AJP: Regulatory, Integrative and Comparative Physiology recent issues

Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure

2008-08-08

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed β-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

AT1 receptor participates in the cardiac hypertrophy induced by resistance training in rats

Barauna, V. G., Magalhaes, F. C., Krieger, J. E., Oliveira, E. M. — 2008-08-08

Resistance training is accompanied by cardiac hypertrophy, but the role of the renin-angiotensin system (RAS) in this response is elusive. We evaluated this question in 36 male Wistar rats divided into six groups: control (n = 6); trained (n = 6); control + losartan (10 mg·kg^–1·day^–1, n = 6); trained + losartan (n = 6); control + high-salt diet (1%, n = 6); and trained + high-salt diet (1%, n = 6). High salt was used to inhibit the systemic RAS and losartan to block the AT₁ receptor. The exercise protocol consisted of: 4 x 12 bouts, 5x/wk during 8 wk, with 65–75% of one repetition maximum. Left ventricle weight-to-body weight ratio increased only in trained and trained + high-salt diet groups (8.5% and 10.6%, P < 0.05) compared with control. Also, none of the pathological cardiac hypertrophy markers, atrial natriuretic peptide, and MHC (-myosin heavy chain)-to-βMHC ratio, were changed. ACE activity was analyzed by fluorometric assay (systemic and cardiac) and plasma renin activity (PRA) by RIA and remained unchanged upon resistance training, whereas PRA decreased significantly with the high-salt diet. Interestingly, using Western blot analysis and RT-PRC, no changes were observed in cardiac AT₂ receptor levels, whereas the AT₁ receptor gene (56%, P < 0.05) and protein (31%, P < 0.05) expressions were upregulated in the trained group. Also, cardiac ANG II concentration evaluated by ELISA remained unchanged (23.27 ± 2.4 vs. 22.01 ± 0.8 pg/mg, P > 0.05). Administration of a subhypotensive dose of losartan prevented left ventricle hypertrophy in response to the resistance training. Altogether, we provide evidence that resistance training-induced cardiac hypertrophy is accompanied by induction of AT₁ receptor expression with no changes in cardiac ANG II, which suggests a local activation of the RAS consistent with the hypertrophic response.

Insulin acts at different CNS sites to decrease acute sucrose intake and sucrose self-administration in rats

Figlewicz, D. P., Bennett, J. L., Aliakbari, S., Zavosh, A., Sipols, A. J. — 2008-08-08

Findings from our laboratory and others have demonstrated that the hormone insulin has chronic effects within the CNS to regulate energy homeostasis and to decrease brain reward function. In this study, we compared the acute action of insulin to decrease intake of a palatable food in two different behavioral tasks—progressive ratios sucrose self-administration and mu opioid-stimulated sucrose feeding—when administered into several insulin-receptive sites of the CNS. We tested insulin efficacy within the medial hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, the nucleus accumbens, and the ventral tegmental area. Administration of insulin at a dose that has no chronic effect on body weight (5 mU) into the ARC significantly suppressed sucrose self-administration (75 ± 5% of paired control). However, although the mu opioid DAMGO, [d-Ala2,N-MePhe4,Gly5-ol]-enkephalin acetate salt, stimulated sucrose intake at all four CNS sites, the ventral tegmental area was the only sensitive site for a direct effect of insulin to antagonize acute (60 min) mu opioid-stimulated sucrose feeding: sucrose intake was 53 ± 8% of DAMGO-induced feeding, when insulin was coadministered with DAMGO. These findings demonstrate that free feeding of sucrose, and motivated work for sucrose, can be modulated within unique sites of the CNS reward circuitry. Further, they support the interpretation that adiposity signals, such as insulin, can decrease different aspects of ingestion of a palatable food, such as sucrose, in an anatomically specific manner.

NEFA minimal model parameters estimated from the oral glucose tolerance test and the meal tolerance test

Boston, R. C., Moate, P. J. — 2008-08-08

The kinetics of nonesterified fatty acid (NEFA) metabolism in humans requires quantification to facilitate understanding of diseases like type 1 and 2 diabetes, metabolic syndrome, and obesity, and the mechanisms underpinning various interventions. Oral glucose tolerance tests (OGTT) and glucose meal tolerance tests (MTT) are potentially useful procedures for enabling quantification of NEFA kinetics because they both cause transitory, but substantial, declines and then rebounds in plasma NEFA concentrations in response to physiologically relevant increases in plasma glucose. The Boston MINIMAL model of NEFA kinetics was developed to analyze data from the intravenous glucose tolerance test (IVGTT), but in this work, we present for the first time its application to modeling NEFA data from both OGTT and MTT studies. This model enables estimation of S_FFA (µmol·l^–1·min^–1) (a parameter describing the maximum rate of lipolysis), and K_FFA (%/min) (a parameter related to NEFA oxidation rate). The model could well describe the trajectories of NEFA concentrations following an OGTT (R² in excess of 0.97) but was not as successful with the MTT (R² > 0.65). Model parameters derived from analysis of OGTT and MTT data were well identified with coefficients of variation generally less than 15%. Type 2 diabetes, body mass index, and dietary treatment (high-fat vs. high-glycemic-index diets) were all shown to have significant effects on model parameters. Modeling plasma NEFA concentrations over 24 h has helped to identify and quantify the extent that periprandial NEFA peaks and nocturnal elevation in plasma NEFA can be accounted for by our model.

Antagonism of corticotrophin-releasing factor receptors in the fourth ventricle modifies responses to mild but not restraint stress

Miragaya, J. R., Harris, R. B. S. — 2008-08-08

Repeated restraint stress (RRS; 3 h of restraint on 3 consecutive days) in rodents produces temporary hypophagia, but a long-term downregulation of body weight. The mild stress (MS) of an intraperitoneal injection of saline and housing in a novel room for 2 h also inhibits food intake and weight gain, but the effects are smaller than for RRS. Previous exposure to RRS exaggerates hypophagia, glucocorticoid release, and anxiety-type behavior caused by MS. Here we tested the involvement of brain stem corticotrophin-releasing factor receptors (CRFR) in mediating energetic and glucocorticoid responses to RRS or MS and in promoting stress hyperresponsiveness in RRS rats. Administration of 1.3 nmol hCRF_(9-41), a nonspecific CRFR antagonist, exaggerated hypophagia and weight loss in both RRS and MS rats, whereas 0.26 nmol had no effect in RRS or MS rats. In contrast, 2 nmol of the nonspecific antagonist astressin had no effect on weight loss or hypersensitivity to subsequent MS in RRS rats, but blocked weight loss and inhibition of food intake caused by MS alone. MS rats infused with 3 nmol antisauvagine-30, a CRFR2 antagonist, did not lose weight in the 48 h after MS, but 0.3 nmol did not prevent weight loss in MS rats. These data suggest that inhibition of food intake and weight loss induced by RRS or by MS involve different pathways, with hindbrain CRFR mediating the effect of MS on body weight and food intake. Hindbrain CRFR do not appear to influence stress-induced corticosterone release in RRS rats.

Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence

2008-08-08

A precise understanding of neural circuits controlling lipid mobilization and thermogenesis remains to be determined. We have been studying the sympathetic nervous system (SNS) contributions to white adipose tissue (WAT) lipolysis largely in Siberian hamsters. Central melanocortins are implicated in the control of the sympathetic outflow to WAT, and, moreover, the melanocortin 4 receptors (MC4-R) appear to be principally involved. We previously found that acute third ventricular melanotan II (MTII; an MC3/4-R agonist) injections increase sympathetic drive (norepinephrine turnover) to interscapular brown adipose tissue (IBAT) and IBAT temperature. Here we tested whether MC4-R mRNA is expressed in IBAT SNS outflow neurons using in situ hybridization for the former and injections of the transneuronal viral retrograde tract tracer, pseudorabies virus (PRV) into IBAT, for the latter. Significant numbers of double-labeled cells for PRV and MC4-R mRNA were found across the neuroaxis (mean of all brain sites ~60%), including the hypothalamic paraventricular nucleus (PVH; ~80%). Acute parenchymal MTII microinjections into the PVH of awake, freely-moving hamsters, using doses below those able to increase IBAT temperature when injected into the third ventricle, increased IBAT temperature for as long as 4 h, as measured by temperature transponders implanted below the tissue. Collectively, these data add significant support to the view that central melanocortins are important in controlling IBAT thermogenesis via the SNS innervation of this tissue, likely through the MC4-Rs.

Ghrelin secretion is not reduced by increased fat mass during diet-induced obesity

Qi, X., Reed, J. T., Wang, G., Han, S., Englander, E. W., Greeley, G. H. — 2008-08-08

Ghrelin is a stomach hormone that stimulates growth hormone (GH) secretion, adiposity, and food intake. Gastric ghrelin production and secretion are regulated by caloric intake; ghrelin secretion increases during fasting, decreases with refeeding, and is reduced by diet-induced obesity. The aim of the present study was to test the hypotheses that 1) an increase in body adiposity will play an inhibitory role in the reduction of gastric ghrelin synthesis and secretion during chronic ingestion of a high-fat (HF) diet and 2) chronic ingestion of an HF diet will suppress the rise in circulating ghrelin levels in response to acute fasting. Adult male Sprague-Dawley rats were fed a standard AIN-76A (~5–12% of calories from fat) or an HF (~45% of calories from fat) diet. The effect of increased adiposity on gastric ghrelin homeostasis was assessed by comparison of stomach ghrelin production and plasma ghrelin levels in obese and nonobese rats fed the HF diet. HF diet-fed, nonobese rats were generated by administration of triiodothyronine to lower body fat accumulation. Our findings indicate that an increased fat mass per se does not exert an inhibitory effect on ghrelin homeostasis during ingestion of the HF diet. Additionally, the magnitude of change in plasma ghrelin in response to fasting was not blunted, indicating that a presumed, endogenous signal for activation of ingestive behavior remains intact, despite excess stored calories in HF-fed rats.

Licking and gaping elicited by microstimulation of the nucleus of the solitary tract

Kinzeler, N. R., Travers, S. P. — 2008-08-08

Intraoral infusions of bitter tastants activate expression of the immediate-early gene c-Fos in neurons located in the medial third of the rostral nucleus of the solitary tract (rNST). The distribution of these neurons is distinct from that activated by sour or sweet stimuli. Bitter stimuli are also distinctive because of their potency for eliciting gaping, an oral reflex that functions to actively reject potentially toxic substances. Glossopharyngeal nerve transection profoundly reduces, whereas decerebration spares, the bitter-evoked Fos-like immunoreactivity (FLI) pattern and gaping, implicating the medial rNST as a substrate for the sensory limb of oral rejection. The present experiment tested this hypothesis using microstimulation (100 Hz, 0.2 ms, 5–40 µA) to activate the rNST in awake rats. NST microstimulation elicited licking and gaping, and gaping was evoked from a restricted rNST region. The results indicated some topographic organization in sites effective for evoking gaping, but, in direct conflict with the hypothesis, lateral sites farther from bitter-evoked FLI were more effective than medial sites centered closer to FLI-expressing neurons. The gape-effective sites resemble locations of bitter-responsive neurons recently observed in neurophysiological recordings. These results indicate that bitter-responsive rNST neurons critical for triggering gaping may not express FLI and imply an alternate function for bitter-responsive neurons that do.

Effects of different intermittent peptide YY (3-36) dosing strategies on food intake, body weight, and adiposity in diet-induced obese rats

Reidelberger, R. D., Haver, A. C., Chelikani, P. K., Buescher, J. L. — 2008-08-08

Chronic administration of anorexigenic substances to experimental animals by injections or continuous infusion typically produces either no effect or a transient reduction in food intake and body weight. Our aim here was to identify an intermittent dosing strategy for intraperitoneal infusion of peptide YY(3-36) [PYY(3-36)] that produces a sustained reduction in daily food intake and adiposity in diet-induced obese rats. Rats (665 ± 10 g body wt, 166 ± 7 g body fat) with intraperitoneal catheters tethered to infusion swivels had free access to a high-fat diet. Vehicle-treated rats (n = 23) had relatively stable food intake, body weight, and adiposity during the 9-wk test period. None of 15 PYY(3-36) dosing regimens administered in succession to a second group of rats (n = 22) produced a sustained 15–25% reduction in daily food intake for >5 days, although body weight and adiposity were reduced across the 9-wk period by 12% (594 ± 15 vs. 672 ± 15 g) and 43% (96 ± 7 vs. 169 ± 9 g), respectively. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was ≥3 h appeared to be due in part to an increase in food intake between infusions. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was < 3 h suggested possible receptor downregulation and tolerance to frequent PYY(3-36) administration; however, food intake significantly increased when PYY(3-36) treatments were discontinued for 1 day following apparent loss in treatment efficacies. Together, these results demonstrate the development of a potent homeostatic response to increase food intake when PYY(3-36) reduces food intake and energy reserves in diet-induced obese rats.

Effect of small intestinal glucose load on plasma ghrelin in healthy men

2008-08-08

Postprandial ghrelin suppression arises from the interaction of meal contents with the small intestine and may relate to elevations in blood glucose and/or plasma insulin. We sought to determine whether the suppression of ghrelin by small intestinal glucose is dependent on the glucose load and can be accounted for by changes in blood glucose and/or plasma insulin. Blood glucose, plasma insulin, and plasma ghrelin levels were measured in 10 healthy males (aged 32 ± 4 yr; body mass index: 25.1 ± 0.4 kg/m²) during intraduodenal glucose infusions at 1 kcal/min (G1), 2 kcal/min (G2), and 4 kcal/min (G4), as well as intraduodenal hypertonic saline (control) for 120 min. There was a progressive decrease in ghrelin with all treatments, control at 45 min and between 90 and 120 min (P < 0.05) and G1 (P < 0.05), G2 (P < 0.0001), and G4 (P < 0.0001) between 30 and 120 min to reach a plateau at ~90 min. There was no difference in plasma ghrelin between G1, G2, or G4. Control suppressed ghrelin to a lesser extent than intraduodenal glucose (P < 0.05). The suppression of ghrelin was not related to rises in blood glucose or plasma insulin. Suppression of ghrelin by intraduodenal glucose in healthy males is apparently independent of the glucose load and unrelated to blood glucose or insulin levels.

