation and NOS uncoupling. Under conditions of NOS blockade, ACh induced vasoconstriction and increased ROS generation that were augmented in arteries from OZR and blunted by COX-2 inhibition and by the ROS scavenger tempol. Hydrogen peroxide (H₂O₂) evoked both endothelium- and vascular smooth muscle (VSM)-dependent contractions, as well as ROS generation that was reduced by COX-2 inhibition. In addition, COX-2 expression was enhanced in both VSM and endothelium of renal arteries from OZR. These results suggest that increased COX-2-dependent vasoconstriction contributes to renal endothelial dysfunction through enhanced (ROS) generation in obesity. COX-2 activity is in turn upregulated by ROS.     

A Blueberry-Enriched Diet Improves Renal Function and Reduces Oxidative Stress in Metabolic Syndrome Animals: Potential Mechanism of TLR4-MAPK Signaling Pathway

Background

Metabolic syndrome (MetS) is characterized by a cluster of health factors that indicate a higher risk for cardio-renal diseases. Recent evidence indicates that antioxidants from berries are alternative to attenuate oxidative stress and inflammation. We tested the hypothesis that inflammation-induced renal damage is triggered by the activation of TLR4, and subsequent modulation of redox-sensitive molecules and mitogen-activated protein kinase (MAPK) pathway.

Methods

Five-week old lean and obese Zucker rats (LZR and OZR) were fed a blueberry-enriched diet or an isocaloric control diet for 15 weeks. A glucose tolerance test and acute renal clearance experiments were performed. Gene and protein expression levels for TLR4, cytokines and phosphorylation of ERK and p38MAPK were measured. Kidney redox status and urinary albumin levels were quantified. Renal pathology was evaluated histologically.

Results

Control OZR exhibited lower glucose tolerance; exacerbated renal function parameters; increased oxidative stress. Gene and protein expression levels of TLR4 were higher and this was accompanied by increased renal pathology with extensive albuminuria and deterioration in antioxidant levels in OZR. In addition, OZR had increased phosphorylation of ERK and p38MAPK. Blueberry-fed OZR exhibited significant improvements in all these parameters compared to OZR.

Conclusion

TLR4-MAPK signaling pathway is a key to the renal structural injury and dysfunction in MetS and blueberry (BB) protect against this damage by inhibiting TLR4.

Significance

This is the first study to put forth a potential mechanism of TLR4-induced kidney damage in a model of MetS and to elucidate a downstream mechanism by which blueberry exert their reno-protective effects.     

Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats

This study determined alterations to hypoxic dilation of isolated skeletal muscle resistance arteries (gracilis arteries; viewed via television microscopy) from obese Zucker rats (OZR) compared with lean Zucker rats (LZR). Hypoxic dilation was reduced in OZR compared with LZR. Endothelium removal and cyclooxygenase inhibition (indomethacin) severely reduced this response in both groups, although nitric oxide synthase inhibition (N(omega)-nitro-L-arginine methyl ester) reduced dilation in LZR only. Treatment of vessels with a PGH(2)-thromboxane A(2) receptor antagonist had no effect on hypoxic dilation in either group. Arterial dilation to arachidonic acid, iloprost, acetylcholine, and sodium nitroprusside was reduced in OZR versus LZR, although dilation to forskolin and aprikalim was unaltered. Treatment of arteries from OZR with oxidative radical scavengers increased dilation to hypoxia and agonists, with no effect on responses in LZR. The restored hypoxic dilation in OZR was abolished by indomethacin. These results suggest that hypoxic dilation of skeletal muscle microvessels from LZR represents the summated effects of prostanoid and nitric oxide release, whereas the impaired response of vessels in OZR may reflect scavenging of PGI(2) by superoxide anion.     

