AJP: Regulatory, Integrative and Comparative Physiology recent issues

Age-dependent regression analysis of male gonadal axis

Keenan, D. M., Veldhuis, J. D. — Fri, 30 Oct 2009 10:13:06 PDT

The mechanisms by which aging progressively depletes testosterone (Te) availability in the male are unknown. Accordingly, the objective was to estimate brain gonadotropin-releasing hormone (GnRH) outflow (release and action), which cannot be observed directly, on the basis of downstream effects on pituitary luteinizing hormone (LH) secretion. LH, in turn, feeds forward on (stimulates) gonadal Te secretion, which then feeds back on (inhibits) GnRH-driven LH secretion. LH and Te concentrations were measured repetitively (every 10 min) over 18 h during graded pharmacological blockade of endogenous GnRH outflow in 24 healthy 20- to 72-yr-old men. Data were analyzed using a new age-dependent regression model of GnRH-LH-Te interactions to estimate pulsatile LH secretion and elimination, GnRH outflow, LH feedforward, and Te feedback. By incorporating regression on age within the dose-response model, we show that aging erodes all three primary forward and reverse pathways linking the brain, pituitary gland, and testes. Aging is associated with concomitant deficits in GnRH -> LH feedforward, LH -> Te feedforward, and Te -> GnRH/LH feedback. The analytical formalism should be generalizable to other ensemble regulatory systems, such as those that control growth, reproduction, stress adaptations, and glucose metabolism.

Effects of contraction on localization of GLUT4 and v-SNARE isoforms in rat skeletal muscle

Rose, A. J., Jeppesen, J., Kiens, B., Richter, E. A. — Fri, 30 Oct 2009 10:13:06 PDT

In skeletal muscle, contractions increase glucose uptake due to a translocation of GLUT4 glucose transporters from intracellular storage sites to the surface membrane. Vesicle-associated membrane proteins (VAMPs) are believed to play an important role in docking and fusion of the GLUT4 transporters at the surface membrane. However, knowledge about which VAMP isoforms colocalize with GLUT4 vesicles in mature skeletal muscle and whether they translocate during muscle contractions is incomplete. The aim of the present study was to further identify VAMP isoforms, which are associated with GLUT4 vesicles and examine which VAMP isoforms translocate to surface membranes in skeletal muscles undergoing contractions. VAMP2, VAMP3, VAMP5, and VAMP7 were enriched in immunoprecipitated GLUT4 vesicles. In response to 20 min of in situ contractions, there was a redistribution of GLUT4 (+64 ± 13%), transferrin receptor (TfR; +75 ± 22%), and insulin-regulated aminopeptidase (IRAP; +70 ± 13%) to fractions enriched in heavy membranes away from low-density membranes (–32 ± 7%; –18 ± 12%; –33 ± 9%; respectively), when compared with the resting contralateral muscle. Similarly, there was a redistribution of VAMP2 (+240 ± 40%), VAMP5 (+79 ± 9%), and VAMP7 (+79 ± 29%), but not VAMP3, to fractions enriched in heavy membranes away from low-density membranes (–49 ± 10%, –54 ± 9%, –14 ± 11%, respectively) in contracted vs. resting muscle. In summary, VAMP2, VAMP3, VAMP5, and VAMP7 coimmunoprecipitate with intracellular GLUT4 vesicles in muscle, and VAMP2, VAMP5, VAMP7, but not VAMP3, translocate to the cell surface membranes similar to GLUT4, TfR, and IRAP in response to muscle contractions. These findings suggest that VAMP2, VAMP5, and VAMP7 may be involved in translocation of GLUT4 during muscle contractions.

Hindbrain leptin receptor stimulation enhances the anorexic response to cholecystokinin

Williams, D. L., Baskin, D. G., Schwartz, M. W. — Fri, 30 Oct 2009 10:13:06 PDT

Leptin is thought to reduce food intake, in part, by increasing sensitivity to satiation signals, including CCK. Leptin action in both forebrain and hindbrain reduces food intake, and forebrain leptin action augments both the anorexic and neuronal activation responses to CCK. Here, we asked whether leptin signaling in hindbrain also enhances these responses to CCK. We found that food intake was strongly inhibited at 30 min after a combination of 4th-intracerebroventricular (4th-icv) leptin injection and intraperitoneal CCK administration, whereas neither hormone affected intake during this period when given alone. Leptin injections targeted directly at the dorsal vagal complex (DVC) similarly enhanced the anorexic response to intraperitoneal CCK. Intra-DVC leptin injection also robustly increased the number of neurons positive for phospho-STAT3 staining in the area surrounding the site of injection, confirming local leptin receptor activation. Conversely, the anorexic response to 4th-icv leptin was completely blocked by IP devazepide, a CCKA-R antagonist, suggesting that hindbrain leptin reduces intake via a mechanism requiring endogenous CCK signaling. We then asked whether hindbrain leptin treatment enhances the dorsomedial hindbrain, hypothalamus, or amygdala c-Fos responses to IP CCK. We found that, in contrast to the effects of forebrain leptin administration, 4th-icv leptin injection had no effect on CCK-induced c-Fos in any structures examined. We conclude that leptin signaling in either forebrain or hindbrain areas can enhance the response to satiation signals and that multiple distinct neural circuits likely contribute to this interaction.

Septic shock induces distinct changes in sympathetic nerve activity to the heart and kidney in conscious sheep

Ramchandra, R., Wan, L., Hood, S. G., Frithiof, R., Bellomo, R., May, C. N. — Fri, 30 Oct 2009 10:13:06 PDT

Sepsis and septic shock are the chief cause of death in intensive care units, with mortality rates between 30 and 70%. In a large animal model of septic shock, we have demonstrated hypotension, increased cardiac output, and tachycardia, together with renal vasodilatation and renal failure. The changes in cardiac sympathetic nerve activity (CSNA) that may contribute to the tachycardia have not been investigated, and the changes in renal SNA (RSNA) that may mediate the changes in renal blood flow and function are unclear. We therefore recorded CSNA and RSNA during septic shock in conscious sheep. Septic shock was induced by administration of Escherichia coli, which caused a delayed hypotension and an immediate, biphasic increase in heart rate (HR) associated with similar changes in CSNA. After E. coli, RSNA decreased for over 3 h, followed by a sustained increase (180%), whereas renal blood flow progressively increased and remained elevated. There was an initial diuresis, followed by oliguria and decreased creatinine clearance. There were differential changes in the range of the arterial baroreflex curves; it was depressed for HR, increased for CSNA, and unchanged for RSNA. Our findings, recording CSNA for the first time in septic shock, suggest that the increase in SNA to the heart is not driven solely by unloading of baroreceptors and that the increase has an important role to increase HR and cardiac output. There was little correlation between the changes in RSNA and renal blood flow, suggesting that the renal vasodilatation was mediated mainly by other mechanisms.

Central overexpression of leptin antagonist reduces wheel running and underscores importance of endogenous leptin receptor activity in energy homeostasis

Matheny, M., Zhang, Y., Shapiro, A., Tumer, N., Scarpace, P. J. — Fri, 30 Oct 2009 10:13:06 PDT

We used recombinant adeno-associated virus (rAAV)-mediated gene delivery to overexpress a mutant of rat leptin yielding a protein that acts as a neutral leptin receptor antagonist. The long-term consequences of this overexpression on body weight homeostasis and physical activity, as assessed by voluntary wheel running (WR), were determined in F344 x Brown Norway (BN) rats. Leptin antagonist overexpression was confirmed by examination of mRNA levels in the hypothalamus. Food consumption and body weight gain were exacerbated in the antagonist group during both chow and high-fat feeding periods over the 192-day experiment. In a second experiment, a lower dose of antagonist vector was used that resulted in no change in food consumption but still increased body weight. The degree of antagonist overexpression was sufficient to partially block signal transducer and activator of transcription 3 (STAT3) phosphorylation due to administration of an acute submaximal dose of leptin. Rats were provided free access to running wheels for 4 days during both the chow and high-fat feeding periods. With both antagonist doses and during both chow and high-fat feeding, WR was substantially less with antagonist overexpression. In contrast, when leptin was overexpressed in the hypothalamus, WR activity was increased by greater than twofold. At death, adiposity and serum leptin levels were greater in the antagonist group. These data indicate that submaximal central leptin receptor blockade promotes obesity and diminishes WR activity. These findings underscore the critical role of unrestrained leptin receptor activity in long-term energy homeostasis and suggest that even minor disruption of leptin receptor function can promote obesity.

Cadmium affects metabolic responses to prolonged anoxia and reoxygenation in eastern oysters (Crassostrea virginica)

Fri, 30 Oct 2009 10:13:06 PDT

Benthic marine organisms such as mollusks are often exposed to periodic oxygen deficiency (due to the tidal exposure and/or seasonal expansion of the oxygen-deficient dead zones) and pollution by metals [e.g., cadmium, (Cd)]. These stressors can strongly affect mollusks' survival; however, physiological mechanisms of their combined effects are not fully understood. We studied the effects of Cd exposure on metabolic responses to prolonged anoxia and subsequent recovery in anoxia-tolerant intertidal mollusks Crassostrea virginica (eastern oysters). Anoxia led to an onset of anaerobiosis indicated by accumulation of l-alanine, acetate, and succinate. Prolonged anoxia (for 6 days) caused a decline in the maximum activity of electron transport chain and ADP-stimulated (state 3) oxygen uptake by mitochondria (MO₂), but no change in the resting (state 4) MO₂ of oyster mitochondria, along with a slight but significant reduction of mitochondrial respiratory control ratio. During reoxygenation, there was a significant overshoot of mitochondrial MO₂ (by up to 70% above the normoxic steady-state values) in control oysters. Mild mitochondrial uncoupling during prolonged shutdown in anoxic tissues and a subsequent strong stimulation of mitochondrial flux during recovery may help to rapidly restore redox status and protect against elevated reactive oxygen species formation in oysters. Exposure to Cd inhibits anaerobic metabolism, abolishes reoxygenation-induced stimulation of mitochondrial MO₂, and leads to oxidative stress (indicated by accumulation of DNA lesions) and a loss of mitochondrial capacity during postanoxic recovery. This may result in increased sensitivity to intermittent hypoxia and anoxia in Cd-exposed mollusks and will have implications for their survival in polluted estuaries and coastal zones.

Meal patterns, satiety, and food choice in a rat model of Roux-en-Y gastric bypass surgery

Fri, 30 Oct 2009 10:13:06 PDT

Gastric bypass surgery efficiently and lastingly reduces excess body weight and reverses type 2 diabetes in obese patients. Although increased energy expenditure may also play a role, decreased energy intake is thought to be the main reason for weight loss, but the mechanisms involved are poorly understood. Therefore, the aim of this study was to characterize the changes in ingestive behavior in a rat model of Roux-en-Y gastric bypass surgery (RYGB). Obese (24% body fat compared with 18% in chow-fed controls), male Sprague-Dawley rats maintained for 15 wk before and 4 mo after RYGB or sham-surgery on a two-choice low-fat/high-fat diet, were subjected to a series of tests assessing energy intake, meal patterning, and food choice. Although sham-operated rats gained an additional 100 g body wt during the postoperative period, RYGB rats lost ~100 g. Intake of a nutritionally complete and palatable liquid diet (Ensure) was significantly reduced by ~50% during the first 2 wk after RYGB compared with sham surgery. Decreased intake was the result of greatly reduced meal size with only partial compensation by meal frequency, and a corresponding increase in the satiety ratio. Similar results were obtained with solid food (regular or high-fat chow) 6 wk after surgery. In 12- to 24-h two-choice liquid or solid diet paradigms with nutritionally complete low- and high-fat diets, RYGB rats preferred the low-fat choice (solid) or showed decreased acceptance for the high-fat choice (liquid), whereas sham-operated rats preferred the high-fat choices. A separate group of rats offered chow only before surgery completely avoided the solid high-fat diet in a choice paradigm. The results confirm anecdotal reports of "nibbling" behavior and fat avoidance in RYGB patients and provide a basis for more mechanistic studies in this rat model.

Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?

Fri, 30 Oct 2009 10:13:06 PDT

This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O₂). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100β, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v_D) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P < 0.05 vs. normoxia) that correlated with the increase in AMS/headache scores (r = –0.50 to –0.90, P < 0.05). This was associated with a reduction in a-v_D and hence net cerebral uptake of plasma nitrite and increased cerebral output of 3-NT (P < 0.05 vs. normoxia) that also correlated against AMS/headache scores (r = 0.74–0.87, P < 0.05). In contrast, hypoxia did not alter the cerebral exchange of S100β and both global cerebral oxidative metabolism (cerebral metabolic rate of oxygen) and neuronal integrity (NSE) were preserved (P > 0.05 vs. normoxia). These findings indicate that hypoxia stimulates cerebral oxidative-nitrative stress, which has broader implications for other clinical models of human disease characterized by hypoxemia. This may prove a risk factor for AMS by a mechanism that appears independent of impaired BBB function and cerebral oxidative metabolism.