Activation of mesolimbic dopamine neurons during novel and daily limited access to palatable food is blocked by the opioid antagonist LY255582

2008-08-08

An analog of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine series (LY255582) exhibits high in vitro binding affinity and antagonist potency for the µ-, -, and -opioid receptors. In vivo, LY255582 exhibits potent effects in reducing food intake and body weight in several rodent models of obesity. In the present study, we evaluated the effects of LY255582 to prevent the consumption of a highly palatable (HP) diet (a high-fat/high-carbohydrate diet) both when the food was novel and following daily limited access to the HP diet. Additionally, we examined the effects of consumption of the HP diet and of LY255582 treatment on mesolimbic dopamine (DA) signaling by in vivo microdialysis. Consumption of the HP diet increased extracellular DA levels within the nucleus accumbens (NAc) shell. Increased DA in the NAc shell was not related to the quantity of the HP diet consumed, and the DA response did not habituate following daily scheduled access to the HP diet. Interestingly, treatment with LY255582 inhibited consumption of the HP diet and the HP diet-associated increase in NAc shell DA levels. Moreover, the increased HP diet consumption observed following daily limited access to the HP diet was completely prevented by LY255582 treatment. LY255582 may be a useful tool in understanding the neural mechanisms involved in the reinforcement mechanisms regulating food intake.

Gastric distension attenuates the hypotensive effect of intraduodenal glucose in healthy older subjects

Gentilcore, D., Meyer, J. H., Rayner, C. K., Horowitz, M., Jones, K. L. — 2008-08-08

Postprandial hypotension occurs frequently, and current management is suboptimal. Recent studies suggest that the magnitude of the fall in postprandial blood pressure (BP) may be attenuated by gastric distension. The aim of this study was to determine the effect of gastric distension on the hypotensive response to intraduodenal (ID) glucose. Eight healthy subjects (5 males, 3 females, aged 65–76 years) received an ID infusion of either 1) 50 g glucose in 300 ml saline (ID glucose) over 60 min (t = 0–60 min), 2) 50 g glucose in 300 ml saline over 60 min and intragastric (4) infusion of 500 ml water between t = 7–10 min (IG water and ID glucose), or 3) ID saline (0.9%) infusion over 60 min and IG infusion of 500 ml water (IG water and ID saline) all followed by ID saline infusion for another 60 min (t = 60–120 min) on three separate days. BP and heart rate (HR) were measured. Gastric emptying (GE) of the IG water was quantified by two-dimensional ultrasonography. Between t = 0–60 min, systolic and diastolic BP was greater (P < 0.05 for both) with IG water and ID saline compared with IG water and ID glucose, and less (P < 0.05 for both) with ID glucose compared with IG water and ID glucose. These effects were evident at relatively low IG volumes (~300 ml). GE was faster with IG water and ID saline when compared with IG water and ID glucose. We conclude that, in healthy older subjects, IG administration of water markedly attenuates the hypotensive response to ID glucose, presumably as a result of gastric distension.

Dietary carbohydrates induce changes in glucosensing capacity and food intake of rainbow trout

Polakof, S., Miguez, J. M., Soengas, J. L. — 2008-08-08

We hypothesize that variations in dietary carbohydrate levels produce changes in glucosensor parameters in previously characterized glucosensing areas (hypothalamus and hindbrain) related with the regulation of food intake of a carnivorous fish species like rainbow trout. Therefore, we fed trout with standard, carbohydrate-free (CF) or high-carbohydrate (HC) diets for 10 days to assess changes in glucosensing system and food intake. Fish fed CF diet displayed hypoglycemia and increased food intake. Fish fed a HC diet displayed hyperglycemia and decreased food intake. Changes in food intake due to dietary carbohydrates were accompanied in hypothalamus and hindbrain of fish fed with HC diet by changes in parameters involved in glucosensing, such as increased glucose, glucose 6-phosphate, and glycogen levels and increased glucokinase (GK), glycogen synthase, and pyruvate kinase activities as well as increased GK and GLUT2 expression. All those results address for the first time in fish, despite the relative intolerance to glucose of carnivorous species, that dietary carbohydrates are important regulators of the glucosensing system in carnivorous fish, suggesting that the information generated by this system can be associated with the changes observed in food intake.

Somatostatin inhibits hepatic growth hormone receptor and insulin-like growth factor I mRNA expression by activating the ERK and PI3K signaling pathways

Hagemeister, A. L., Sheridan, M. A. — 2008-08-08

Previously, we reported that somatostatins (SS) inhibit organismal growth by reducing hepatic growth hormone (GH) sensitivity and by inhibiting insulin-like growth factor I (IGF-I) production. In this study, we used hepatocytes isolated from rainbow trout to elucidate the mechanism(s) associated with the extrapituitary growth-inhibiting actions of SS. SS-14, a predominant SS isoform, stimulated tyrosine phosphorylation of several endogenous proteins, including extracellular signal-regulated kinase (ERK), a member the mitogen-activated protein kinase (MAPK) family, and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K). SS-14 specifically stimulated the phosphorylation of both ERK 1/2 and Akt in a concentration-dependent fashion. This activation occurred within 5–15 min, then subsided after 1 h. The ERK inhibitor U0126 retarded SS-14-stimulated phosphorylation of ERK 1/2, whereas the PI3K inhibitor LY294002 blocked SS-14-stimulated phosphorylation of Akt. SS-14-inhibited expression of GH receptor (GHR) mRNA was blocked by U0126 but not by LY294002. By contrast, U1026 had no effect on SS-14 inhibition of GH-stimulated IGF-I mRNA expression, whereas LY294002 partially blocked the inhibition of GH-stimulated IGF-I mRNA expression by SS-14. These results indicate that SS-14-inhibited GHR expression is mediated by the ERK signaling pathway and that the PI3K/Akt pathway mediates, at least in part, SS-14 inhibition of GH-stimulated IGF-I expression.

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

2008-08-08

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2^–/–), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 µM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2^–/– mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2^–/– mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2^–/– mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2^–/– mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.

Quercetin reduces susceptibility to influenza infection following stressful exercise

Davis, J. M., Murphy, E. A., McClellan, J. L., Carmichael, M. D., Gangemi, J. D. — 2008-08-08

Exercise stress is associated with increased risk for upper respiratory tract infection. We have shown that exercise stress can increase susceptibility to infection. Quercetin, a flavonoid present in a wide variety of fruits and vegetables, has been reported to inhibit infectivity and replication of a broad spectrum of viruses and may offset the increase in susceptibility to infection associated with stressful exercise. This study examined the effects of quercetin feedings on susceptibility to the influenza virus A/Puerto Rico/8/34 (H1N1) following stressful exercise. Mice were randomly assigned to one of four treatment groups: exercise-placebo, exercise-quercetin, control-placebo, or control-quercetin. Exercise consisted of a run to fatigue (~140 min) on a treadmill for 3 consecutive days. Quercetin (12.5 mg/kg) was administered via gavage for 7 days before viral challenge. At 30 min after the last bout of exercise or rest, mice (n = 23–30) were intranasally inoculated with a standardized dose of influenza virus (0.04 hemagglutinating units). Mice were monitored daily for morbidity (time to sickness), symptom severity, and mortality (time to death) for 21 days. Exercise stress was associated with an increased susceptibility to infection [morbidity, mortality, and symptom severity on days 5–7 (P < 0.05)]; quercetin offset the increase in susceptibility to infection [morbidity, mortality, and symptom severity on days 5–7 (P < 0.05)] that was associated with stressful exercise. These data suggest that short-term quercetin feedings may prove to be an effective strategy to lessen the impact of stressful exercise on susceptibility to respiratory infection.