Reduced constrictor reactivity balances impaired vasodilation in the mesenteric circulation of the obese Zucker rat

Obesity causes whole body insulin resistance and impaired vasodilation to nitric oxide (NO). Because NO is a major contributor to the regulation of mesenteric blood flow, the mesenteric circulation of obese animals is faced with reduced capacity to increase flow and increased demand for flow associated with elevated consumption of food. This study hypothesized that insulin resistance impairs NO-mediated dilation but that constrictor reactivity would be reduced to compensate in obese animals. We further hypothesized that elevated superoxide levels caused impaired responses to NO in insulin resistance. Vasodilator reactivity and vasoconstrictor reactivity of mesenteric resistance arteries from lean (LZR) and obese (OZR) Zucker rats were examined in vitro using videomicroscopy. Insulin resistance independent of obesity was induced via fructose feeding in LZR (FF-LZR). Endothelium-dependent NO-mediated dilation was reduced in OZR and FF-LZR compared with LZR. Impairments in NO-mediated dilation were reversed with 1 mM tempol, a SOD mimetic. Constrictor reactivity to norepinephrine was reduced in OZR but not in FF-LZR relative to LZR. Basal mesenteric vascular resistance was similar in LZR and OZR despite impaired NO-dependent dilation in OZR. Mesenteric vascular resistance was increased in FF-LZR relative to LZR. These data indicate that there is reduced constrictor reactivity in OZR that may offset the impaired NO-mediated dilation and preserve mesenteric blood flow in hyperphagic, obese animals.     

Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

Objectives

The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications.

Methods

Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O₂ ^.–), NA and ATP releases were also determined.

Results

EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O₂ ^.– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals.

Conclusion

Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.     

Oxidant stress and constrictor reactivity impair cerebral artery dilation in obese Zucker rats

This study tested the hypothesis that evolution of the metabolic syndrome in obese Zucker rats (OZR) leads to impaired dilator reactivity of cerebral resistance arteries vs. responses determined in lean Zucker rats (LZR). Middle cerebral arteries (MCA) from 17-wk-old male LZR and OZR were isolated and cannulated with glass micropipettes. Vascular reactivity was assessed in response to challenge with ACh, sodium nitroprusside (SNP), reductions and elevations in Po2, 5-HT, and increased intralumenal pressure. Vessels were treated with the free radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) to assess the role of superoxide production in altering reactivity, and passive vascular wall mechanics was assessed in each vessel. Vascular superoxide production was assessed in isolated arteries using fluorescence microscopy. Vessel dilation to ACh and hypoxia was impaired in OZR vs. LZR, although responses to SNP were normal. Vessel constriction to 5-HT, elevated Po2, and elevated intralumenal pressure was enhanced in OZR vs. LZR. Fluorescence microscopy demonstrated an increased superoxide production in arteries of OZR vs. LZR, correctable by incubation with tempol. Although treatment of vessels from OZR with tempol improved dilation to ACh and hypoxia, constrictor responses to 5-HT, elevated Po2, and pressure were not altered by tempol treatment. Indexes of vessel wall mechanics were comparable between groups. These results suggest that vasodilator reactivity of MCA of OZR in response to endothelium-dependent dilator stimuli is impaired vs. LZR and that this may represent a reduced bioavailability of signaling molecules due to oxidant scavenging. However, oxidative stress-independent increases in myogenic tone and constrictor reactivity may contribute to blunted dilator responses of cerebral microvessels.     

Augmented adrenergic vasoconstriction in hypertensive diabetic obese Zucker rats

This study examined skeletal muscle microvessel reactivity to constrictor stimuli in obese (OZR) versus lean Zucker rats (LZR). Gracilis arteries from both rat groups were isolated, cannulated with glass micropipettes, and viewed via television microscopy. Changes in vessel diameter were measured with a video micrometer. Arterial constriction to norepinephrine was elevated in OZR versus LZR, although vasoconstrictor reactivity to endothelin and angiotensin II was unaltered. Differences in reactivity between vessels of LZR and OZR were not explained by the loss of either endothelial nitric oxide synthase or beta-adrenergic receptor function. Reactivity of in situ cremasteric arterioles of OZR to norepinephrine was elevated versus LZR. Treatment with prazosin increased the diameter of in vivo gracilis arteries of OZR to levels determined in LZR and also normalized blood pressure in OZR. These results suggest that the constrictor reactivity of skeletal muscle microvessels in OZR is heightened in response to alpha-adrenergic stimuli and that development of diabetes in OZR may be associated with impaired skeletal muscle perfusion and hypertension due to microvessel hyperreactivity in response to sympathetic stimulation.     

Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats

This study characterized myogenic activation of skeletal muscle (gracilis) resistance arteries from lean (LZR) and obese Zucker rats (OZR). Arteries from OZR exhibited increased myogenic activation versus LZR; this increase was impaired by endothelium denudation or nitric oxde synthase inhibition. Treatment of vessels with 17-octadecynoic acid impaired responses in both strains by comparable amounts. Dihydroethidine microfluorography indicated elevated vascular superoxide levels in OZR versus LZR; immunohistochemistry demonstrated elevated vascular nitrotyrosine levels in OZR, indicating increased peroxynitrite presence. Vessel treatment with oxidative radical scavengers (polythylene glycol-superoxide dismutase/catalase) or inhibition of Ca(2+)-activated K(+) (K(Ca)) channels (iberiotoxin) did not alter myogenic activation in LZR but normalized activation in OZR. Application of peroxynitrite to vessels of OZR caused a greater vasoconstriction versus LZR; the response was impaired in OZR by elevated intraluminal pressure and was abolished in both strains by iberiotoxin. These results suggest that enhanced myogenic activation of gracilis arteries of OZR versus LZR 1) is not due to alterations in cytochrome P-450 contribution, and 2) may be due to elevated peroxynitrite levels inhibiting K(Ca) channels following increased intraluminal pressure.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.     

Deregulation of Hepatic Insulin Sensitivity Induced by Central Lipid Infusion in Rats Is Mediated by Nitric Oxide

Background

Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis.

Methodology

We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats).

Principal Findings

Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid).

Conclusions/Significance

We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.     

Impaired hemorrhage tolerance in the obese Zucker rat model of metabolic syndrome.

As obese Zucker rats (OZR) manifesting the metabolic syndrome exhibit enhanced vascular adrenergic constriction and potentially an enhanced adrenergic activity vs. lean Zucker rats (LZR), this study tested the hypothesis that OZR exhibit an improved tolerance to progressive hemorrhage. Preliminary experiments indicated that, corrected for body mass, total blood volume was reduced in OZR vs. LZR. Anesthetized LZR and OZR had a cremaster muscle prepared for in situ videomicroscopy and had renal, splanchnic, hindlimb, and skeletal muscle perfusion monitored with flow probes. Arterial pressure, arteriolar reactivity to norepinephrine, and tissue/organ perfusion were monitored after either infusion of phentolamine or successive withdrawals of 10% total blood volume. Phentolamine infusion indicated that regional adrenergic tone under control conditions differs substantially between LZR and OZR, whereas with hemorrhage OZR exhibit decompensation in arterial pressure before LZR. Renal, distal hindlimb, and skeletal muscle perfusion decreased more rapidly and to a greater extent in OZR vs. LZR after hemorrhage. In contrast, hemorrhage-induced reductions in splanchnic perfusion in OZR lagged behind those in LZR, although a similar maximum reduction was ultimately attained. With increasing hemorrhage, cremasteric arteriolar tone increased more in OZR than LZR, and this increase in active tone was entirely due to an elevated adrenergic contribution. Norepinephrine-induced arteriolar constriction was greater in OZR vs. LZR under control conditions and during hemorrhage, with arterioles from OZR demonstrating early closure vs. LZR. These results suggest that a combination of reduced blood volume and elevated peripheral adrenergic constriction contribute to impaired hemorrhage tolerance in OZR.     

Alterations in Perivascular Sympathetic and Nitrergic Innervation Function Induced by Late Pregnancy in Rat Mesenteric Arteries

Background and Purpose

We investigated whether pregnancy was associated with changed function in components of perivascular mesenteric innervation and the mechanism/s involved.