Role of hypoleptinemia during cold adaptation in Brandt's voles (Lasiopodomys brandtii)

Tang, G.-B., Cui, J.-G., Wang, D.-H. — Fri, 30 Oct 2009 10:13:06 PDT

Brandt's voles Lasiopodomys brandtii exhibit large increases in nonshivering thermogenesis to cope with chronic cold exposure, resulting in compensatory hyperphagia and fat mobilization. These physiological events are accompanied by a remarkable reduction in serum leptin levels. However, the role of hypoleptinemia in cold adaptation in this species is still unknown. In the present study, we tested the hypothesis that hypoleptinemia contributes to increases in food intake and brown adipose tissue (BAT) thermogenesis by modifying hypothalamic neuropeptides in cold-exposed Brandt's voles. Adult male voles were transferred to 5°C for 28 days. Accompanied by a decrease in serum leptin levels, hypothalamic agouti-related protein (AgRP) mRNA levels were significantly increased, but there were no changes in the long form of leptin receptor (Ob-Rb), suppressor of cytokine signaling 3 (SOCS3), neuropeptide Y (NPY) mRNA, proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated peptide (CART) mRNA levels in the hypothalamus. When cold-exposed voles were returned to warm (23°C) for 28 days, body mass, food intake, serum leptin, and AgRP mRNA were restored to control levels. Leptin administration in cold-exposed voles decreased food intake as well as hypothalamic AgRP mRNA levels. There were no significant effects of leptin administration on hypothalamic Ob-Rb, SOCS3, NPY, POMC, CART mRNA, and uncoupling protein 1 levels under cold conditions. These results suggest that hypoleptinemia partially contributes to cold-induced hyperphagia, which might involve the elevation of hypothalamic AgRP gene expression.

High-fat diets induce a rapid loss of the insulin anorectic response in the amygdala

Boghossian, S., Lemmon, K., Park, M., York, D. A. — Fri, 30 Oct 2009 10:13:06 PDT

Intracerebroventricular insulin decreases food intake (FI). The central bed nucleus of the amygdala (CeA), as other regions of the brain regulating feeding behavior, expresses insulin receptors. Our objectives were to show an insulin anorectic response in the amygdala, study the effect of high-fat diets on this response, and map the neural network activated by CeA insulin using c-Fos immunohistochemistry. Sprague-Dawley (SD) rats fitted with unilateral CeA cannulas were adapted to a low-fat (LFD) diet before they were fed a high-fat diet (HFD). Their feeding response to CeA saline or insulin (8 mU) was tested after 24 h, 72 h, or 7 days of being on a HFD. In a second experiment, SD rats were fed the HFD for 3, 7, or 49 days and were then refed with the LFD. They were tested for their insulin response before and after an HFD and every 3 days for the following weeks. Insulin tolerance tests were performed in a parallel group of rats. The CeA insulin stimulation c-Fos expression was studied to identify the distribution of activated neuronal populations. Feeding an HFD for 72 h or more induced a CeA, but not peripheral, insulin resistance, which was slowly reversed by LFD refeeding. The duration of HFD feeding determined the time frame for reversal of the insulin resistance. CeA insulin increased c-Fos in multiple brain regions, including the arcuate nucleus/paraventricular nucleus region of the hypothalamus. We conclude that the amygdala may be an important site for insulin regulation of food intake and may have a significant role in determining susceptibility to HFD-induced obesity.

Intraventricular injection of antibodies to {beta}1-integrins generates pressure gradients in the brain favoring hydrocephalus development in rats

Nagra, G., Koh, L., Aubert, I., Kim, M., Johnston, M. — Fri, 30 Oct 2009 10:13:06 PDT

In some tissues, the injection of antibodies to the β₁-integrins leads to a reduction in interstitial fluid pressure, indicating an active role for the extracellular matrix in tissue pressure regulation. If perturbations of the matrix occur in the periventricular area of the brain, a comparable lowering of interstitial pressures may induce transparenchymal pressure gradients favoring ventricular expansion. To examine this concept, we measured periventricular (parenchymal) and ventricular pressures with a servo-null micropipette system (2-µm tip) in adult Wistar rats before and after anti-integrin antibodies or IgG/IgM isotype controls were injected into a lateral ventricle. In a second group, the animals were kept for 2 wk after similar injections and after euthanization, the brains were removed and assessed for hydrocephalus. In experiments in which antibodies to β₁-integrins (n = 10) but not isotype control IgG/IgM (n = 7) were injected, we observed a decline in periventricular pressures relative to the preinjection values. Under similar circumstances, ventricular pressures were elevated (n = 10) and were significantly greater than those in the periventricular interstitium. We estimated ventricular to periventricular pressure gradients of up to 4.3 cmH₂O. In the chronic preparations, we observed enlarged ventricles in many of the animals that received injections of anti-integrin antibodies (21 of 29 animals; 72%) but not in any animal receiving the isotype controls. We conclude that modulation/disruption of β₁-integrin-matrix interactions in the brain generates pressure gradients favoring ventricular expansion, suggesting a novel mechanism for hydrocephalus development.

Eicosapentaenoic acid attenuates arthritis-induced muscle wasting acting on atrogin-1 and on myogenic regulatory factors

Fri, 30 Oct 2009 10:13:06 PDT

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that has anti-inflammatory and anticachectic actions. The aim of this work was to elucidate whether EPA administration is able to prevent an arthritis-induced decrease in body weight and muscle wasting in rats. Arthritis was induced by intradermal injection of Freund's adjuvant; 3 days later, nine rats received 1 g/kg EPA or coconut oil daily. All rats were killed 15 days after adjuvant injection. EPA administration decreased the external signs of arthritis and paw volume as well as liver TNF- mRNA. EPA did not modify arthritis-induced decrease in food intake or body weight gain. However, EPA treatment prevented arthritis-induced increase in muscle TNF- and atrogin-1, whereas it attenuated the decrease in gastrocnemius weight and the increase in MuRF1 mRNA. Arthritis not only decreased myogenic regulatory factors but also increased PCNA, MyoD, and myogenin mRNA in the gastrocnemius. Western blot analysis showed that changes in protein content followed the pattern seen with mRNA. In the control rats, EPA administration increased PCNA and MyoD mRNA and protein. In arthritic rats, EPA did not modify the stimulatory effect of arthritis on these myogenic regulatory factors. The results suggest that in experimental arthritis, in addition to its anti-inflammatory effect, EPA treatment attenuates muscle wasting by decreasing atrogin-1 and MuRF1 gene expression and increasing the transcription factors that regulate myogenesis.

Insulin-like growth factor-I and genetic effects on indexes of protein degradation in response to feed deprivation in rainbow trout (Oncorhynchus mykiss)

Cleveland, B. M., Weber, G. M., Blemings, K. P., Silverstein, J. T. — Fri, 30 Oct 2009 10:13:06 PDT

This study determined the effect of genetic variation, feed deprivation, and insulin-like growth factor-I (IGF-I) on weight loss, plasma IGF-I and growth hormone, and indexes of protein degradation in eight full-sibling families of rainbow trout. After 2 wk of feed deprivation, fish treated with IGF-I lost 16% less (P < 0.05) wet weight than untreated fish. Feed deprivation increased growth hormone (P < 0.05) and decreased IGF-I (P < 0.05), but hormone levels were not altered by IGF-I. Plasma 3-methylhistidine concentrations were not affected by IGF-I but were decreased after 2 wk (P < 0.05) and increased after 4 wk (P < 0.05) of feed deprivation. In white muscle, transcript abundance of genes in the ubiquitin-proteasome, lysosomal, and calpain- and caspase-dependent pathways were affected by feed deprivation (P < 0.05). IGF-I prevented the feed deprivation-induced upregulation of MAFbx (F-box) and cathepsin transcripts and reduced abundance of proteasomal mRNAs (P < 0.05), suggesting that reduction of protein degradation via these pathways may be partially responsible for the IGF-I-induced reduction of weight loss. Family variations in gene expression, IGF-I concentrations, and weight loss during fasting suggest genetic variation in the fasting response, with considerable impact on regulation of proteolytic pathways. These data indicate that nutrient availability, IGF-I, and genetic variation affect weight loss, in part through alterations of proteolytic pathways in rainbow trout, and that regulation of genes within these pathways is coordinated in a way that supports a similar physiological response.

Synchrotron imaging of the grasshopper tracheal system: morphological and physiological components of tracheal hypermetry

Fri, 30 Oct 2009 10:13:06 PDT

As grasshoppers increase in size during ontogeny, they have mass specifically greater whole body tracheal and tidal volumes and ventilation than predicted by an isometric relationship with body mass and body volume. However, the morphological and physiological bases to this respiratory hypermetry are unknown. In this study, we use synchrotron imaging to demonstrate that tracheal hypermetry in developing grasshoppers (Schistocerca americana) is due to increases in air sacs and tracheae and occurs in all three body segments, providing evidence against the hypothesis that hypermetry is due to gaining flight ability. We also assessed the scaling of air sac structure and function by assessing volume changes of focal abdominal air sacs. Ventilatory frequencies increased in larger animals during hypoxia (5% O₂) but did not scale in normoxia. For grasshoppers in normoxia, inflated and deflated air sac volumes and ventilation scaled hypermetrically. During hypoxia (5% O₂), many grasshoppers compressed air sacs nearly completely regardless of body size, and air sac volumes scaled isometrically. Together, these results demonstrate that whole body tracheal hypermetry and enhanced ventilation in larger/older grasshoppers are primarily due to proportionally larger air sacs and higher ventilation frequencies in larger animals during hypoxia. Prior studies showed reduced whole body tracheal volumes and tidal volume in late-stage grasshoppers, suggesting that tissue growth compresses air sacs. In contrast, we found that inflated volumes, percent volume changes, and ventilation were identical in abdominal air sacs of late-stage fifth instar and early-stage animals, suggesting that decreasing volume of the tracheal system later in the instar occurs in other body regions that have harder exoskeleton.

Effects of maternal genotype and diet on offspring glucose and fatty acid-sensing ventromedial hypothalamic nucleus neurons

Le Foll, C., Irani, B. G., Magnan, C., Dunn-Meynell, A., Levin, B. E. — Fri, 30 Oct 2009 10:13:06 PDT

Maternal obesity accentuates offspring obesity in dams bred to develop diet-induced obesity (DIO) on a 31% fat, high-sucrose, high-energy (HE) diet but has no effect on offspring of diet-resistant (DR) dams. Also, only DIO dams become obese when they and DR dams are fed HE diet throughout gestation and lactation. We assessed glucose and oleic acid (OA) sensitivity of dissociated ventromedial hypothalamic nucleus (VMN) neurons from 3- to 4-wk old offspring of DIO and DR dams fed chow or HE diet using fura-2 calcium imaging to monitor intracellular calcium fluctuations as an index of neuronal activity. Offspring of DIO dams fed chow had ~2-fold more glucose-inhibited (GI) neurons than did DR offspring. This difference was eliminated in offspring of DIO dams fed HE diet. At 2.5 mM glucose, offspring of chow-fed DIO dams had more GI neurons that were either excited or inhibited by OA than did DR offspring. Maternal HE diet intake generally increased the percentage of neurons that were excited and decreased the percentage that were inhibited by OA in both DIO and DR offspring. However, this effect was more pronounced in DIO offspring. These data, as well as concentration-dependent differences in OA sensitivity, suggest that genotype, maternal obesity, and dietary content can all affect the sensitivity of offspring VMN neurons to glucose and long-chain fatty acids. Such altered sensitivities may underlie the propensity of DIO offspring to become obese when fed high-fat, high-sucrose diets.

Recovery from renal ischemia-reperfusion injury is associated with altered renal hemodynamics, blunted pressure natriuresis, and sodium-sensitive hypertension

Fri, 30 Oct 2009 10:13:06 PDT

The present studies evaluated intrarenal hemodynamics, pressure natriuresis, and arterial blood pressure in rats following recovery from renal ischemia-reperfusion (I/R) injury. Acute I/R injury, induced by 40 min of bilateral renal arterial occlusion, resulted in an increase in plasma creatinine that resolved within a week. Following 5 wk of recovery on a 0.4% NaCl diet, the pressure-natriuresis response was assessed in anesthetized rats in which the kidney was denervated and extrarenal hormones were administered intravenously. Increasing renal perfusion pressure (RPP) from 107 to 141 mmHg resulted in a fourfold increase in urine flow and sodium excretion in sham control rats. In comparison, pressure diuresis and natriuresis were significantly attenuated in post-I/R rats. In sham rats, glomerular filtration rate (GFR) averaged 1.6 ± 0.2 ml·min^–1·g kidney weight^–1 and renal blood flow (RBF) averaged 7.8 ± 0.7 ml·min^–1·g kidney weight^–1 at RPP of 129 mmHg. Renal cortical blood flow, measured by laser-Doppler flowmetry, was well autoregulated whereas medullary blood flow and renal interstitial hydrostatic pressure increased directly with elevated RPP in sham rats. In contrast, GFR and RBF were significantly reduced whereas medullary perfusion and interstitial pressure demonstrated an attenuated response to RPP in post-I/R rats. Further experiments demonstrated that conscious I/R rats develop hypertension when sodium intake is increased. The present data indicate that the pressure-natriuretic-diuretic response in I/R rats is blunted because of a decrease in GFR and RBF and the depressed pressure-dependent increase in medullary blood flow and interstitial pressure.