Sampling of cardiovascular data; how often and how much?

Guild, S.-J., Barrett, C. J., McBryde, F. D., Van Vliet, B. N., Malpas, S. C. — 2008-08-08

Long-term measurement of cardiovascular variables by telemetry in laboratory animals has become indispensable in recent years. However, limited battery life and management of large volumes of recorded data are major drawbacks. These limitations can often be overcome by intermittent sampling of data. The question is, how much data does one need to collect to accurately reflect the underlying average value? To investigate this, 24-h continuous recordings of rabbit heart rate, arterial pressure, and integrated renal sympathetic nerve activity (RSNA) were resampled using a variety of protocols that differed with respect to the number of individual sampling periods used and the total amount of time that was sampled. The absolute percentage errors of estimates of the daily mean, standard deviation, and interquartile range were calculated for each sampling protocol. A similar analysis was repeated using arterial pressure data from rats. The results show that the number of sampling periods spread throughout the day had more effect than the total amount of data recorded. For example, just 2 h of total sampling time spread over 12 evenly spaced 10-min periods gave estimates of the daily mean of blood pressure and heart rate with < 1% error and RSNA with < 3% error. We show that accurate estimates of the daily mean of arterial pressure, heart rate, and RSNA can all be made using scheduled recording, and we recommend recording a minimum of 2 h/day spread over a number of periods throughout the day.

Sex-specific differences in chromosome-dependent regulation of vascular reactivity in female consomic rat strains from a SS x BN cross

Kunert, M. P., Dwinell, M. R., Drenjancevic Peric, I., Lombard, J. H. — 2008-08-08

High-throughput studies in the Medical College of Wisconsin Program for Genomic Applications (Physgen) were designed to link chromosomes with physiological function in consomic strains derived from a cross between Dahl salt-sensitive SS/JrHsdMcwi (SS) and Brown Norway normotensive BN/NHsdMcwi (BN) rats. The specific goal of the vascular protocol was to characterize the responses of aortic rings from these strains to vasoconstrictor and vasodilator stimuli (phenylephrine, acetylcholine, sodium nitroprusside, and bath hypoxia) to identify chromosomes that either increase or decrease vascular reactivity to these vasoactive stimuli. Because previous studies demonstrated sex-specific quantitative trait loci (QTLs) related to regulation of cardiovascular phenotypes in an F2 cross between the parental strains, males and females of each consomic strain were included in all experiments. As there were significant sex-specific differences in aortic sensitivity to vasoconstrictor and vasodilator stimuli compared with the parental SS strain, we report the results of the females separately from the males. There were also sex-specific differences in aortic ring sensitivity to these vasoactive stimuli in consomic strains that were fed a high-salt diet (4% NaCl) for 3 wk to evaluate salt-induced changes in vascular reactivity. Differences in genetic architecture could contribute to sex-specific differences in the development and expression of cardiovascular diseases via differential regulation and expression of genes. Our findings are the first to link physiological traits with specific chromosomes in female SS rats and support the idea that sex is an important environmental variable that plays a role in the expression and regulation of genes.

Sex-related changes in cardiac function following myocardial infarction in mice

Shioura, K. M., Geenen, D. L., Goldspink, P. H. — 2008-08-08

Recent awareness of cardiovascular diseases as a number one killer of the middle-aged women has prompted interest in sex differences leading to heart failure (HF). Therefore, we evaluated cardiac function in female and male mice following myocardial infarction (MI) using the Millar pressure-volume (P-V) conductance system in vivo, at time points corresponding to early (2 wk), late compensatory hypertrophy (4 wk), and decompensation (10 wk) to HF. A significant deterioration of the load dependent and independent hemodynamic measurements occurred in both female and male mice during the early phase of hypertrophy. Later, compensatory hypertrophy was marked by a normalization of volumes to control levels in females compared with males. The most notable differences between sexes occurred in the measurements of cardiac contractility during the decompensation to HF. In females, there was a significant improvement in contractility compared with males, which was apparent in the load-independent measurements of preload recruitable stroke work (10 wk post-MI, female = 48.7 ± 8.0 vs. male = 25.2 ± 1.8 mmHg, P < 0.05) and maximum dP/dt vs. maximum end-diastolic volume (10 wk post-MI, female=359 ± 58 vs. male=149 ± 28 mmHg·s^–1·µl^–1, P < 0.05). Despite these differences, there were no differences in the heart weight to body weight ratio and infarct size between the sexes. These data demonstrate that compensatory hypertrophy is associated with an improvement in contractility and a delayed decompensation to HF in females. However, compensatory hypertrophy in males appears to be undermined by a steady decline in contractility associated with decompensation to HF.

Central G-alpha subunit protein-mediated control of cardiovascular function, urine output, and vasopressin secretion in conscious Sprague-Dawley rats

Wainford, R. D., Kurtz, K., Kapusta, D. R. — 2008-08-08

The role(s) of central G-proteins in the regulation of cardiovascular and renal function is unknown. We examined how inhibition/downregulation of central Gi/Go, Gz or Gq proteins altered the characteristic cardiovascular (depressor), renal excretory (diuretic), and plasma AVP (inhibitory) responses to intracerebroventricular injection of nociceptin/orphanin FQ (N/OFQ) in rats. Before investigation, rats were pretreated intracerebroventricularly with saline vehicle (5 µl, 48 h, n = 6), pertussis toxin (PTX; 48-h, 1 µg, n = 6), or Gz, Gq, or scrambled oligodeoxynucleotide (ODN) (25 µg, 24 h, n = 6 per group). On the study day, intracerebroventricular N/OFQ (5.5 nmol) or vehicle (5 µl) was injected into pretreated conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were recorded, and urine was collected for 90 min. In vehicle or scrambled ODN groups, intracerebroventricular N/OFQ decreased MAP and HR and produced water diuresis (sensitive to UFP-101, N/OFQ receptor antagonist). The hypotension and bradycardia, but not diuresis, to N/OFQ were abolished in PTX-pretreated rats. In contrast, intracerebroventricular ODN pretreatment markedly blunted (Gz) or augmented (Gq) the diuresis to intracerebroventricular N/OFQ. In separate studies, the action of central N/OFQ to decrease plasma AVP levels in naïve water-restricted rats was differentially altered by intracerebroventricular Gz ODN (blunted) and Gq ODN (augmented) pretreatment. These studies demonstrate central Gi/Go activity mediates intracerebroventricular N/OFQ's cardiovascular depressor function. Alternatively, central Gz (inhibitory) and Gq (stimulatory) activity differentially modulates AVP release to control the pattern of diuresis to intracerebroventricular N/OFQ. These findings highlight the novel selective central G-subunit protein-mediated control of cardiovascular vs. renal excretory function.