Experimental Approach

We used superior mesenteric arteries from female Sprague-Dawley rats divided into two groups: control rats (in oestrous phase) and pregnant rats (20 days of pregnancy). Modifications in the vasoconstrictor response to electrical field stimulation (EFS) were analysed in the presence/absence of phentolamine (alpha-adrenoceptor antagonist) or L-NAME (nitric oxide synthase-NOS- non-specific inhibitor). Vasomotor responses to noradrenaline (NA), and to NO donor DEA-NO were studied, NA and NO release measured and neuronal NOS (nNOS) expression/activation analysed.

Key Results

EFS induced a lower frequency-dependent contraction in pregnant than in control rats. Phentolamine decreased EFS-induced vasoconstriction in segments from both experimental groups, but to a greater extent in control rats. EFS-induced vasoconstriction was increased by L-NAME in arteries from both experimental groups. This increase was greater in segments from pregnant rats. Pregnancy decreased NA release while increasing NO release. nNOS expression was not modified but nNOS activation was increased by pregnancy. Pregnancy decreased NA-induced vasoconstriction response and did not modify DEA-NO-induced vasodilation response.

Conclusions and Implications

Neural control of mesenteric vasomotor tone was altered by pregnancy. Diminished sympathetic and enhanced nitrergic components both contributed to the decreased vasoconstriction response to EFS during pregnancy. All these changes indicate the selective participation of sympathetic and nitrergic innervations in vascular adaptations produced during pregnancy.     

Hepatic-dependent and -independent insulin actions are impaired in the obese Zucker rat model

Objective: Whole‐body insulin sensitivity (IS) depends on a hepatic pathway, involving parasympathetic activation and hepatic nitric oxide (NO) production. Both atropine and N‐monomethyl‐l ‐arginine (l ‐NMMA, NO synthase inhibitor) induce insulin resistance (IR). IR is associated with obesity. Because NO action was shown to be impaired in animal models of obesity, such as the obese Zucker rat (OZR), we tested the hypothesis that the hepatic‐dependent pathway is diminished in OZR, resulting in IR. Research Methods and Procedures: Lean Zucker rats (LZRs) were used as OZR controls. IS was evaluated in terms of glucose disposal [milligrams of glucose per kilogram of body weight (bw)]. Two groups were submitted to two protocols. First, a control clamp was followed by a post‐atropine (3 mg/kg intravenously) clamp. Second, after the control clamp, l ‐NMMA (0.73 mg/kg intraportally) was given, and a second clamp was performed. Hepatic‐dependent IS was assessed by subtracting the response after atropine or l ‐NMMA from the basal response. Results: In the first protocol, basal IS was lower in OZR than in LZR (OZR, 73.7 ± 14.2; LZR, 289.2 ± 24.7 mg glucose/kg bw; p < 0.001), and atropine decreased IS in the same proportion for both groups (OZR, 41.3 ± 8.0%; LZR, 40.1 ± 6.5%). Equally, in the second protocol, OZR presented lower IS (OZR, 79.3 ± 1.6; LZR, 287.4 ± 22.7 mg glucose/kg bw; p < 0.001). l ‐NMMA induced IS inhibition in both groups (OZR, 48.3 ± 6.6%; LZR, 46.4 ± 4.1%), similar to that after atropine. Discussion: We show that the IR in OZR is due to similar impairment of both hepatic‐dependent and ‐independent components of insulin action, suggesting the existence of a defect common to both pathways.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats

This study determined alterations to nitric oxide (NO)-dependent dilation of skeletal muscle arterioles from obese (OZR) versus lean Zucker rats (LZR). In situ cremaster muscle arterioles from both groups were viewed via television microscopy, and vessel dilation was measured with a video micrometer. Arteriolar dilation to acetylcholine and sodium nitroprusside was reduced in OZR versus LZR, although dilation to aprikalim was unaltered. NO-dependent flow-induced arteriolar dilation (via parallel microvessel occlusion) was attenuated in OZR, impairing arteriolar ability to regulate wall shear rate. Vascular superoxide levels, as assessed by dihydroethidine fluorescence, were elevated in OZR versus LZR. Treatment of cremaster muscles of OZR with the superoxide scavengers polyethylene glycol-superoxide dismutase and catalase improved arteriolar dilation to acetylcholine and sodium nitroprusside and restored flow-induced dilation and microvascular ability to regulate wall shear rate. These results suggest that NO-dependent dilation of skeletal muscle microvessels in OZR is impaired due to increased levels of superoxide. Taken together, these data suggest that the development of diabetes and hypertension in OZR may be associated with an impaired skeletal muscle perfusion via an elevated vascular oxidant stress.     