Enhanced angiotensin-mediated excitation of renal sympathetic nerve activity within the paraventricular nucleus of anesthetized rats with heart failure

Zheng, H., Li, Y.-F., Wang, W., Patel, K. P. — Fri, 30 Oct 2009 10:13:06 PDT

Chronic heart failure (HF) is characterized by increased sympathetic drive. Enhanced angiotensin II (ANG II) activity may contribute to the increased sympathoexcitation under HF condition. The present study examined sympathoexcitation by 1) the effects of ANG II in the paraventricular nucleus (PVN) on renal sympathetic nerve activity (RSNA), and 2) the altered ANG II type 1 (AT₁) receptor expression during HF. Left coronary artery ligation was used to induce HF. In the anesthetized Sprague-Dawley rats, microinjection of ANG II (0.05–1 nmol) into the PVN increased RSNA, mean arterial pressure (MAP), and heart rate (HR) in both sham-operated and HF rats. The responses of RSNA and HR were significantly enhanced in rats with HF compared with sham rats (RSNA: 64 ± 8% vs. 33 ± 4%, P < 0.05). Microinjection of AT₁ receptor antagonist losartan into the PVN produced a decrease of RSNA, MAP, and HR in both sham and HF rats. The RSNA and HR responses to losartan in HF rats were significantly greater (RSNA: –25 ± 4% vs. –13 ± 1%, P < 0.05). Using RT-PCR and Western blot analysis, we found that there were significant increases in the AT₁ receptor mRNA (186 ± 39%) and protein levels (88 ± 20%) in the PVN of rats with HF (P < 0.05). The immunofluorescence of AT₁ receptors was significantly higher in the PVN of rats with HF. These data support the conclusion that an increased angiotensinergic activity on sympathetic regulation, due to the upregulation of ANG II AT₁ receptors within the PVN, may contribute to the elevated sympathoexcitation that is observed during HF.

Chemotherapy-induced kaolin intake is increased by lesion of the lateral parabrachial nucleus of the rat

Horn, C. C., De Jonghe, B. C., Matyas, K., Norgren, R. — Fri, 30 Oct 2009 10:13:07 PDT

Anticancer agents, such as cisplatin, stimulate nausea, vomiting, and behaviors indicative of malaise. Rats and mice do not possess a vomiting response, and, therefore, in these species, the ingestion of kaolin clay (a pica response) has been used as an index of malaise. In the rat, cisplatin-induced kaolin intake is inhibited by antiemetic treatments. In addition, cisplatin activates vagal afferent fibers in the gut, and kaolin intake induced by cisplatin is largely dependent on an intact vagus. Nevertheless, little is known about the brain pathways controlling pica. We investigated the role of the lateral parabrachial nucleus (lPBN), a major visceral afferent link between the hindbrain and forebrain, in cisplatin-induced c-Fos expression and pica. Injection of cisplatin (6 mg/kg ip) produced c-Fos expression in the ventrolateral (external) lPBN, a region receiving viscerosensory input. In rats with bilateral ibotenic acid lPBN lesions, cisplatin treatment substantially increased kaolin intake compared with controls (~30 g vs. ~5 g, respectively, over 24 h). Food intake was reduced by cisplatin treatment and by apomorphine, an emetic agent that acts centrally. Unlike cisplatin, however, apomorphine stimulated kaolin intake to a similar degree in both the lesioned and control rats, suggesting that lPBN damage neither produces nonspecific effects nor enhances malaise in general. These data suggest that lPBN-lesioned animals not only demonstrate pica after cisplatin treatment, but, in fact, show an exaggerated response that is greatly in excess of any treatment known to produce kaolin intake in rats.

Time-dependent effects of short-term training on muscle metabolism during the early phase of exercise

Fri, 30 Oct 2009 10:13:07 PDT

In this study, we investigated the hypothesis that the metabolic adaptations observed during steady-state exercise soon after the onset of training would be displayed during the nonsteady period of moderate exercise and would occur in the absence of increases in peak aerobic power (Vo_2peak) and in muscle oxidative potential. Nine untrained males [age = 20.8 ± 0.70 (SE) yr] performed a cycle task at 62% Vo_2peak before (Pre-T) and after (Post-T) training for 2 h/day for 5 days at task intensity. Tissue samples extracted from the vastus lateralis at 0 min (before exercise) and at 10, 60, and 180 s of exercise, indicated that at Pre-T, reductions (P < 0.05) in phosphocreatine and increases (P < 0.05) in creatine, inorganic phosphate, calculated free ADP, and free AMP occurred at 60 and 180 s but not at 10 s. At Post-T, the concentrations of all metabolites were blunted (P < 0.05) at 60 s. Training also reduced (P < 0.05) the increase in lactate and the lactate-to-pyruvate ratio observed during exercise at Pre-T. These adaptations occurred in the absence of change in Vo_2peak (47.8 ± 1.7 vs. 49.2 ± 1.7 ml·kg^–1·min^–1) and in the activities (mol·kg protein^–1·h^–1) of succinic dehydrogenase (3.48 ± 0.21 vs. 3.77 ± 0.35) and citrate synthase (7.48 ± 0.61 vs. 8.52 ± 0.65) but not cytochrome oxidase (70.8 ± 5.1 vs. 79.6 ± 6.6 U/g protein; P < 0.05). It is concluded that the tighter metabolic control observed following short-term training is initially expressed during the nonsteady state, probably as a result of increases in oxidative phosphorylation that is not dependent on changes in Vo_2peak while the role of oxidative potential remains uncertain.

Effects and mechanisms of gastrointestinal electrical stimulation on slow waves: a systematic canine study

Sun, Y., Song, G.-Q., Yin, J., Lei, Y., Chen, J. D. Z. — Fri, 30 Oct 2009 10:13:07 PDT

The aims of this study were to determine optimal pacing parameters of electrical stimulation on different gut segments and to investigate effects and possible mechanisms of gastrointestinal electrical stimulation on gut slow waves. Twelve female hound-mix dogs were used in this study. A total of six pairs of electrodes were implanted on the stomach, duodenum, and ascending colon. Bilateral truncal vagotomy was performed in six of the dogs. One experiment was designed to study the effects of the pacing frequency on the entrainment of gut slow waves. Another experiment was designed to study the modulatory effects of the vagal and sympathetic pathways on gastrointestinal pacing. The frequency of slow waves was 4.88 ± 0.23 cpm (range, 4–6 cpm) in the stomach and 19.68 ± 0.31 cpm (range, 18–22 cpm) in the duodenum. There were no consistent or dominant frequencies of the slow waves in the colon. The optimal parameters to entrain slow waves were: frequency of 1.1 intrinsic frequency (IF; 10% higher than IF) and pulse width of 150–450 ms (mean, 320.0 ± 85.4 ms) for the stomach, and 1.1 IF and 10–20 ms for the small intestine. Electrical stimulation was not able to alter colon slow waves. The maximum entrainable frequency was 1.27 IF in the stomach and 1.21 IF in the duodenum. Gastrointestinal pacing was not blocked by vagotomy nor the application of an - or β-adrenergic receptor antagonist; whereas the induction of gastric dysrhythmia with electrical stimulation was completely blocked by the application of the - or β-adrenergic receptor antagonist. Gastrointestinal pacing is achievable in the stomach and small intestine but not the colon, and the maximal entrainable frequency of the gastric and small intestinal slow waves is about 20% higher than the IF. The entrainment of slow waves with gastrointestinal pacing is not modulated by the vagal or sympathetic pathways, suggesting a purely peripheral or muscle effect.

Opioid microinjection into raphe magnus modulates cardiorespiratory function in mice and rats

Fri, 30 Oct 2009 10:13:07 PDT

The raphe magnus (RM) participates in opioid analgesia and contains pain-modulatory neurons with respiration-related discharge. Here, we asked whether RM contributes to respiratory depression, the most prevalent lethal effect of opioids. To investigate whether opioidergic transmission in RM produces respiratory depression, we microinjected a mu-opioid receptor agonist, DAMGO, or morphine into the RM of awake rodents. In mice, opioid microinjection produced sustained decreases in respiratory rate (170 to 120 breaths/min), as well as heart rate (520 to 400 beats/min). Respiratory sinus arrhythmia, indicative of enhanced parasympathetic activity, was prevalent in mice receiving DAMGO microinjection. We performed similar experiments in rats but observed no changes in breathing rate or heart rate. Both rats and mice experienced significantly more episodes of bradypnea, indicative of impaired respiratory drive, after opioid microinjection. During spontaneous arousals, rats showed less tachycardia after opioid microinjection than before microinjection, suggestive of an attenuated sympathetic tone. Thus, activation of opioidergic signaling within RM produces effects beyond analgesia, including the unwanted destabilization of cardiorespiratory function. These adverse effects on homeostasis consequent to opioid microinjection imply a role for RM in regulating the balance of sympathetic and parasympathetic tone.

pH regulating transporters in neurons from various chemosensitive brainstem regions in neonatal rats

Fri, 30 Oct 2009 10:13:07 PDT

We studied the membrane transporters that mediate intracellular pH (pH_i) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pH_i recovery from an NH₄Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO₂) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na⁺/H⁺ exchange (NHE). Recovery in RTN neurons was blocked ~50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO₃-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ~65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na⁺ and increased by external HCO₃^–. On the basis of these findings, pH_i recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO₃-dependent transporter in LC neurons. Thus, pH_i recovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons.

Rainbow trout genetically selected for greater muscle fat content display increased activation of liver TOR signaling and lipogenic gene expression

Skiba-Cassy, S., Lansard, M., Panserat, S., Medale, F. — Fri, 30 Oct 2009 10:13:07 PDT

Genetic selection is commonly used in farm animals to manage body fat content. In rainbow trout, divergent selection for low or high muscle fat content leads to differences in utilization of dietary energy sources between the fat muscle line (FL) and the lean muscle line (LL). To establish whether genetic selection on muscle fat content affects the hepatic insulin/nutrient signaling pathway, we analyzed this pathway and the expression of several metabolism-related target genes in the livers of the two divergent lines under fasting and then refeeding conditions. Whereas glycemia returned to basal level 24 h after refeeding in FL trout, it remained elevated in the LL trout. Target of rapamycin (TOR) protein was more abundant in the livers of FL trout than in LL trout, and refeeding activation of the hepatic TOR signaling pathway (TOR, S6K1, and S6) was therefore enhanced. Genes related to glycolysis (glucokinase and pyruvate kinase) and gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) were only slightly affected by refeeding and genetic selection. Refeeding stimulated expression of lipogenic genes and the sterol-responsive element binding protein (SREBP1), and expression of fatty acid synthase, glucose-6-phosphate dehydrogenase, and serine dehydratase was predominant in the livers of FL fish compared with LL fish. In agreement with recent findings linking TOR to lipogenesis control, we concluded that genetic selection for muscle fat content resulted in overactivation of the TOR signaling pathway-associated lipogenesis and probably also improved utilization of glucose.

Recurrent restriction of sleep and inadequate recuperation induce both adaptive changes and pathological outcomes

Everson, C. A., Szabo, A. — Fri, 30 Oct 2009 10:13:07 PDT

Chronic restriction of a basic biological need induces adaptations to help meet requisites for survival. The adaptations to chronic restriction of sleep are unknown. A single episode of 10 days of partial sleep loss in rats previously was shown to be tolerated and to result in increased food intake and loss of body weight as principal signs. The purpose of the present experiment was to investigate the extent to which adaptation to chronic sleep restriction would ameliorate short-term effects and result in a changed internal phenotype. Rats were studied during 10 wk of multiple periods of restricted and unrestricted sleep to allow adaptive changes to develop. Control rats received the same ambulatory requirements only consolidated into periods that lessened interruptions of their sleep. The results indicate a latent period of relatively stable food and water intake without weight gain, followed by a dynamic phase marked by enormous increases in food and water intake and progressive loss of body weight, without malabsorption of calories. Severe consequences ensued, marked especially by changes to the connective tissues, and became fatal for two individuals. The most striking changes to internal organs in sleep-restricted rats included lengthening of the small intestine, decreased size of adipocytes, and increased incidence of multilocular adipocytes. Major organs accounted for an increased proportion of total body mass. These changes to internal tissues appear adaptive in response to high energy production, decomposition of lipids, and increased need to absorb nutrients, but ultimately insufficient to compensate for inadequate sleep.

Effect of consecutive repeated sprint and resistance exercise bouts on acute adaptive responses in human skeletal muscle

Fri, 30 Oct 2009 10:13:07 PDT

We examined acute molecular responses in skeletal muscle to repeated sprint and resistance exercise bouts. Six men [age, 24.7 ± 6.3 yr; body mass, 81.6 ± 7.3 kg; peak oxygen uptake, 47 ± 9.9 ml·kg^–1·min^–1; one repetition maximum (1-RM) leg extension 92.2 ± 12.5 kg; means ± SD] were randomly assigned to trials consisting of either resistance exercise (8 x 5 leg extension, 80% 1-RM) followed by repeated sprints (10 x 6 s, 0.75 N·m torque·kg^–1) or vice-versa. Muscle biopsies from vastus lateralis were obtained at rest, 15 min after each exercise bout, and following 3-h recovery to determine early signaling and mRNA responses. There was divergent exercise order-dependent phosphorylation of p70 S6K (S6K). Specifically, initial resistance exercise increased S6K phosphorylation (~75% P < 0.05), but there was no effect when resistance exercise was undertaken after sprints. Exercise decreased IGF-I mRNA following 3-h recovery (~50%, P = 0.06) independent of order, while muscle RING finger mRNA was elevated with a moderate exercise order effect (P < 0.01). When resistance exercise was followed by repeated sprints PGC-1 mRNA was increased (REX1-SPR2; P = 0.02) with a modest distinction between exercise orders. Repeated sprints may promote acute interference on resistance exercise responses by attenuating translation initiation signaling and exacerbating ubiquitin ligase expression. Indeed, repeated sprints appear to generate the overriding acute exercise-induced response when undertaking concurrent repeated sprint and resistance exercise. Accordingly, we suggest that sprint-activities are isolated from resistance training and that adequate recovery time is considered within periodized training plans that incorporate these divergent exercise modes.