Moderate zinc restriction during fetal and postnatal growth of rats: effects on adult arterial blood pressure and kidney

2008-08-08

Intrauterine and postnatal zinc restriction may result in an adverse environment for the development of cardiovascular and renal systems. This study evaluated the effects of moderate zinc deficiency during fetal life, lactation, and/or postweaning growth on systolic blood pressure, renal function, and morphology in adult life. Female Wistar rats received low (8 ppm) or control (30 ppm) zinc diets from the beginning of pregnancy up to weaning. After weaning, male offspring of each group of mothers were fed low or control zinc diet. Systolic blood pressure, creatinine clearance, proteinuria, renal morphology, renal apoptosis. and renal oxidative stress state were evaluated after 60 days. Zinc deficiency during pre- and postweaning growth induced an increase in systolic blood pressure and a decrease in the glomerular filtration rate associated with a reduction in the number and size of nephrons. Activation of renal apoptosis, reduction in catalase activity, glutathione peroxidase activity, and glutathione levels and increase in lipid peroxidation end products could explain these morphometric changes. Zinc deficiency through pre- and postweaning growth induced more pronounced renal alteration than postweaning zinc deficiency. These animals showed signs of renal fibrosis, proteinuria, increased renal apoptosis, and higher lipid peroxidation end products. A control diet during postweaning growth did not totally overcome renal oxidative stress damage, apoptosis, and fibrosis induced by zinc deficiency before weaning. In conclusion, zinc deficiency during a critical period of renal development and maturation could induce functional and morphological alterations that result in elevated blood pressure and renal dysfunction in adult life.

Temporal asymmetries of short-term heart period variability are linked to autonomic regulation

2008-08-08

We exploit time reversibility analysis, checking the invariance of statistical features of a series after time reversal, to detect temporal asymmetries of short-term heart period variability series. Reversibility indexes were extracted from 22 healthy fetuses between 16th to 40th wk of gestation and from 17 healthy humans (aged 21 to 54, median = 28) during graded head-up tilt with table inclination angles randomly selected inside the set {15, 30, 45, 60, 75, 90}. Irreversibility analysis showed that nonlinear dynamics observed in short-term heart period variability are mostly due to asymmetric patterns characterized by bradycardic runs shorter than tachycardic ones. These temporal asymmetries were 1) more likely over short temporal scales than over longer, dominant ones; 2) more frequent during the late period of pregnancy (from 25th to 40th week of gestation); 3) significantly present in healthy humans at rest in supine position; 4) more numerous during 75 and 90° head-up tilt. Results suggest that asymmetric patterns observable in short-term heart period variability might be the result of a fully developed autonomic regulation and that an important shift of the sympathovagal balance toward sympathetic predominance (and vagal withdrawal) can increase their presence.

Hepatic translation control in the late-gestation fetal rat

Gruppuso, P. A., Tsai, S.-W., Boylan, J. M., Sanders, J. A. — 2008-08-08

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4A1 and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin.

Repeated ethanol exposure during late gestation decreases nephron endowment in fetal sheep

2008-08-08

Maternal alcohol consumption during pregnancy can affect fetal development, but little is known about the effects on the developing kidney. Our objectives were to determine the effects of repeated ethanol exposure during the latter half of gestation on glomerular (nephron) number and expression of key genes involved in renal development or function in the ovine fetal kidney. Pregnant ewes received daily intravenous infusion of ethanol (0.75 g/kg, n = 5) or saline (control, n = 5) over 1 h from 95 to 133 days of gestational age (DGA; term is ~147 DGA). Maternal and fetal arterial blood samples were taken before and after the start of the daily ethanol infusions for determination of blood ethanol concentration (BEC). Necropsy was performed at 134 DGA, and fetal kidneys were collected for determination of total glomerular number using the physical disector/fractionator technique; at this gestational age nephrogenesis is completed in sheep. Maximal maternal and fetal BECs of 0.12 ± 0.01 g/dl (mean ± SE) and 0.11 ± 0.01 g/dl, respectively, were reached 1 h after starting maternal ethanol infusions. Ethanol exposure had no effect on fetal body weight, kidney weight, or the gene expression of members of the renin-angiotensin system, insulin-like growth factors, and sodium channels. However, fetal glomerular number was lower after ethanol exposure (377,585 ± 8,325) than in controls (423,177 ± 17,178, P < 0.001). The data demonstrate that our regimen of fetal ethanol exposure during the latter half of gestation results in an 11% reduction in nephron endowment without affecting the overall growth of the kidney or fetus or the expression of key genes involved in renal development or function. A reduced nephron endowment of this magnitude could have important implications for the cardiovascular health of offspring during postnatal life.

High multivitamin intake by Wistar rats during pregnancy results in increased food intake and components of the metabolic syndrome in male offspring

2008-08-08

The effect of high multivitamin intake during pregnancy on the metabolic phenotype of rat offspring was investigated. Pregnant Wistar rats (n = 10 per group) were fed the AIN-93G diet with the recommended vitamin (RV) content or a 10-fold increase [high vitamin (HV) content]. In experiment 1, male and female offspring were followed for 12 wk after weaning; in experiment 2, only males were followed for 28 wk. Body weight (BW) was measured weekly. Every 4 wk, after an overnight fast, food intake over 1 h was measured 30 min after a gavage of glucose or water. An oral glucose tolerance test was performed every 3–5 wk. Postweaning fasting glucose, insulin, ghrelin, glucagon-like peptide-1, and systolic blood pressure were measured. No difference in BW at birth or litter size was observed. Food intake was greater in males born to HV dams (P < 0.05), and at 28 wk after weaning, BW was 8% higher (P < 0.05) and fat pad mass was 27% higher (P < 0.05). Food intake reduction after the glucose preload was nearly twofold less in males born to HV dams at 12 wk after weaning (P < 0.05). Fasting glucose, insulin, and ghrelin were 11%, 62%, and 41% higher in males from HV dams at 14 wk after weaning (P < 0.05). Blood glucose response was 46% higher at 23 wk after weaning (P < 0.01), and systolic blood pressure was 16% higher at 28 wk after weaning (P < 0.05). In conclusion, high multivitamin intake during pregnancy programmed the male offspring for the development of the components of metabolic syndrome in adulthood, possibly by its effects on central mechanisms of food intake control.

Early fetal hypoxia leads to growth restriction and myocardial thinning

Ream, M., Ray, A. M., Chandra, R., Chikaraishi, D. M. — 2008-08-08

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired O₂ from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of E11.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18–24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate.

Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss

Ramadoss, J., Lunde, E. R., Ouyang, N., Chen, W.-J. A., Cudd, T. A. — 2008-08-08

Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258 ± 10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

Resistance exercise decreases eIF2B{varepsilon} phosphorylation and potentiates the feeding-induced stimulation of p70S6K1 and rpS6 in young men

2008-08-08

We investigated the effect of resistance exercise and feeding on the activation of signaling proteins involved in translation initiation. Nine young men (23.7 ± 0.41 yr; BMI = 25.5 ± 1.0 kg/m²; means ± SE) were tested twice after they performed a strenuous bout of unilateral resistance exercise, such that their contralateral leg acted as a nonexercised comparator, in either the fasted and fed [1,000 kJ, each 90 min (3 doses): 10 g protein, 41 g carbohydrate, 4 g fat] states. Muscle biopsies were obtained 6 h postexercise from both legs, resulting in four experimental conditions: rest-fasted, rest-fed, exercise-fasted, and exercise-fed. Feeding increased PKB/Akt (Ser473) phosphorylation (P < 0.05), while exercise increased the phosphorylation of Akt and the downstream 70 kDa S6 protein kinase (p70^S6K1, Thr389) and ribosomal protein S6 (rpS6, Ser235/236, Ser240/244; all P < 0.05). The combination of resistance exercise and feeding increased the phosphorylation of p70^S6K1 (Thr389) and rpS6 (Ser240/244) above exercise alone (P < 0.05). Exercise also reduced phosphorylation of the catalytic epsilon subunit of eukaryotic initiation factor 2B (eIF2B, Ser540; P < 0.05). Mammalian target of rapamycin (mTOR, Ser2448), glycogen synthase kinase-3β (GSK-3β, Ser9), and focal adhesion kinase (FAK, Tyr^576/577) phosphorylation were unaffected by either feeding or resistance exercise (all P > 0.14). In summary, feeding resulted in phosphorylation of Akt, while resistance exercise stimulated phosphorylation of Akt, p70^S6K1, rpS6, and dephosphorylation eIF2B with a synergistic effect of feeding and exercise on p70^S6K1 and its downstream target rpS6. We conclude that resistance exercise potentiates the effect of feeding on the phosphorylation and presumably activation of critical proteins involved in the regulation of muscle protein synthesis in young men.