Mitochondrial apoptotic signaling is elevated in cardiac but not skeletal muscle in the obese Zucker rat and is reduced with aerobic exercise

Mitochondrial apoptosis and apoptotic signaling modulations by aerobic training were studied in cardiac and skeletal muscles of obese Zucker rats (OZR), a rodent model of metabolic syndrome. Comparisons were made between left ventricle, soleus, and gastrocnemius muscles from OZR (n = 16) and aged-matched lean Zucker rats (LZR; n = 16) that were untrained (n = 8) or aerobically trained on a treadmill for 9 wk (n = 8). Cardiac Bcl-2 protein expression levels were approximately 50% lower in the OZR compared with the LZR, with no difference in either of the skeletal muscles. Bax protein expression levels were similar in skeletal muscles of the OZR compared with the LZR. Furthermore, mitochondrial apoptotic signaling was not different in skeletal muscles of OZR and LZR groups. However, there was an approximate sevenfold increase in the Bax protein accumulation in the myocardial mitochondrial-rich protein fraction of the OZR compared with the LZR. Additionally, there was an increase in cytosolic cytochrome c released from the mitochondria, caspase-9 and caspase-3 activity, with a corresponding elevation in DNA fragmentation in the cardiac muscles of the OZR compared with the LZR. Exercise training reduced cardiac Bax protein levels, the mitochondrial localization of Bax, cytosolic cytochrome c, caspase activity, and DNA fragmentation in cardiac muscles of the OZR after exercise, with no change in the skeletal muscles. These data show that mitochondrial apoptosis is elevated in the cardiac but not skeletal muscles of the OZR, but aerobic exercise training was effective in reducing cardiac mitochondrial apoptotic signaling.     

Effects of Intracerebroventricular Infusion of Leptin in Obese Zucker Rats

AL-BARAZANJI, KAMAL A, ROBIN E BUCKINGHAM, JONATHAN RS ARCH, ANDREA HAYNES, DANUTA E MOSSAKOWSKA, DIANE L McBAY, STEPHEN D HOLMES, MARK T McHALE, XIN-MIN WANG, ISRAEL S GLOGER. Effects of intracerebro-ventricular infusion of leptin in obese Zucker rats. The obese Zucker rat (OZR) exhibits a missense mutation in the cDNA for the leptin receptor, producing a single amino acid substitution in the extracellular domain of the receptor. A mutation in the leptin receptor gene of the db/db mouse prevents the synthesis of the long splice variant of the receptor. The possibility that the OZR, like the db/db mouse, is refractory to the actions of murine leptin was tested by infusing the protein intracerebroventricularly via a minipump for 7 days. Lean Zucker rats (LZR) infused with leptin acted as positive controls, and other groups of OZR and LZR were infused with vehicle. In LZR, leptin reduced body-weight and food intake and increased brown adipose tissue (BAT) temperature. Plasma corticosterone increased (61%) in these rats, and plasma triglycerides fell (78%). Leptin treatment improved tolerance to an oral glucose load (16% reduction in the area under the blood glucose curve) while lowering plasma insulin. In OZR, the actions of leptin were blunted. Food intake was slightly, but not significantly, reduced. Although there was a reduction in the rate of increase in body mass, the effect of leptin was about half that seen in LZR. BAT temperature and glucose tolerance were unchanged. In contrast to the elevated plasma corticosterone seen in LZR, leptin reduced the level of this hormone (27%) in OZR. In OZR and LZR treated with leptin, the plasma leptin levels were increased 24-fold and 47-fold, respectively. The results suggest that leptin retains some efficacy in OZR, although these rats are less responsive than LZR.