Aerobic exercise training improves whole muscle and single myofiber size and function in older women

Fri, 30 Oct 2009 10:13:07 PDT

To comprehensively assess the influence of aerobic training on muscle size and function, we examined seven older women (71 ± 2 yr) before and after 12 wk of cycle ergometer training. The training program increased (P < 0.05) aerobic capacity by 30 ± 6%. Quadriceps muscle volume, determined by magnetic resonance imaging (MRI), was 12 ± 2% greater (P < 0.05) after training and knee extensor power increased 55 ± 7% (P < 0.05). Muscle biopsies were obtained from the vastus lateralis to determine size and contractile properties of individual slow (MHC I) and fast (MHC IIa) myofibers, myosin light chain (MLC) composition, and muscle protein concentration. Aerobic training increased (P < 0.05) MHC I fiber size 16 ± 5%, while MHC IIa fiber size was unchanged. MHC I peak power was elevated 21 ± 8% (P < 0.05) after training, while MHC IIa peak power was unaltered. Peak force (Po) was unchanged in both fiber types, while normalized force (Po/cross-sectional area) was 10% lower (P < 0.05) for both MHC I and MHC IIa fibers after training. The decrease in normalized force was likely related to a reduction (P < 0.05) in myofibrillar protein concentration after training. In the absence of an increase in Po, the increase in MHC I peak power was mediated through an increased (P < 0.05) maximum contraction velocity (Vo) of MHC I fibers only. The relative proportion of MLC_1s (Pre: 0.62 ± 0.01; Post: 0.58 ± 0.01) was lower (P < 0.05) in MHC I myofibers after training, while no differences were present for MLC_2s and MLC_3f isoforms. These data indicate that aerobic exercise training improves muscle function through remodeling the contractile properties at the myofiber level, in addition to pronounced muscle hypertrophy. Progressive aerobic exercise training should be considered a viable exercise modality to combat sarcopenia in the elderly population.

Thermogenic activity of the Ca2+-ATPase from blue marlin heater organ: regulation by KCl and temperature

da Costa, D. C. F., Landeira-Fernandez, A. M. — Fri, 30 Oct 2009 10:13:07 PDT

This work shows that vesicles derived from the blue marlin heater organ retain a sarcoplasmic reticulum (SR) Ca²⁺-ATPase that can interconvert different forms of energy. During the hydrolysis of ATP part of the energy is always converted into heat, and the other part can be converted into work (Ca²⁺ transport) or heat, depending on the temperature and the presence of KCl in the reaction medium. At 15°C, where KCl stimulates the activity approximately threefold, measurements of the amount of heat released per mole of ATP hydrolyzed (H^cal) show similar values (approximately –11 kcal/mol) in the presence or absence of a Ca²⁺ gradient. At 25°C, KCl activates the enzyme to the same extent as at 15°C, but inhibits the production of extra heat by SR Ca²⁺-ATPase when a Ca²⁺ gradient is built up across the membrane. The H^cal values found in the presence of a Ca²⁺-gradient were –26.2 ± 2.9 kcal/mol (n = 7) in control experiments and –16.1 ± 1.5 (n = 14) in the presence of 100 mM KCl. At 35°C, KCl has a smaller effect (~1.5-fold) on activating the enzyme. Similar to SR Ca²⁺-ATPase from mammals, at this temperature the enzyme produces almost twice the amount of heat per mole of ATP hydrolyzed in the presence of a Ca²⁺ gradient and KCl has no effect at all on this increment. These data suggest that the marlin SR Ca²⁺-ATPase may play an important role in heater organ thermogenesis and that KCl has the potential for regulating the heat production catalyzed by the enzyme.

Calcium-dependent phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells

Ortiz-Capisano, M. C., Liao, T.-D., Ortiz, P. A., Beierwaltes, W. H. — Fri, 30 Oct 2009 10:13:07 PDT

Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by calcium. We previously showed JG cells contain a calcium sensing receptor (CaSR), which, when stimulated, decreases cAMP formation and inhibits renin release. We hypothesize CaSR activation decreases cAMP and renin release, in part, by stimulating a calcium calmodulin-activated phosphodiesterase 1 (PDE1). We incubated our primary culture of JG cells with two selective PDE1 inhibitors [8-methoxymethil-IBMX (8-MM-IBMX; 20 µM) and vinpocetine (40 µM)] and the calmodulin inhibitor W-7 (10 µM) and measured cAMP and renin release. Stimulation of the JG cell CaSR with the calcimimetic cinacalcet (1 µM) resulted in decreased cAMP from a basal of 1.13 ± 0.14 to 0.69 ± 0.08 pM/mg protein (P < 0.001) and in renin release from 0.89 ± 0.16 to 0.38 ± 0.08 µg ANG I/ml·h^–1·mg protein^–1 (P < 0.001). However, the addition of 8-MM-IBMX with cinacalcet returned both cAMP (1.10 ± 0.19 pM/mg protein) and renin (0.57 ± 0.16 µg ANG I/ml·h^–1·mg protein^–1) to basal levels. Similar results were obtained with vinpocetine, and also with W-7. Combining 8-MM-IBMX and W-7 had no additive effect. To determine which PDE1 isoform is involved, we performed Western blot analysis for PDE1A, B, and C. Only Western blot analysis for PDE1C showed a characteristic band apparent at 80 kDa. Immunofluorescence showed cytoplasmic distribution of PDE1C and renin in the JG cells. In conclusion, PDE1C is expressed in isolated JG cells, and contributes to calcium's inhibitory modulation of renin release from JG cells.

Maternal hemoglobin level and fetal outcome at low and high altitudes

Gonzales, G. F., Steenland, K., Tapia, V. — Fri, 30 Oct 2009 10:13:07 PDT

Both, low (<7 g/dl) and high (>14.5 g/dl), maternal hemoglobin (Hb) levels have been related to poor fetal outcome. Most studies have been done at low altitude (LA). Here, we have sought to determine whether this relationship exists at both high and low altitude, and also whether there is an adverse effect of high altitude (HA) on fetal outcome independent of level of maternal hemoglobin. The study is based on a retrospective multicenter analysis of 35,449 pregnancies at LA and six other cities above 3000 meters. In analyses of all women at both LA and HA, those with Hb <9 g/dl had odds ratios (ORs) and 95% confidence intervals (CI) of 4.4 (CI: 2.8–6.7), 2.5 (CI: 1.9–3.2), and 1.4 (CI: 1.1–1.9) for stillbirths, preterm, and small for gestational age (SGA) births, respectively, compared with women with 11–12.9 g/dl of Hb, after adjustment for confounders. These risks by hemoglobin level differed little between women at LA and HA, suggesting that no correction of the definition of anemia is necessary for women at HA. Women living at high altitude with hemoglobin >15.5 g/dl had higher risks for stillbirths (OR: 1.3; CI: 1.05–1.3), preterm (OR: 1.5; CI 1.3–1.8), and SGA births (OR: 2.1, CI 1.8–2.3). There was also a significant adverse effect of living at HA, independent of hemoglobin level for all three outcomes (OR: 3.9, 1.7, and 2.3; CI: 2.8–5.2, 1.5–1.9, and 2.1–2.5) for stillbirths, preterms, and SGA respectively, after adjusting for hemoglobin level. Both, high and low maternal hemoglobin levels were related to poor pregnancy outcome, with similar effect of low hemoglobin in both LA and HA. Our data suggest, that maternal hemoglobin above 11 g/dl but below 13 g/dl is the area of minimal risk of poor adverse outcomes. Living at HA had an adverse effect independent of hemoglobin level.

Functional significance of muscarinic receptor expression within the proximal and distal rat vagina

Basha, M., LaBelle, E. F., Northington, G. M., Wang, T., Wein, A. J., Chacko, S. — Fri, 30 Oct 2009 10:13:07 PDT

Information regarding the role of cholinergic nerves in mediating vaginal smooth muscle contraction is sparse, and in vitro studies of the effects of muscarinic agonists on vaginal smooth muscle are discrepant. The goal of this study was to determine the expression of muscarinic receptors in the vaginal wall of the rat. In addition, we sought to determine the effect of the muscarinic receptor agonist carbachol on contractility and inositol phosphate production of the proximal and distal rat vaginal muscularis. RT-PCR analysis indicated that both M₂ and M₃ receptor transcripts were expressed within the proximal and distal rat vagina. Carbachol dose-dependently (10^–7–10^–4 M) contracted the rat vaginal muscularis with a greater maximal contractile response in the proximal vagina (P < 0.01) compared with the distal vagina. The contractile responses of the rat vaginal muscularis to carbachol were dose dependently inhibited by the M₃ antagonist para-fluoro-hexahydrosiladefenidol, and a pK_B of 7.78 and 7.95 was calculated for the proximal and distal vagina, respectively. Inositol phosphate production was significantly increased in both regions of the vagina following 20-min exposure to 50 µM carbachol with higher levels detected in the proximal vagina compared with the distal (P < 0.05). Preliminary experiments indicated the presence of M₂ and M₃ receptors in the human vaginal muscularis as well as contraction of human vaginal muscularis to carbachol, indicating that our animal studies are relevant to human tissue. Our results provide strong evidence for the functional significance of M₃ receptor expression in the vaginal muscularis.

Mice lacking the G protein {gamma}3-subunit show resistance to opioids and diet induced obesity

Schwindinger, W. F., Borrell, B. M., Waldman, L. C., Robishaw, J. D. — Fri, 30 Oct 2009 10:13:07 PDT

Contributing to the obesity epidemic, there is increasing evidence that overconsumption of high-fat foods may be analogous to drug addiction in that the palatability of these foods is associated with activation of specific reward pathways in the brain. With this perspective, we report that mice lacking the G protein ₃-subunit (Gng3^–/– mice) show resistance to high-fat diet-induced weight gain over the course of a 12-wk study. Compared with Gng3^+/+ controls, female Gng3^–/– mice exhibit a 40% reduction in weight gain and a 53% decrease in fat pad mass, whereas male Gng3^–/– mice display an 18% reduction in weight gain and no significant decrease in fat pad mass. The basis for the lowered weight gain is related to reduced food consumption for female and male Gng3^–/– mice of 13% and 14%, respectively. Female Gng3^–/– mice also show a lesser preference for high-fat chow than their female Gng3^+/+ littermates, suggesting an attenuated effect on a reward pathway associated with overconsumption of fat. One possible candidate is the µ-opioid receptor (Oprm1) signaling cascade. Supporting a defect in this signaling pathway, Gng3^–/– mice show marked reductions in both acute and chronic morphine responsiveness, as well as increases in endogenous opioid mRNA levels in reward-related regions of the brain. Taken together, these data suggest that the decreased weight gain of Gng3^–/– mice may be related to a reduced rewarding effect of the high-fat diet resulting from a defect in Oprm1 signaling and loss of the G protein ₃-subunit.

Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells

Zisa, D., Shabbir, A., Mastri, M., Suzuki, G., Lee, T. — Fri, 30 Oct 2009 10:13:07 PDT

Skeletal muscle produces a myriad of mitogenic factors possessing cardiovascular regulatory effects that can be explored for cardiac repair. Given the reported findings that VEGF may modulate muscle regeneration, we investigated the therapeutic effects of chronic injections of low doses of human recombinant VEGF-A₁₆₅ (0.1–1 µg/kg) into the dystrophic hamstring muscle in a hereditary hamster model of heart failure and muscular dystrophy. In vitro, VEGF stimulated proliferation, migration, and growth factor production of cultured C2C12 skeletal myocytes. VEGF also induced production of HGF, IGF2, and VEGF by skeletal muscle. Analysis of skeletal muscle revealed an increase in myocyte nuclear [531 ± 12 VEGF 1 µg/kg vs. 364 ± 19 for saline (number/mm²) saline] and capillary [591 ± 80 VEGF 1 µg/kg vs. 342 ± 21 for saline (number/mm²)] densities. Skeletal muscle analysis revealed an increase in Ki67⁺ nuclei in the VEGF 1 µg/kg group compared with saline. In addition, VEGF mobilized c-kit⁺, CD31⁺, and CXCR4⁺ progenitor cells. Mobilization of progenitor cells was consistent with higher SDF-1 concentrations found in hamstring, plasma, and heart in the VEGF group. Echocardiogram analysis demonstrated improvement in left ventricular ejection fraction (0.60 ± 0.02 VEGF 1 µg/kg vs. 0.45 ± 0.01 mm for saline) and an attenuation in ventricular dilation [5.59 ± 0.12 VEGF 1 µg/kg vs. 6.03 ± 0.09 for saline (mm)] 5 wk after initiating therapy. Hearts exhibited higher cardiomyocyte nuclear [845 ± 22 VEGF 1 µg/kg vs. 519 ± 40 for saline (number/mm²)] and capillary [2,159 ± 119 VEGF 1 µg/kg vs. 1,590 ± 66 for saline (number/mm²)] densities. Myocardial analysis revealed ~2.5 fold increase in Ki67+ cells and ~2.8-fold increase in c-kit⁺ cells in the VEGF group, which provides evidence for cardiomyocyte regeneration and progenitor cell expansion. This study provides novel evidence of a salutary effect of VEGF in the cardiomyopathic hamster via induction of myogenic growth factor production by skeletal muscle and mobilization of progenitor cells, which resulted in attenuation of cardiomyopathy and repair of the heart.