Mild endotoxemia, NF-{kappa}B translocation, and cytokine increase during exertional heat stress in trained and untrained individuals

Selkirk, G. A., McLellan, T. M., Wright, H. E., Rhind, S. G. — 2008-08-08

This study examined endotoxin-mediated cytokinemia during exertional heat stress (EHS). Subjects were divided into trained [TR; n = 12, peak aerobic power (Vo_2peak) = 70 ± 2 ml·kg lean body mass^–1·min^–1] and untrained (UT; n = 11, Vo_2peak = 50 ± 1 ml·kg lean body mass^–1·min^–1) groups before walking at 4.5 km/h with 2% elevation in a climatic chamber (40°C, 30% relative humidity) wearing protective clothing until exhaustion (Exh). Venous blood samples at baseline and 0.5°C rectal temperature increments (38.0, 38.5, 39.0, 39.5, and 40.0°C/Exh) were analyzed for endotoxin, lipopolysaccharide binding protein, circulating cytokines, and intranuclear NF-B translocation. Baseline and Exh samples were also stimulated with LPS (100 ng/ml) and cultured in vitro in a 37°C water bath for 30 min. Phenotypic determination of natural killer cell frequency was also determined. Enhanced blood (104 ± 6 vs. 84 ± 3 ml/kg) and plasma volumes (64 ± 4 vs. 51 ± 2 ml/kg) were observed in TR compared with UT subjects. EHS produced an increased concentration of circulating endotoxin in both TR (8 ± 2 pg/ml) and UT subjects (15 ± 3 pg/ml) (range: not detected to 32 pg/ml), corresponding with NF-B translocation and cytokine increases in both groups. In addition, circulating levels of tumor necrosis factor- and IL-6 were also elevated combined with concomitant increases in IL-1 receptor antagonist in both groups and IL-10 in TR subjects only. Findings suggest that the threshold for endotoxin leakage and inflammatory activation during EHS occurs at a lower temperature in UT compared with TR subjects and support the endotoxin translocation hypothesis of exertional heat stroke, linking endotoxin tolerance and heat tolerance.

Phase I dynamics of cardiac output, systemic O2 delivery, and lung O2 uptake at exercise onset in men in acute normobaric hypoxia

2008-08-08

We tested the hypothesis that vagal withdrawal plays a role in the rapid (phase I) cardiopulmonary response to exercise. To this aim, in five men (24.6 ± 3.4 yr, 82.1 ± 13.7 kg, maximal aerobic power 330 ± 67 W), we determined beat-by-beat cardiac output (Q), oxygen delivery (Qa_O₂), and breath-by-breath lung oxygen uptake (Vo₂) at light exercise (50 and 100 W) in normoxia and acute hypoxia (fraction of inspired O₂ = 0.11), because the latter reduces resting vagal activity. We computed Q from stroke volume (Q_st, by model flow) and heart rate (f_H, electrocardiography), and Qa_O₂ from Q and arterial O₂ concentration. Double exponentials were fitted to the data. In hypoxia compared with normoxia, steady-state f_H and Q were higher, and Q_st and Vo₂ were unchanged. Qa_O₂ was unchanged at rest and lower at exercise. During transients, amplitude of phase I (A₁) for Vo₂ was unchanged. For f_H, Q and Qa_O₂, A₁ was lower. Phase I time constant (₁) for Qa_O₂ and Vo₂ was unchanged. The same was the case for Q at 100 W and for f_H at 50 W. Q_st kinetics were unaffected. In conclusion, the results do not fully support the hypothesis that vagal withdrawal determines phase I, because it was not completely suppressed. Although we can attribute the decrease in A₁ of f_H to a diminished degree of vagal withdrawal in hypoxia, this is not so for Q_st. Thus the dual origin of the phase I of Q and Qa_O₂, neural (vagal) and mechanical (venous return increase by muscle pump action), would rather be confirmed.

Fiber-specific responses of muscle glycogen repletion in fasted rats physically active during recovery from high-intensity physical exertion

Raja, G., Brau, L., Palmer, T. N., Fournier, P. A. — 2008-08-08

Mild physical activity performed immediately after a bout of intense exercise in fasting humans results in net glycogen breakdown in their slow oxidative (SO) muscle fibers and glycogen repletion in their fast twitch (FT) fibers. Because several animal species carry a low proportion of SO fibers, it is unclear whether they can also replenish glycogen in their FT fibers under these conditions. Given that most skeletal muscles in rats are poor in SO fibers (<5%), this issue was examined using groups of 24-h fasted Wistar rats (n = 10) that swam for 3 min at high intensity with a 10% weight followed by either a 60-min rest (passive recovery, PR) or a 30-min swim with a 0.5% weight (active recovery, AR) preceding a 30-min rest. The 3-min sprint caused 61–79% glycogen fall across the muscles examined, but not in the soleus (SOL). Glycogen repletion during AR without food was similar to PR in the white gastrocnemius (WG), where glycogen increased by 71%, and less than PR in both the red and mixed gastrocnemius (RG, MG). Glycogen fell by 26% during AR in the SOL. Following AR, glycogen increased by 36%, 87%, and 37% in the SOL, RG, and MG, respectively, and this was accompanied by the sustained activation of glycogen synthase and inhibition of glycogen phosphorylase in the RG and MG. These results suggest that mammals with a low proportion of SO fibers can also replenish the glycogen stores of their FT fibers under extreme conditions combining physical activity and fasting.

Elevation in heat shock protein 72 mRNA following contractions in isolated single skeletal muscle fibers

Stary, C. M., Walsh, B. J., Knapp, A. E., Brafman, D., Hogan, M. C. — 2008-08-08

The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n = 26) or not stimulated to contract (REST; n = 14). The relative mean developed tension of EX fibers decreased to 29 ± 7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 (n = 9), 2 (n = 8), or 4 h (n = 9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac -actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly (P < 0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5 ± 7.5-fold) or cardiac -actin (14.7 ± 4.3-fold), although not at the 1- or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac -actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources.

Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice

Soliz, J., Soulage, C., Borter, E., van Patot, M. T., Gassmann, M. — 2008-08-08

Proteins harboring a Per-Arnt-Sim (PAS) domain are versatile and allow archaea, bacteria, and plants to sense oxygen partial pressure, as well as light intensity and redox potential. A PAS domain associated with a histidine kinase domain is found in FixL, the oxygen sensor molecule of Rhizobium species. PASKIN is the mammalian homolog of FixL, but its function is far from being understood. Using whole body plethysmography, we evaluated the ventilatory response to acute and chronic hypoxia of homozygous deficient male and female PASKIN mice (Paskin^–/–). Although only slight ventilatory differences were found in males, female Paskin^–/– mice increased ventilatory response to acute hypoxia. Unexpectedly, females had an impaired ability to reach ventilatory acclimatization in response to chronic hypoxia. Central control of ventilation occurs in the brain stem respiratory centers and is modulated by catecholamines via tyrosine hydroxylase (TH) activity. We observed that TH activity was altered in male and female Paskin^–/– mice. Peripheral chemoreceptor effects on ventilation were evaluated by exposing animals to hyperoxia (Dejours test) and domperidone, a peripheral ventilatory stimulant drug directly affecting the carotid sinus nerve discharge. Male and female Paskin^–/– had normal peripheral chemosensory (carotid bodies) responses. In summary, our observations suggest that PASKIN is involved in the central control of hypoxic ventilation, modulating ventilation in a gender-dependent manner.

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

2008-08-08

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca²⁺-induced mitochondrial Ca²⁺ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124 ± 47 vs. 30.4 ± 6.8 pmol Ca²⁺/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121 ± 60 vs. 40 ± 10 nmol Ca²⁺/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca²⁺ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56 ± 4 vs. 111 ± 11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca²⁺-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca²⁺ dynamics between fiber types.

Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors

2008-08-08

O₂ chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O₂-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H₂S) metabolism is involved in O₂ sensing in vascular smooth muscle. Here, we examined the possibility that H₂S is an O₂ sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O₂ sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H₂S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H₂S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H₂S-synthesizing enzymes, cystathionine β-synthase and cystathionine -lyase, was present in branchial tissue. Homogenized gills produced H₂S enzymatically, and H₂S production was inhibited by O₂, whereas mitochondrial H₂S consumption was O₂ dependent. Ambient hypoxia did not affect plasma H₂S in unanesthetized trout, but produced a Po₂-dependent increase in a sulfide moiety suggestive of increased H₂S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H₂S, produced a similar ~10-mV depolarization. These studies are consistent with H₂S involvement in O₂ sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H₂S concentration ([H₂S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H₂S] to Po₂ and may provide an exquisitely sensitive, yet simple, O₂ sensor in a variety of tissues.

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

2008-08-08

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8 x 13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22 ± 4 to 41 ± 1% between 2 and 24 h reperfusion and remained unchanged at 48 (38 ± 6%) and 72 (40 ± 1%) h reperfusion (P < 0.05; n = 4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44 ± 2 to 22 ± 2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P < 0.05; n = 4–5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca²⁺ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P < 0.05; n = 6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.

Hormonal and metabolic rhythms associated with the daily scheduled nursing in rabbit pups

2008-08-08

Young rabbits are nursed every 24 h for a period of 3–5 min. As a consequence, pups are synchronized to this nursing event; this synchronization is characterized by increased locomotor activity and a peaking of core temperature and plasma corticosterone in anticipation of the daily meal. Ghrelin is a hormone suggested to play a role in meal initiation and to promote food intake. The present study explored the role of ghrelin in food-entrained conditions. Newborn rabbits were maintained in constant darkness and nursed once daily at 1000 by the lactating dam. On postnatal day 7, rabbits were killed at six different time points to complete a 24-h cycle. All pups developed locomotor rhythms entrained by mealtime and exhibited anticipatory activity. Food-entrained rhythms in plasma corticosterone and free fatty acids were observed even if two meals were omitted. In contrast, daily food-driven rhythms in stomach weight, plasma glucose, liver glycogen, and ghrelin did not persist when two meals were omitted. Peak ghrelin levels were observed at the moment in the cycle when the stomach weight was lowest, i.e., before initiation of anticipation. The present data are in agreement with previous data from rabbit pups maintained in light-dark conditions and provide evidence that 7- to 9-day-old rabbits in constant darkness can exhibit metabolic and hormonal rhythms mainly driven by the restricted daily nursing.

Effects of age on thermoregulatory responses during cold exposure in a nonhuman primate, Microcebus murinus

2008-08-08

Cold resistance appears altered with aging. Among existing hypotheses, the impaired capacity in response to cold could be related to an altered regulation of plasma IGF-1 concentration. The combined effects of age and cold exposure were studied in a short-living primate, the gray mouse lemur (Microcebus murinus), which adjusts its energy balance using a daily torpor phase, to avoid high energy cost of normothermia maintenance. Changes in body mass, core temperature, locomotor activity, and caloric intake were monitored under 9-day exposures to 25°C and 12°C in captive animals in winter conditions. Short-term (after 2 days) and long-term (after 9 days) cold-induced changes in IGF-1 levels were also evaluated. In thermoneutral conditions (25°C), general characteristics of the daily rhythm of core temperature were preserved with age. At 12°C, age-related changes were mainly characterized by a deeper hypothermia and an increased frequency of torpor phases, associated with a loss of body mass. A short-term cold-induced decrease in plasma IGF-1 levels was observed. IGF-1 levels returned to basal values after 9 days of cold exposure. No significant effect of age could be evidenced on IGF-1 response. However, IGF-1 levels of cold-exposed aged animals were negatively correlated with the frequency of daily torpor. Responses exhibited by aged mouse lemurs exposed to cold revealed difficulties in the maintenance of normothermia and energy balance and might involve modulations of IGF-1 levels.

Teleost fish osmoregulation: what have we learned since August Krogh, Homer Smith, and Ancel Keys

Evans, D. H. — 2008-08-08

In the 1930s, August Krogh, Homer Smith, and Ancel Keys knew that teleost fishes were hyperosmotic to fresh water and hyposmotic to seawater, and, therefore, they were potentially salt depleted and dehydrated, respectively. Their seminal studies demonstrated that freshwater teleosts extract NaCl from the environment, while marine teleosts ingest seawater, absorb intestinal water by absorbing NaCl, and excrete the excess salt via gill transport mechanisms. During the past 70 years, their research descendents have used chemical, radioisotopic, pharmacological, cellular, and molecular techniques to further characterize the gill transport mechanisms and begin to study the signaling molecules that modulate these processes. The cellular site for these transport pathways was first described by Keys and is now known as the mitochondrion-rich cell (MRC). The model for NaCl secretion by the marine MRC is well supported, but the model for NaCl uptake by freshwater MRC is more unsettled. Importantly, these ionic uptake mechanisms also appear to be expressed in the marine gill MRC, for acid-base regulation. A large suite of potential endocrine control mechanisms have been identified, and recent evidence suggests that paracrines such as endothelin, nitric oxide, and prostaglandins might also control MRC function.

Long-lasting breaches in the bladder epithelium lead to storage dysfunction with increase in bladder PGE2 levels in the rat

2008-08-08

Increase in bladder mucosal permeability can be reproduced by intravesical administration of protamine sulfate (PS); however, the influence of PS once administered into the bladder disappears within several days. We developed a chronic animal model of urothelial injury using PS. Insertion of a polyethylene catheter through the bladder dome was performed in female Wistar rats. The other end of the catheter was connected to an osmotic pump for continuous delivery of PS or vehicle for 2 wk. Urinary frequency (UF) and voided volume (VV) were measured in the metabolic cage. The fifth group of rats received a high dose of PS (10 mg/ml) for 2 wk and were followed for a further 2 wk without PS. The sixth group received a high dose of PS for 2 wk and loxoprofen (0.1 mg·kg^–1·day^–1) for 4 wk. UF was increased, and VV was reduced in rats treated with a high dose of PS but not changed in rats treated with a vehicle or a low dose of PS (1 mg/ml). UF was further increased in the fifth group, while unchanged in the sixth group. Histological sections in rats treated with a high dose of PS demonstrated a loss of the upper layer of urothelial cells and an increased number of mast cells. PGE₂ level in the bladder was significantly elevated in the fifth group. These results indicate that chronic urotherial injury leads to an increase in UF and a decrease in VV. Increased PGE₂ level in the bladder is likely to be associated with long-lasting storage dysfunction.