Maternal low-protein diet alters pancreatic islet mitochondrial function in a sex-specific manner in the adult rat

Theys, N., Bouckenooghe, T., Ahn, M.-T., Remacle, C., Reusens, B. — Fri, 30 Oct 2009 10:13:08 PDT

Mitochondrial dysfunction may be a long-term consequence of a poor nutritional environment during early life. Our aim was to investigate whether a maternal low-protein (LP) diet may program mitochondrial dysfunction in islets of adult progeny before glucose intolerance ensues. To address this, pregnant Wistar rats were fed isocaloric diets containing either 20% protein (control) or 8% protein (LP diet) throughout gestation. From birth, offspring received the control diet. The mitochondrial function was analyzed in islets of 3-mo-old offspring. Related to their basal insulin release, cultured islets from both male and female LP offspring presented a lower response to glucose challenge and a blunted ATP production compared with control offspring. The expression of malate dehydrogenase as well as the subunit 6 of the ATP synthase encoded by mitochondrial genome (mtDNA) was lower in these islets, reducing the capacity of ATP production through the Krebs cycle and oxidative phosphorylation. However, mtDNA content was unchanged in LP islets compared with control. Several consequences of protein restriction during fetal life were more marked in male offspring. Only LP males showed an increased reactive oxygen species production associated with a higher expression of mitochondrial subunits of the electron transport chain NADH-ubiquinone oxireductase subunit 4L, an overexpression of peroxisome proliferator-activated receptor- and uncoupling protein-2, and a strongly reduced beta-cell mass. In conclusion, mitochondrial function is clearly altered in islets from LP adult offspring in a sex-specific manner. That may provide a cellular explanation for the earlier development of glucose intolerance in male than in female offspring of dams fed an LP diet.

Ischemia-induced brain damage is enhanced in human renin and angiotensinogen double-transgenic mice

Chen, S., Li, G., Zhang, W., Wang, J., Sigmund, C. D., Olson, J. E., Chen, Y. — Fri, 30 Oct 2009 10:13:08 PDT

To investigate the role of brain angiotensin II (ANG II) in the pathogenesis of injury following ischemic stroke, mice overexpressing renin and angiotensinogen (R+A+) and their wild-type control animals (R–A–) were used for experimental ischemia studies. Focal brain ischemia was induced by middle cerebral artery occlusion (MCAO). The severity of ischemic injury was determined by measuring neurological deficits and histological damage at 24 and 48 h after MCAO, respectively. To exclude the influence of blood pressure and local collateral blood flow, brain slices were used for oxygen and glucose deprivation (OGD) studies. The severity of OGD-induced damage was determined by measuring indicators of tissue swelling and cell death, the intensity of the intrinsic optical signal (IOS), and the number of propidium iodide (PI) staining cells, respectively. Results showed 1) R+A+ mice showed higher neurological deficit score (3.8 ± 0.5 and 2.5 ± 0.3 for R+A+ and R–A–, respectively, P < 0.01) and larger infarct volume (22.2 ± 1.6% and 14.1 ± 1.2% for R+A+ and R–A–, respectively, P < 0.01); 2) The R+A+ brain slices showed more severe tissue swelling and cell death in the cortex (IOS: 140 ± 6% and 114 ± 10%; PI: 139 ± 20 cells/field and 39 ± 9 cells/field for R+A+ and R–A–, respectively, P < 0.01); 3) treatment with losartan (20 µmol/l) abolished OGD-induced exaggeration of cell injury seen in R+A+ mice. The data indicate that activation of ANG II/AT₁ signaling is harmful to brain exposed to ischemia.

Peripheral osmotic stimulation inhibits the brain's innate immune response to microdialysis of acidic perfusion fluid adjacent to supraoptic nucleus

Summy-Long, J. Y., Hu, S. — Fri, 30 Oct 2009 10:13:08 PDT

During the brain's innate immune response microglia, astroglia and ependymal cells resolve/repair damaged tissue and control infection. Released interleukin-1β (IL-1β) reaching cerebroventricles stimulates circumventricular organs (CVOs; subfornical organ, SFO; organum vasculosum lamina terminalis, OVLT), the median preoptic nucleus (MePO), and magnocellular and parvocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei. Hypertonic saline (HS) also activates these osmosensory CVOs and neuroendocrine systems, but, in contrast to IL-1β, inhibits the peripheral immune response. To examine whether the brain's innate immune response is attenuated by osmotic stimulation, sterile acidic perfusion fluid was microdialyzed (2 µl/min) in the SON area of conscious rats for 6 h with sterile HS (1.5 M NaCl) injected subcutaneously (15 ml/kg) at 5 h. Immunohistochemistry identified cytokine sources (IL-1β⁺; OX-42⁺ microglia) and targets (IL-1R⁺; inducible cyclooxygenase, COX-2⁺; c-Fos⁺) near the probe, in CVOs, MePO, ependymal cells, periventricular hypothalamus, SON, and PVN. Inserting the probe stimulated magnocellular neurons (c-Fos⁺; SON; PVN) via the MePO (c-Fos⁺), a response enhanced by HS. Microdialysis activated microglia (OX-42⁺; amoeboid/hypertrophied; IL-1β⁺) in the adjacent SON and bilaterally in perivascular areas of the PVN, periventricular hypothalamus and ependyma, coincident with c-Fos expression in ependymal cells and COX-2 in the vasculature. These microglial responses were attenuated by HS, coincident with activating parvocellular and magnocellular neuroendocrine systems and elevating circulating IL-1β, oxytocin, and vasopressin. Acidosis-induced cellular injury from microdialysis activated the brain's innate immune response by a mechanism inhibited by peripheral osmotic stimulation.

Inhibition of biliverdin reductase increases ANG II-dependent superoxide levels in cultured renal tubular epithelial cells

Fri, 30 Oct 2009 10:13:08 PDT

Induction of heme oxygenase-1 (HO-1) in the renal medulla increases carbon monoxide and bilirubin production and decreases ANG II-mediated superoxide production. The goal of this study was to determine the importance of increases in bilirubin to the antioxidant effects of HO-1 induction in cultured mouse thick ascending loop of Henle (TALH) and inner medullary collecting duct (IMCD3) cells. Bilirubin levels were decreased by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR), which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH or IMCD-3 cells with BVR siRNA (50 or 100 nM) resulted in an 80% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 ± 5 to 23 ± 4 nM (n = 4). We then determined the effects of inhibition of BVR on ANG II-mediated superoxide production. Superoxide production induced by ANG II (10^–9 M) significantly increased in both TALH and IMCD-3 cells. Treatment of TALH cells with BVR siRNA resulted in a significant increase in ouabain-sensitive rubidium uptake from 95 ± 6 to 122 ± 5% control (n = 4, P < 0.05). Lastly, inhibition of BVR with siRNA did not prevent the decrease in superoxide levels observed in cells pretreated with the HO-1 inducer, hemin. We conclude that decreased levels of cellular bilirubin increase ANG II-mediated superoxide production and sodium transport; however, increases in bilirubin are not necessary for HO-1 induction to attenuate ANG II-mediated superoxide production.

Whey protein potentiates the intestinotrophic action of glucagon-like peptide-2 in parenterally fed rats

Liu, X., Murali, S. G., Holst, J. J., Ney, D. M. — Fri, 30 Oct 2009 10:13:08 PDT

Glucagon-like peptide-2 (GLP-2) is a nutrient-regulated intestinotrophic hormone derived from proglucagon in the distal intestine. Enteral nutrients (EN) potentiate the action of GLP-2 to reverse parenteral nutrition (PN)-induced mucosal hypoplasia. The objective was to determine what enteral protein component, casein, soy, or whey protein, potentiates the intestinal growth response to GLP-2 in rats with PN-induced mucosal hypoplasia. Rats received PN and continuous intravenous infusion of GLP-2 (100 µg/kg/day) for 7 days. Six EN groups received PN+GLP-2 for days 1–3 and partial PN+GLP-2 plus EN for days 4–7. EN was provided by ad libitum intake of a semielemental liquid diet with different protein sources: casein, hydrolyzed soy, whey protein concentrate (WPC), and hydrolyzed WPC+casein. Controls received PN+GLP-2 alone. EN induced significantly greater jejunal sucrase activity and gain of body weight, and improved feed efficiency compared with PN+GLP-2 alone. EN induced greater ileal proglucagon expression, increased plasma concentration of bioactive GLP-2 by 35%, and reduced plasma dipeptidyl peptidase IV (DPP-IV) activity compared with PN+GLP-2 alone, P < 0.05. However, only whey protein, and not casein or soy, potentiated the ability of GLP-2 to reverse PN-induced mucosal hypoplasia and further increase ileal villus height, crypt depth, and mucosa cellularity compared with PN+GLP-2 alone, P < 0.05. The ability of whey protein to induce greater mucosal surface area was associated with decreased DPP-IV activity in ileum and colon compared with casein, soy, or PN+GLP-2 alone, P < 0.05. In conclusion, whey protein potentiates the action of GLP-2 to reverse PN-induced mucosal hypoplasia in association with decreased intestinal DPP-IV activity.

Tissue-specific PAI-1 gene expression and glycosylation pattern in insulin-resistant old rats

Fri, 30 Oct 2009 10:13:08 PDT

Increased levels of plasminogen activator inhibitor-1 (PAI-1) have been associated with obesity, aging, insulin resistance, and type 2 diabetes, conditions that contribute to increased cardiovascular risk. PAI-1 is expressed in a variety of tissues, but the cellular origin of plasma PAI-1 is unknown. To link insulin resistance, aging, and cardiovascular disease, we examined the expression and glycosylation pattern of PAI-1 in liver and white adipose tissue (WAT) from adult (3 mo) and insulin-resistant old (24 mo) Wistar rats. Glycosylated PAI-1 protein was also purified by affinity chromatography from endothelial culture supernatans to analyze its inhibitory activity. We also analyzed the contribution of adipocytes and stromal vascular cells from WAT to PAI-1 levels with aging. Aging caused a significant increase of PAI-1 mRNA (P < 0.001) in WAT that was predominantly due to the adipocytes and not to stroma-vascular cells, while there was no modification in liver from aged rats. Moreover, PAI-1 expression increased during preadipocyte differentiation (P < 0.001). Furthermore, we found a tissue-dependent PAI-1 glycosylation pattern: adipose tissue only expresses the glycosylated PAI-1 form, whereas the liver mainly expresses the nonglycosylated form. Finally, we also found evidences suggesting that the glycosylated PAI-1 form shows higher inhibitory activity than the nonglycosylated. Our data suggest that WAT may be a major source of the elevated plasma levels of PAI-1 in insulin-resistant old rats. Additionally, the high degree of PAI-1 glycosylation and activity, together with the significant increase in visceral fat in old rats, may well contribute to an increased cardiovascular risk associated with insulin-resistant states.

Evolutionary structural and functional conservation of an ortholog of the GLUT2 glucose transporter gene (SLC2A2) in zebrafish

Fri, 30 Oct 2009 10:13:08 PDT

In mammals, GLUT2 plays an essential role in glucose homeostasis. From an evolutionary perspective, relatively little is known about the biology of GLUT2, or other GLUTs, in nonmammalian vertebrates. Here, we have conducted studies to functionally characterize GLUT2 in zebrafish. First, we cloned the zebrafish ortholog of GLUT2 (zfGLUT2) encoding a protein of 504 amino acids with high-sequence identity to other known vertebrate GLUT2 proteins. The zfGLUT2 gene consists of 11 exons and 10 introns, spanning 20 kb and mapping to a region of chromosome 2 that exhibits conserved synteny with human chromosome 3. When expressed in Xenopus oocytes, zfGLUT2 transported 2-deoxyglucose (2-DG) with similar affinity than mammalian GLUT2 (K_m of 11 mM). Transport of 2-DG was competed mostly by d-fructose and d-mannose and was inhibited by cytochalasin B. During early development, zfGLUT2 expression was detected already at 10 h postfertilization and remained elevated in 5-day larvae, when it was clearly localized to the liver and intestinal bulb. In the adult, zfGLUT2 expression was highest in testis, brain, skin, kidney, and intestine, followed by liver and muscle. In the intestine, zfGLUT2 transcripts were detected in absorptive enterocytes, and its mRNA levels were altered by fasting and refeeding, suggesting that its expression in the intestine may be regulated by the nutritional status. These results indicate that the structure and function of GLUT2 has been remarkably well conserved during vertebrate evolution and open the way for the use of zebrafish as a model species in which to study the biology and pathophysiology of GLUT2.

Physiological response to extreme fasting in subantarctic fur seal (Arctocephalus tropicalis) pups: metabolic rates, energy reserve utilization, and water fluxes

Verrier, D., Groscolas, R., Guinet, C., Arnould, J. P. Y. — Fri, 30 Oct 2009 10:13:08 PDT

Surviving prolonged fasting requires various metabolic adaptations, such as energy and protein sparing, notably when animals are simultaneously engaged in energy-demanding processes such as growth. Due to the intermittent pattern of maternal attendance, subantarctic fur seal pups have to repeatedly endure exceptionally long fasting episodes throughout the 10-mo rearing period while preparing for nutritional independence. Their metabolic responses to natural prolonged fasting (33.4 ± 3.3 days) were investigated at 7 mo of age. Within 4–6 fasting days, pups shifted into a stage of metabolic economy characterized by a minimal rate of body mass loss (0.7%/day) and decreased resting metabolic rate (5.9 ± 0.1 ml O₂·kg^–1·day^–1) that was only 10% above the level predicted for adult terrestrial mammals. Field metabolic rate (289 ± 10 kJ·kg^–1·day^–1) and water influx (7.9 ± 0.9 ml·kg^–1·day^–1) were also among the lowest reported for any young otariid, suggesting minimized energy allocation to behavioral activity and thermoregulation. Furthermore, lean tissue degradation was dramatically reduced. High initial adiposity (>48%) and predominant reliance on lipid catabolism likely contributed to the exceptional degree of protein sparing attained. Blood chemistry supported these findings and suggested utilization of alternative fuels, such as β-hydroxybutyrate and de novo synthesized glucose from fat-released glycerol. Regardless of sex and body condition, pups tended to adopt a convergent strategy of extreme energy and lean body mass conservation that appears highly adaptive for it allows some tissue growth during the repeated episodes of prolonged fasting they experience throughout their development.

Natural antibodies and complement modulate intimal thickening after arterial injury

Fri, 30 Oct 2009 10:13:08 PDT

Immune factors are involved in modulating neointimal response to arterial wall injury, but the role of individual immune effectors in this response remains unclear. Using a carotid cuff injury model in mice, we tested the role of immunoglobulin isotypes in modulating intimal thickening by using adoptive transfer of splenocytes from WT mice, or the direct administration of IgG or IgM into immune-deficient Rag-1–/– [Rag-1 knockout (Rag-1KO)] mice. The direct role of complement was also tested by depletion of complement. Splenocytes from WT mice were isolated and adoptively transferred to Rag-1KO mice subjected to carotid cuff arterial injury. Transfer of splenocytes to Rag-1KO mice resulted in increased serum IgM and IgG within 48 h and were comparable to WT levels by 21 days after injury. Splenocyte transfer in Rag-1KO decreased intimal area by 40% compared with Rag-1KO mice without cell transfer. To further differentiate the relative contribution of IgM or IgG in reducing intimal thickening, additional groups of Rag-1KO mice were subjected to injury and given intravenous injections of pooled mouse IgG or IgM. Both IgG and IgM treatment significantly reduced intimal thickening compared with untreated Rag-1KO mice. Immunoglobulin treatments modified serum complement C3 profile and decreased C3 presence in injured arteries. Depletion of C3 using cobra venom factor in Rag-1KO mice significantly decreased intimal thickening. Our results identify the direct role of natural IgG and IgM, and complement in the modulation of neointimal response to arterial injury.

Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells

Fri, 30 Oct 2009 10:13:08 PDT

Hypocretins/orexins act through two receptor subtypes: OX₁ and OX₂. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX₂ receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12–24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX₂ receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca²⁺ but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca²⁺, as well as PKC-ERK1/2 signaling.

Endolymphatic sac is involved in the regulation of hydrostatic pressure of cochlear endolymph

Inamoto, R., Miyashita, T., Akiyama, K., Mori, T., Mori, N. — Fri, 30 Oct 2009 10:13:08 PDT

To clarify the role of the endolymphatic sac (ES) in the regulation of endolymphatic pressure, the effects of isoproterenol, a β-adrenergic receptor agonist, and acetazolamide, a potent carbonic anhydrase inhibitor, both of which decrease ES direct current potential on cochlear hydrostatic pressure, were examined in guinea pigs. When isoproterenol was applied intravenously, hydrostatic pressures of cochlear endolymph and perilymph were significantly increased with no change in endocochlear potential or the hydrostatic pressure of cerebrospinal fluid. Acetazolamide produced no marked change in the hydrostatic pressure of cochlear endolymph. In ears with an obstructed ES, the action of isoproterenol on the hydrostatic pressure of cochlear endolymph and perilymph was suppressed. These results suggest that the ES may regulate the hydrostatic pressure of the endolymphatic system via the action of the agents such as catecholamines on the ES.

Insulin-like growth factor-II regulates maternal hemodynamic adaptation to pregnancy in rats

Van Mieghem, T., van Bree, R., Van Herck, E., Deprest, J., Verhaeghe, J. — Fri, 30 Oct 2009 10:13:09 PDT

The relationship between maternal plasma volume (PV) expansion and fetal growth is well established, but the underlying mechanisms remain unclear. Here, we examined the influence of maternal body weight and fetoplacental mass on gestational PV increment in the rat. Because IGF-I and IGF-II have growth-promoting and vasoactive properties, their relationship to PV expansion and fetoplacental growth was also studied. In normal rats, the gradual expansion of PV (+35% at day 22, i.e., term) was accompanied by a rise in circulating IGF-II (+45%) and a considerable drop in IGF-I (–73%). Increased maternal body weight induced by an obesogenic diet did not influence PV and circulating IGFs compared with rats on the standard diet. Combining the results from both diets, circulating IGF-II was the principal correlate of PV. A second experiment examined the effect of fetoplacental mass reduction by surgically removing half of the gestational sacs at day 16. This procedure reduced maternal PV and circulating IGF-II at term by 14% and 20%, respectively. We then investigated the effect of a constant infusion of IGF-II (1 mg·kg^–1·day^–1) from day 16, which raised circulating IGF-II by 38% and found increased PV (+19%) and a larger placental trophospongial area (+29%) at term. Our results indicate that the placenta, the primary source of IGF-II synthesis in pregnancy, drives PV expansion, and that IGF-II is among the regulatory factors of the gestational PV increment. Further studies should clarify whether IGF-II directly affects vascular function and/or indirectly promotes the secretion of placenta-derived vasoactive substances.

Corrigendum

Fri, 30 Oct 2009 10:13:09 PDT

Corrigendum

Fri, 30 Oct 2009 10:13:09 PDT

Modulation of myocellular fat stores: lipid droplet dynamics in health and disease

Meex, R. C. R., Schrauwen, P., Hesselink, M. K. C. — Fri, 25 Sep 2009 09:51:58 PDT

Storage of fatty acids as triacylglycerol (TAG) occurs in almost all mammalian tissues. Whereas adipose tissue is by far the largest storage site of fatty acids as TAG, subcellular TAG-containing structures—referred to as lipid droplets (LD)—are also present in other tissues. Until recently, LD were considered inert storage sites of energy dense fats. Nowadays, however, LD are increasingly considered dynamic functional organelles involved in many intracellular processes like lipid metabolism, vesicle trafficking, and cell signaling. Next to TAG, LD also contain other neutral lipids such as diacylglycerol. Furthermore, LD are coated by a monolayer of phospholipids decorated with a variety of proteins regulating the delicate balance between LD synthesis, growth, and degradation. Disturbances in LD-coating proteins may result in disequilibrium of TAG synthesis and degradation, giving rise to insulin-desensitizing lipid intermediates, especially in insulin-responsive tissues like skeletal muscle. For a proper and detailed understanding, more information on processes and players involved in LD synthesis and degradation is necessary. This, however, is hampered by the fact that research on LD dynamics in (human) muscle is still in its infancy. A rapidly expanding body of knowledge on LD dynamics originates from studies in other tissues and other species. Here, we aim to review the involvement of LD-coating proteins in LD formation and degradation (LD dynamics) and to extrapolate this knowledge to human skeletal muscle and to explore the role of LD dynamics in myocellular insulin sensitivity.

In situ blood oxygen analysis for a truly free-living deep-diving animal. Focus on "Extreme hypoxemic tolerance and blood oxygen depletion in diving elephant seals"

Fedak, M. — Fri, 25 Sep 2009 09:51:58 PDT

Extreme hypoxemic tolerance and blood oxygen depletion in diving elephant seals

Meir, J. U., Champagne, C. D., Costa, D. P., Williams, C. L., Ponganis, P. J. — Fri, 25 Sep 2009 09:51:58 PDT

Species that maintain aerobic metabolism when the oxygen (O₂) supply is limited represent ideal models to examine the mechanisms underlying tolerance to hypoxia. The repetitive, long dives of northern elephant seals (Mirounga angustirostris) have remained a physiological enigma as O₂ stores appear inadequate to maintain aerobic metabolism. We evaluated hypoxemic tolerance and blood O₂ depletion by 1) measuring arterial and venous O₂ partial pressure (Po₂) during dives with a Po₂/temperature recorder on elephant seals, 2) characterizing the O₂-hemoglobin (O₂-Hb) dissociation curve of this species, 3) applying the dissociation curve to Po₂ profiles to obtain %Hb saturation (So₂), and 4) calculating blood O₂ store depletion during diving. Optimization of O₂ stores was achieved by high venous O₂ loading and almost complete depletion of blood O₂ stores during dives, with net O₂ content depletion values up to 91% (arterial) and 100% (venous). In routine dives (>10 min) Pv_O₂ and Pa_O₂ values reached 2–10 and 12–23 mmHg, respectively. This corresponds to So₂ of 1–26% and O₂ contents of 0.3 (venous) and 2.7 ml O₂/dl blood (arterial), demonstrating remarkable hypoxemic tolerance as Pa_O₂ is nearly equivalent to the arterial hypoxemic threshold of seals. The contribution of the blood O₂ store alone to metabolic rate was nearly equivalent to resting metabolic rate, and mean temperature remained near 37°C. These data suggest that elephant seals routinely tolerate extreme hypoxemia during dives to completely utilize the blood O₂ store and maximize aerobic dive duration.

Sustained stimulation of vasopressin and oxytocin release by ATP and phenylephrine requires recruitment of desensitization-resistant P2X purinergic receptors

Gomes, D. A., Song, Z., Stevens, W., Sladek, C. D. — Fri, 25 Sep 2009 09:51:58 PDT

Coexposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine [PE; an 1-adrenergic receptor (1-AR) agonist] induces an extended elevation in vasopressin and oxytocin (VP/OT) release. New evidence is presented that this extended response is mediated by recruitment of desensitization-resistant ionotropic purinergic receptor subtypes (P2X-Rs): 1) Antagonists of the P2X2/3 and P2X7-Rs truncated the sustained VP/OT release induced by ATP+PE but did not alter the transient response to ATP alone. 2) The P2X2/3 and P2X7-R antagonists did not alter either ATP or ATP+PE-induced increases in [Ca²⁺]_i. 3) P2X2/3 and P2X7-R agonists failed to elevate [Ca²⁺]_i, while ATP--S, an agonist for P2X2-Rs increased [Ca²⁺]_i and induced a transient increase in VP/OT release. 4) A P2Y1-R antagonist did not prevent initiation of the synergistic, sustained stimulation of VP/OT release by ATP+PE but did reduce its duration. Thus, the desensitization-resistant P2X2/3 and P2X7-R subtypes are required for the sustained, synergistic hormone response to ATP+PE, while P2X2-Rs are responsible for the initial activation of Ca²⁺-influx by ATP and ATP stimulation of VP/OT release. Immunohistochemistry, coimmunoprecipitation, and Western blot analysis confirmed the presence of P2X2 and P2X3, P2X2/3, and P2X7-R protein, respectively in SON. These findings support the hypothesis that concurrent activation of P2X2-R and 1-AR induces calcium-driven recruitment of P2X2/3 and 7-Rs, allowing sustained activation of a homeostatic circuit. Recruitment of these receptors may provide sustained release of VP during dehydration and may be important for preventing hemorrhagic and septic shock.

Dietary palmitate and linoleate oxidations, oxidative stress, and DNA damage differ according to season in mouse lemurs exposed to a chronic food deprivation

Fri, 25 Sep 2009 09:51:58 PDT

This study investigated the extent to which the increase in torpor expression in the grey mouse lemur, due to graded food restriction, is modulated by a trade-off between a whole body sparing of polyunsaturated dietary fatty acids and the related oxidative stress generated during daily torpor. We measured changes in torpor frequency, total energy expenditure (TEE), linoleate (polyunsaturated fatty acid) and palmitate (saturated fatty acid) oxidation, hexanoyl-lysine (HEL; the product of linoleate peroxidation), and 8-hydroxydeoxyguanosine (8OHdG; a marker of DNA damage). Animals under summer-acclimated long days (LD) or winter-acclimated short days (SD) were exposed to a 40% (LD40 and SD40) and 80% (LD80 and SD80) 35-day calorie restriction (CR). During CR, all groups reduced their body mass, but LD80 animals reached survival-threatened levels at day 22 and were then excluded from the CR trial. Only SD mouse lemurs increased their torpor frequency with CR and displayed a decrease in their TEE adjusted for fat-free mass. After CR, SD40 mouse lemurs shifted the dietary fatty acid oxidation toward palmitate and spared linoleate. Such a shift was not observed in LD animals and during severe CR, during which oxidation of both dietary fatty acids was increased. Concomitantly, HEL increased in both LD40 and SD80 groups, whereas DNA damage was only seen in SD80 food-restricted animals. HEL correlated positively with linoleate oxidation confirming in vivo the substrate/product relationship demonstrated in vitro, and negatively with TEE adjusted for fat-free mass, suggesting higher oxidative stress associated with increased torpor expression. This suggests a seasonal-dependant, cost-benefit trade-off between maximizing torpor propensity and minimizing oxidative stress that is associated with a shift toward sparing of dietary polyunsaturated fatty acids that is dependent upon the expression of a winter phenotype.