Inflammation is associated with a decrease of lipogenic factors in omental fat in women

Poulain-Godefroy, O., Lecoeur, C., Pattou, F., Fruhbeck, G., Froguel, P. — 2008-07-09

Obesity is characterized by systemic low-grade inflammation in which adipose tissue, especially the omental depot, is thought to play a key role. We have previously shown that inflammation impairs 3T3-L1 preadipocyte cell line differentiation. To explore whether this interaction also takes place in vivo, the expression of several genes related to inflammation and adipocyte differentiation was assessed in human samples. Paired adipose tissue biopsies (from omental and subcutaneous depots) were obtained from 24 women: 6 lean normoglycemic and 18 obese volunteers with different glycemic states (normoglycemic, glucose-intolerant, or type 2 diabetic). The expression levels of CD14, IL-18, leptin, adiponectin, sterol regulatory element binding transcription factor 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPAR), pre-B-cell colony enhancing factor 1 (PBEF1) (or visfatin), glycerol-3-phosphate dehydrogenase 1 (soluble) (GPD1), lipoprotein lipase (LPL), fatty acid binding protein 4, adipocyte (FABP4), and hypoxia-inducible factor 1 were determined by quantitative real-time PCR. CD14 and IL-18 were overexpressed in omental adipose tissue compared with the subcutaneous depot, irrespective of the subject's obesity or diabetes status. A significant decrease of LPL, GPD1, and leptin expression was observed in omental tissue, and an inverse correlation between expression of CD14 and IL-18 and that of PPAR, LPL, and FABP4 was observed. The underexpression of omental lipogenic markers was more accentuated in the presence of glucose intolerance. Furthermore, adiponectin and SREBP1 expression was also significantly decreased in omental tissue of type 2 diabetic patients. PBEF1 and HIF1 expression remained comparable in all samples. Therefore, in humans, inflammation is increased in the omental depot, as evidenced by CD14 and IL-18 expression. In this localization, the inflammatory state is associated with a decreased expression of lipogenic markers, which is more pronounced in diabetic subjects.

Baroreflex control of lumbar and renal sympathetic nerve activity in conscious rats

Kanbar, R., Chapuis, B., Orea, V., Barres, C., Julien, C. — 2008-07-09

This study compared the baroreflex control of lumbar and renal sympathetic nerve activity (SNA) in conscious rats. Arterial pressure (AP) and lumbar and renal SNA were simultaneously recorded in six freely behaving rats. Pharmacological estimates of lumbar and renal sympathetic baroreflex sensitivity (BRS) were obtained by means of the sequential intravenous administration of sodium nitroprusside and phenylephrine. Sympathetic BRS was significantly (P < 0.05) lower for lumbar [3.0 ± 0.4 normalized units (NU)/mmHg] than for renal (7.6 ± 0.6 NU/mmHg) SNA. During a 219-min baseline period, spontaneous lumbar and renal BRS were continuously assessed by computing the gain of the transfer function relating AP and SNA at heart rate frequency over consecutive 61.4-s periods. The transfer gain was considered only when coherence between AP and SNA significantly differed from zero, which was verified in 99 ± 1 and 96 ± 3% of cases for lumbar and renal SNA, respectively. When averaged over the entire baseline period, spontaneous BRS was significantly (P < 0.05) lower for lumbar (1.3 ± 0.2 NU/mmHg) than for renal (2.3 ± 0.3 NU/mmHg) SNA. For both SNAs, spontaneous BRS showed marked fluctuations (variation coefficients were 26 ± 2 and 28 ± 2% for lumbar and renal SNA, respectively). These fluctuations were positively correlated in five of six rats (R = 0.44 ± 0.06; n = 204 ± 8; P < 0.0001). We conclude that in conscious rats, the baroreflex control of lumbar and renal SNA shows quantitative differences but is modulated in a mostly coordinated way.

Dynamic transcriptomic response to acute hypertension in the nucleus tractus solitarius

2008-07-09

Baroreceptor afferents project to the cardiovascular region of the nucleus tractus solitarius (cvNTS), and their cvNTS target neurons may play a role in governing the sensitivity and operating range of the arterial baroreceptor reflex (baroreflexes). Recent studies have shown differential gene and protein expression in the cvNTS in response to changed arterial pressure. However, the extent of these responses is unknown. Therefore, we collected differential global gene expression data in a time series following acute hypertension in awake, freely moving rats. To acquire statistically significant results and place them in functional context, we overcame several quality control requirements and developed novel analytical approaches. The physiologically new findings from the study are that acute hypertension causes very extensive, time-varying gene regulatory changes, many involving neuronal function-specific genes and systems of genes. We use standard genomic analysis methods to manage the large data sets and to develop results such as heat maps to examine patterns and clusters in the gene regulation. We used the Gene Ontology categories to provide functional context. To place our findings in the context of the relevant literature, we developed two graphical representations of the networks implicated, linking receptors and channels to signaling pathways. The results point to the multivariate complexity of the response and implicate a group of receptors as candidates for mediating nucleus tractus solitarius baroreflex function in hypertension by identifying concurrent upregulation of receptor genes. We were able to make transcription factor binding predictions and record dysregulation of heart rate correlated with the transcriptional response.

Dynamic time-varying analysis of heart rate and blood pressure variability in cats exposed to short-term chronic intermittent hypoxia

Rey, S., Tarvainen, M. P., Karjalainen, P. A., Iturriaga, R. — 2008-07-09

Chronic intermittent hypoxia (CIH) contributes to the development of hypertension in patients with obstructive sleep apnea and animal models. However, the early cardiovascular changes that precede CIH-induced hypertension are not completely understood. Nevertheless, it has been proposed that one of the possible contributing mechanisms to CIH-induced hypertension is a potentiation of carotid body (CB) hypoxic chemoreflexes. Therefore, we studied the dynamic responses of heart rate, blood pressure, and their variabilities during acute exposure to different levels of hypoxia after CIH short-term preconditioning (4 days) in cats. In addition, we measured baroreflex sensitivity (BRS) on the control of heart rate by noninvasive techniques. To assess the relationships among these indexes and CB chemoreflexes, we also recorded CB chemosensory discharges. Our data show that short-term CIH reduced BRS, potentiated the increase in heart rate induced by acute hypoxia, and was associated with a dynamic shift of heart rate variability (HRV) spectral indexes toward the low-frequency band. In addition, we found a striking linear correlation (r = 0.97) between the low-to-high frequency ratio of HRV and baseline. CB chemosensory discharges in the CIH-treated cats. Thus, our results suggest that cyclic hypoxic stimulation of the CB by short-term CIH induces subtle but clear selective alterations of HRV and BRS in normotensive cats.