FAT/CD36-null mice reveal that mitochondrial FAT/CD36 is required to upregulate mitochondrial fatty acid oxidation in contracting muscle

Fri, 25 Sep 2009 09:51:58 PDT

The plasma membrane fatty acid transport protein FAT/CD36 is also present at the mitochondria, where it may contribute to the regulation of fatty acid oxidation, although this has been challenged. Therefore, we have compared enzyme activities and rates of mitochondrial palmitate oxidation in muscles of wild-type (WT) and FAT/CD36 knockout (KO) mice, at rest and after muscle contraction. In WT and KO mice, carnitine palmitoyltransferase-I, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase activities did not differ in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria of WT and FAT/CD36 KO mice. Basal palmitate oxidation rates were lower (P < 0.05) in KO mice (SS –18%; IMF –13%). Muscle contraction increased fatty acid oxidation (+18%) and mitochondrial FAT/CD36 protein (+16%) in WT IMF but not in WT SS, or in either mitochondrial subpopulation in KO mice. This revealed that the difference in IMF mitochondrial fatty acid oxidation between WT and KO mice can be increased ~2.5-fold from 13% under basal conditions to 35% during muscle contraction. The FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), inhibited palmitate transport across the plasma membrane in WT, but not in KO mice. In contrast, SSO bound to mitochondrial membranes and reduced palmitate oxidation rates to a similar extent in both WT and KO mitochondria (~80%; P < 0.05). In addition, SSO reduced state III respiration with succinate as a substrate, without altering mitochondrial coupling (P/O ratios). Thus, while SSO inhibits FAT/CD36-mediated palmitate transport at the plasma membrane, SSO has undefined effects on mitochondria. Nevertheless, the KO animals reveal that FAT/CD36 contributes to the regulation of mitochondrial fatty acid oxidation, which is especially important for meeting the increased metabolic demands during muscle contraction.

Nitric oxide dilates rat retinal blood vessels by cyclooxygenase-dependent mechanisms

Fri, 25 Sep 2009 09:51:58 PDT

It has been suggested that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent mechanisms in the ocular vasculature; however, the importance of the pathway in regulating retinal circulation in vivo remains to be elucidated. Therefore, we investigated the role of COX-dependent mechanisms in NO-induced vasodilation of retinal blood vessels in thiobutabarbital-anesthetized rats with and without neuronal blockade (tetrodotoxin treatment). Fundus images were captured with a digital camera that was equipped with a special objective lens. The retinal vascular response was assessed by measuring changes in diameter of the retinal blood vessel. The localization of COX and soluble guanylyl cyclase in rat retina was examined using immunohistochemistry. The NO donors (sodium nitroprusside and NOR3) increased the diameter of the retinal blood vessels but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a nonselective COX inhibitor, or SC-560, a selective COX-1 inhibitor, markedly attenuated the vasodilation of retinal arterioles, but not the depressor response, to the NO donors. However, both the vascular responses to NO donors were unaffected by the selective COX-2 inhibitors NS-398 and nimesulide. Indomethacin did not change the retinal vascular and depressor responses to hydralazine, 8-(4-chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (a membrane-permeable cGMP analog) and 8-(4-chlorophenylthio)-adenosine-3', 5'-cyclic monophosphate (a membrane-permeable cAMP analog). Treatment with SQ 22536, an adenylyl cyclase inhibitor, but not ODQ, a soluble guanylyl cyclase inhibitor, significantly attenuated the NOR3-induced vasodilation of retinal arterioles. The COX-1 immunoreactivity was found in retinal blood vessels. The retinal blood vessel was faintly stained for soluble guanylyl cyclase, although the apparent immunoreactivities on mesenteric and choroidal blood vessels were observed. These results suggest that NO exerts a substantial part of its dilatory effect via a mechanism that involves COX-1-dependent pathway in rat retinal vasculature.

Long-term ventilatory adaptation and ventilatory response to hypoxia in plateau pika (Ochotona curzoniae): role of nNOS and dopamine

Fri, 25 Sep 2009 09:51:58 PDT

We assessed ventilatory patterns and ventilatory responses to hypoxia (HVR) in high-altitude (HA) plateau pikas, repetitively exposed to hypoxic burrows, and control rats. We evaluated the role of neuronal nitric oxide synthase (nNOS) and dopamine by using S-methyl-l-thiocitrulline (SMTC) inhibitor and haloperidol antagonist, respectively. Ventilation (Vi) was measured using a whole body plethysmograph in conscious pikas (n = 9) and low-altitude (LA) rats (n = 7) at different Pi_O₂ (56, 80, 111, 150, and 186 mmHg) and in HA acclimatized rats (n = 9, 8 days at 4,600 m) at two different Pi_O₂ (56 and 80 mmHg). The effects of NaCl, SMTC, and haloperidol on ventilatory patterns were assessed in pikas at Pi_O₂ = 56 and 80 mmHg. We observed a main species effect with larger Vi, tidal volume (VT), inspiratory time/total time (T_i/T_tot), and a lower expiratory time in pikas than in LA rats. Pikas had also a larger VT and lower respiratory frequency compared with HA rats in hypoxia. HVR of pikas and rats were not statistically different. In pikas, SMTC induced a significant increase in Vi and VT for a Pi_O₂ of 56 mmHg, but had no effect for a PiO₂ of 80 mmHg, i.e., the living altitude of pikas. In pikas, haloperidol injection had no effect on any ventilatory parameter. Long-term ventilatory adaptation in pikas is mainly due to an improvement in respiratory pattern (VT and T_i/T_tot) with no significant improvement in HVR. The sensitivity to severe acute hypoxia in pikas seems to be regulated by a peripheral nNOS mechanism.

Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio)

Perry, S. F., Vulesevic, B., Grosell, M., Bayaa, M. — Fri, 25 Sep 2009 09:51:58 PDT

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial Cl^–/HCO₃^– exchange in adult zebrafish (Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [Cl^–] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5–10 days of exposure relative to fish raised in normal water [Cl^–] (0.4 mmol/l); transferring fish to Cl^–-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHCO₃ (10–12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and 10 days of transfer that was associated with a significant 40% increase in Cl^– uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHCO₃] water, concurrent with increases in Cl^– uptake and SLC26 mRNA levels, suggests a role for these anion transporters in Cl^– uptake and acid-base regulation owing to their Cl^–/HCO₃^– exchange activities.

The role of the neural sympathetic and parasympathetic systems in diurnal and sleep state-related cardiovascular rhythms in the late-gestation ovine fetus

Jensen, E. C., Bennet, L., Guild, S.-J., Booth, L. C., Stewart, J., Gunn, A. J. — Fri, 25 Sep 2009 09:51:58 PDT

The efferent mechanisms mediating the well-known diurnal cardiovascular rhythms in the late-gestation fetus are only partially understood. In the present study, we evaluated the contribution of the parasympathetic and sympathetic nervous systems (SNS) to these rhythms. Chronically instrumented fetal sheep at a mean (SE) of 122 (1) days gestation (term is 147 days) underwent either chemical sympathectomy with 6-hydroxydopamine the day after surgery (n = 8), vagotomy at surgery (n = 8), or were sham controls (n = 8). Fetal heart rate (HR), fetal HR variability (HRV), mean arterial blood pressure (MAP), carotid blood flow (CaBF), electrocorticogram (ECoG) activity, and nuchal activity were measured continuously for 24 h. Changes between sleep states were determined in a 6-h interval. Control fetal sheep showed consistent diurnal rhythms in fetal HR, HRV, MAP, and CaBF, with maximal activity in the evening, but not in nuchal activity. Sympathectomy was associated with a significant reduction of both fetal HR and HRV, while vagotomy was associated with a fall in fetal HRV (P < 0.05) but no change in HR. Despite this, most animals in the two intervention groups still showed diurnal rhythms for fetal HR, HRV, MAP, and CaBF, although peak HR may have been delayed in the sympathectomy group (mean 02:22 vs. 23:54 h in controls, P = 0.06). There was no effect of either intervention on sleep state cycling, although state-related cardiovascular rhythms were significantly modulated. These data indicate that, neither the SNS nor vagal activity, in isolation at least, is essential for generating cardiovascular diurnal rhythms in the late-gestation fetus.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist

Fri, 25 Sep 2009 09:51:58 PDT

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC₅₀ = 0.69 ± 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-l-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI₂ generation but not that of cAMP or recruitment of β-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

Endotoxin depresses heart rate variability in mice: cytokine and steroid effects

Fri, 25 Sep 2009 09:51:58 PDT

Heart rate variability (HRV) falls in humans with sepsis, but the mechanism is not well understood. We utilized a mouse model of endotoxemia to test the hypothesis that cytokines play a role in abnormal HRV during sepsis. Adult male C57BL/6 mice underwent surgical implantation of probes to continuously monitor electrocardiogram and temperature or blood pressure via radiotelemetry. Administration of high-dose LPS (Escherichia coli LPS, 10 mg/kg, n = 10) caused a biphasic response characterized by an early decrease in temperature and heart rate at 1 h in some mice, followed by a prolonged period of depressed HRV in all mice. Further studies showed that LPS doses as low as 0.01 mg/kg evoked a significant decrease in HRV. With high-dose LPS, the initial drops in temperature and HR were temporally correlated with peak expression of TNF 1 h post-LPS, whereas maximal depression in HRV coincided with peak levels of multiple other cytokines 3–9 h post-LPS. Neither hypotension nor hypothermia explained the HRV response. Pretreatment with dexamethasone prior to LPS significantly blunted expression of 7 of the 10 cytokines studied and shortened the duration of depressed HRV by about half. Interestingly, dexamethasone treatment alone caused a dramatic increase in both low- and high-frequency HRV. Administration of recombinant TNF caused a biphasic response in HR and HRV similar to that caused by LPS. Understanding the role of cytokines in abnormal HRV during sepsis could lead to improved strategies for detecting life-threatening nosocomial infections in intensive care unit patients.

Hibernation induces pentobarbital insensitivity in medulla but not cortex

Hengen, K. B., Behan, M., Carey, H. V., Jones, M. V., Johnson, S. M. — Fri, 25 Sep 2009 09:51:58 PDT

The 13-lined ground squirrel (Ictidomys tridecemlineatus), a hibernating species, is a natural model of physiological adaption to an extreme environment. During torpor, body temperature drops to 0–4°C, and the cortex is electrically silent, yet the brain stem continues to regulate cardiorespiratory function. The mechanisms underlying selective inhibition in the brain during torpor are not known. To test whether altered GABAergic function is involved in regional and seasonal differences in neuronal activity, cortical and medullary slices from summer-active (SA) and interbout aroused (IBA) squirrels were placed in a standard in vitro recording chamber. Silicon multichannel electrodes were placed in cortex, ventral respiratory column (VRC), and nucleus tractus solitarius (NTS) to record spontaneous neuronal activity. In slices from IBA squirrels, bath-applied pentobarbital sodium (300 µM) nearly abolished cortical neuronal activity, but VRC and NTS neuronal activity was unaltered. In contrast, pentobarbital sodium (300 µM) nearly abolished all spontaneous cortical, VRC, and NTS neuronal activity in slices from SA squirrels. Muscimol (20 µM; GABA_A receptor agonist) abolished all neuronal activity in cortical and medullary slices from both IBA and SA squirrels, thereby demonstrating the presence of functional GABA_A receptors. Pretreatment of cortical slices from IBA squirrels with bicuculline (100 µM; GABA_A receptor antagonist) blocked pentobarbital-dependent inhibition of spontaneous neuronal activity. We hypothesize that GABA_A receptors undergo a seasonal modification in subunit composition, such that cardiorespiratory neurons are uniquely unaffected by surges of an endogenous positive allosteric modulator.

Calcineurin plays a modulatory role in loading-induced regulation of type I myosin heavy chain gene expression in slow skeletal muscle

Fri, 25 Sep 2009 09:51:58 PDT

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine (T₃)] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + T₃ was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg·kg^–1·day^–1) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + T₃ (P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression.

Maternal and postweaning diet interaction alters hypothalamic gene expression and modulates response to a high-fat diet in male offspring

Page, K. C., Malik, R. E., Ripple, J. A., Anday, E. K. — Fri, 25 Sep 2009 09:51:58 PDT

Epidemiological data and results from animal studies indicate that imbalances in maternal nutrition impact the expression of metabolic disorders in the offspring. We tested the hypothesis that consumption of excess saturated fats during pregnancy and lactation contributes to adult metabolic dysfunction and that these disturbances can be further influenced by the postweaning diet. Adult male offspring from chow-fed dams were compared with males from dams fed a diet high in saturated fat (45 kcal/100 kcal) before mating, pregnancy, and lactation. Offspring were weaned to a standard chow diet or high fat diet. Animals were killed at 120 days after a 24-h fast. Body weight, energy intake, fat deposition, serum leptin, and insulin were significantly higher in offspring from control or high-fat dams if fed a high-fat diet from weaning to adulthood. Only fat-fed offspring from fat-fed dams were hyperglycemic. Leptin receptor, proopiomelanocortin, and neuropeptide Y (NPY) were also significantly increased in offspring exposed to excess saturated fat during gestation and into adulthood, whereas NPY₁ receptor was downregulated. Signal transducer and activator of transcription 3 mRNA level was significantly higher in offspring from high-fat-fed dams compared with controls; however, no change was detected in cocaine and amphetamine-regulated transcript or suppressor of cytokine signaling 3. An increase in agouti-related protein expression did not reach significance. A significant reduction in phosphatidylinositol 3-kinase regulatory subunit (p85) coupled to an upregulation of protein kinase B was observed in offspring from high-fat-fed dams transitioned to chow food, whereas p85 expression was significantly increased in high-fat offspring weaned to the high-fat diet. These data support the hypothesis that early life exposure to excess fat is associated with changes in hypothalamic regulation of body weight and energy homeostasis and that postweaning diet influences development of metabolic dysfunction and obesity.

Jugular venous pooling during lowering of the head affects blood pressure of the anesthetized giraffe

Fri, 25 Sep 2009 09:51:58 PDT

How blood flow and pressure to the giraffe's brain are regulated when drinking remains debated. We measured simultaneous blood flow, pressure, and cross-sectional area in the carotid artery and jugular vein of five anesthetized and spontaneously breathing giraffes. The giraffes were suspended in the upright position so that we could lower the head. In the upright position, mean arterial pressure (MAP) was 193 ± 11 mmHg (mean ± SE), carotid flow was 0.7 ± 0.2 l/min, and carotid cross-sectional area was 0.85 ± 0.04 cm². Central venous pressure (CVP) was 4 ± 2 mmHg, jugular flow was 0.7 ± 0.2 l/min, and jugular cross-sectional area was 0.14 ± 0.04 cm² (n = 4). Carotid arterial and jugular venous pressures at head level were 118 ± 9 and –7 ± 4 mmHg, respectively. When the head was lowered, MAP decreased to 131 ± 13 mmHg, while carotid cross-sectional area and flow remained unchanged. Cardiac output was reduced by 30%, CVP decreased to –1 ± 2 mmHg (P < 0.01), and jugular flow ceased as the jugular cross-sectional area increased to 3.2 ± 0.6 cm² (P < 0.01), corresponding to accumulation of ~1.2 l of blood in the veins. When the head was raised, the jugular veins collapsed and blood was returned to the central circulation, and CVP and cardiac output were restored. The results demonstrate that in the upright-positioned, anesthetized giraffe cerebral blood flow is governed by arterial pressure without support of a siphon mechanism and that when the head is lowered, blood accumulates in the vein, affecting MAP.

Control of respiration in flight muscle from the high-altitude bar-headed goose and low-altitude birds

Scott, G. R., Richards, J. G., Milsom, W. K. — Fri, 25 Sep 2009 09:51:58 PDT

Bar-headed geese fly at altitudes of up to 9,000 m on their biannual migration over the Himalayas. To determine whether the flight muscle of this species has evolved to facilitate exercise at high altitude, we compared the respiratory properties of permeabilized muscle fibers from bar-headed geese and several low-altitude waterfowl species. Respiratory capacities were assessed for maximal ADP stimulation (with single or multiple inputs to the electron transport system) and cytochrome oxidase excess capacity (with an exogenous electron donor) and were generally 20–40% higher in bar-headed geese when creatine was present. When respiration rates were extrapolated to the entire pectoral muscle mass, bar-headed geese had a higher mass-specific aerobic capacity. This may represent a surplus capacity that counteracts the depressive effects of hypoxia on mitochondrial respiration. However, there were no differences in activity for mitochondrial or glycolytic enzymes measured in homogenized muscle. The [ADP] leading to half-maximal stimulation (K_m) was approximately twofold higher in bar-headed geese (10 vs. 4–6 µM), and, while creatine reduced K_m by 30% in this species, it had no effect on K_m in low-altitude birds. Mitochondrial creatine kinase may therefore contribute to the regulation of oxidative phosphorylation in flight muscle of bar-headed geese, which could promote efficient coupling of ATP supply and demand. However, this was not based on differences in creatine kinase activity in isolated mitochondria or homogenized muscle. The unique differences in bar-headed geese existed without prior exercise or hypoxia exposure and were not a result of phylogenetic history, and may, therefore, be important evolutionary specializations for high-altitude flight.

Identification of two glucocorticoid response elements in the promoter region of the ubiquitous isoform of glutamine synthetase in gulf toadfish, Opsanus beta

Esbaugh, A. J., Walsh, P. J. — Fri, 25 Sep 2009 09:51:58 PDT

Unlike most teleosts, gulf toadfish have the capacity to switch from ammoniotely to ureotely as the predominate means of nitrogen excretion during periods of stress. The switch to ureotely is a result of increased glutamine synthetase (GS) mRNA expression/enzyme activity in the liver and muscle, which is initiated by cortisol. Cortisol typically affects gene expression through the action of cortisol-activated transcription factors, such as glucocorticoid receptors, which bind to glucocorticoid response elements (GRE) in the upstream regulatory region of genes. The purpose of the present study was to identify the GRE responsible for increased GS gene expression during crowding/confinement in gulf toadfish using an in vivo luciferase reporter assay. Upstream promoter regions for both the ubiquitous and gill GS isoforms were amplified by PCR. Additionally, an intron was amplified from the ubiquitous GS isoform that suggested the possibility of two discreet transcripts for the mitochondrial and cytoplasmic proteins. When tested via in vivo reporter assays, both the cytoplasmic and mitochondrial ubiquitous GS promoters showed increased luciferase activity during crowding vs. noncrowded controls; the gill GS promoter showed no effects in response to crowding. In silico analysis of the mitochondrial and cytoplasmic ubiquitous GS promoter constructs showed an overlapping section of 565 bp containing two potential GREs. Mutation of either site alone had no effect on luciferase activity vs. wild-type controls. However, when both sites were mutated a significant decrease in luciferase activity was observed. We conclude that two functional GREs combine to confer cortisol-inducible GS expression in the liver of gulf toadfish.

Is active sweating during heat acclimation required for improvements in peripheral sweat gland function?

Buono, M. J., Numan, T. R., Claros, R. M., Brodine, S. K., Kolkhorst, F. W. — Fri, 25 Sep 2009 09:51:58 PDT

We investigated whether the eccrine sweat glands must actively produce sweat during heat acclimation if they are to adapt and increase their capacity to sweat. Eight volunteers received intradermal injections of BOTOX, to prevent neural stimulation and sweat production of the sweat glands during heat acclimation, and saline injections as a control in the contralateral forearm. Subjects performed 90 min of moderate-intensity exercise in the heat (35°C, 40% relative humidity) on 10 consecutive days. Heat acclimation decreased end-exercise heart rate (156 ± 22 vs. 138 ± 17 beats/min; P = 0.0001) and rectal temperature (38.2 ± 0.3 vs. 37.9 ± 0.3°C; P = 0.0003) and increased whole body sweat rate (0.70 ± 0.29 vs. 1.06 ± 0.50 l/h; P = 0.030). During heat acclimation, there was no measurable sweating in the BOTOX-treated forearm, but the control forearm sweat rate during exercise increased 40% over the 10 days (P = 0.040). Peripheral sweat gland function was assessed using pilocarpine iontophoresis before and after heat acclimation. Before heat acclimation, the pilocarpine-induced sweat rate of the control and BOTOX-injected forearms did not differ (0.65 ± 0.20 vs. 0.66 ± 0.22 mg·cm^–2·min^–1). However, following heat acclimation, the pilocarpine-induced sweat rate in the control arm increased 18% to 0.77 ± 0.21 mg·cm^–2·min^–1 (P = 0.021) but decreased 52% to 0.32 ± 0.18 mg·cm^–2·min^–1 (P < 0.001) in the BOTOX-treated arm. Using complete chemodenervation of the sweat glands, coupled with direct cholinergic stimulation via pilocarpine iontophoresis, we demonstrated that sweat glands must be active during heat acclimation if they are to adapt and increase their capacity to sweat.

Induction of hepatic cyclooxygenase-2 by hyperhomocysteinemia via nuclear factor-{kappa}B activation

Wu, N., Siow, Y. L., O, K. — Fri, 25 Sep 2009 09:51:58 PDT

Hyperhomocysteinemia, an elevation of blood homocysteine (Hcy), is a metabolic disorder associated with dysfunction of multiple organs. Apart from endothelial dysfunction, Hcy can cause hepatic lipid accumulation and liver injury. However, the mechanism responsible for Hcy-induced liver injury is poorly understood. The aim of this study was to investigate the regulation of cyclooxygenase-2 (COX-2), a proinflammatory factor, expression in the liver during the initial phase of hyperhomocysteinemia. Sprague-Dawley rats were fed a high-methionine diet for 1 or 4 wk. Serum and liver concentrations of Hcy were significantly elevated after 1 or 4 wk of dietary treatment. COX-2 mRNA and protein levels were significantly elevated in the liver of hyperhomocysteinemic rats. The induction of COX-2 expression was more prominent in 1-wk hyperhomocysteinemic rats than that in the 4-wk group. EMSA revealed an activation of NF-B in the same liver tissue in which COX-2 was induced. Administration of a NF-B inhibitor to hyperhomocysteinemic rats effectively abolished hepatic COX-2 expression, inhibited the formation of inflammatory foci, and improved liver function. Further investigation revealed that oxidative stress due to increased superoxide generation was responsible for increased phosphorylation and degradation of IB leading to NF-B activation in the liver. Administration of 4-hydroxy-tetramethyl-piperidine-1-oxyl, an SOD mimetic, to hyperhomocysteinemic rats not only inhibited NF-B activation but also prevented hepatic COX-2 induction and improved liver function. These results suggest that hyperhomocysteinemia-induced COX-2 expression is mediated via NF-B activation. Increased oxidative stress and inflammatory response may contribute to liver injury associated with hyperhomocysteinemia.

MitoQ administration prevents endotoxin-induced cardiac dysfunction

Supinski, G. S., Murphy, M. P., Callahan, L. A. — Fri, 25 Sep 2009 09:51:58 PDT

Sepsis elicits severe alterations in cardiac function, impairing cardiac mitochondrial and pressure-generating capacity. Currently, there are no therapies to prevent sepsis-induced cardiac dysfunction. We tested the hypothesis that administration of a mitochondrially targeted antioxidant, 10-(6'-ubiquinonyl)-decyltriphenylphosphonium (MitoQ), would prevent endotoxin-induced reductions in cardiac mitochondrial and contractile function. Studies were performed on adult rodents (n = 52) given either saline, endotoxin (8 mg·kg^–1·day^–1), saline + MitoQ (500 µM), or both endotoxin and MitoQ. At 48 h animals were killed and hearts were removed for determination of either cardiac mitochondrial function (using polarography) or cardiac pressure generation (using the Langendorf technique). We found that endotoxin induced reductions in mitochondrial state 3 respiration rates, the respiratory control ratio, and ATP generation. Moreover, MitoQ administration prevented each of these endotoxin-induced abnormalities, P < 0.001. We also found that endotoxin produced reductions in cardiac pressure-generating capacity, reducing the systolic pressure-diastolic relationship. MitoQ also prevented endotoxin-induced reductions in cardiac pressure generation, P < 0.01. One potential link between mitochondrial and contractile dysfunction is caspase activation; we found that endotoxin increased cardiac levels of active caspases 9 and 3 (P < 0.001), while MitoQ prevented this increase (P < 0.01). These data demonstrate that MitoQ is a potent inhibitor of endotoxin-induced mitochondrial and cardiac abnormalities. We speculate that this agent may prove a novel therapy for sepsis-induced cardiac dysfunction.

Attenuation of peripheral salt taste responses and local immune function contralateral to gustatory nerve injury: effects of aldosterone

Guagliardo, N. A., West, K. N., McCluskey, L. P., Hill, D. L. — Fri, 25 Sep 2009 09:51:59 PDT

Dietary sodium restriction coupled with axotomy of the rat chorda tympani nerve (CTX) results in selectively attenuated taste responses to sodium salts in the contralateral, intact chorda tympani nerve. Converging evidence indicates that sodium deficiency also diminishes the activated macrophage response to injury on both the sectioned and contralateral, intact sides of the tongue. Because a sodium-restricted diet causes a robust increase in circulating aldosterone, we tested the hypothesis that changes in neurophysiological and immune responses contralateral to the CTX could be mimicked by aldosterone administration instead of the low-sodium diet. Taste responses in rats with CTX and supplemental aldosterone for 4–6 days were similar to rats with CTX and dietary sodium restriction. Responses to sodium salts were as much as 50% lower compared with sham-operated and vehicle-supplemented rats. The group-related functional differences were eliminated with lingual application of amiloride, suggesting that a major transduction pathway affected was through epithelial sodium channels. Consistent with the functional results, few macrophages were observed on either side of the tongue in rats with CTX and aldosterone. In contrast, macrophages were elevated on both sides of the tongue in rats with CTX and the vehicle. These results show that sodium deficiency or administration of aldosterone suppresses the immune response to neural injury, resulting in attenuation of peripheral gustatory function. They also show a potential key link among downstream consequences of sodium imbalance, taste function, and immune activity.

The role of nitric oxide in the development of neurogenic pulmonary edema in spinal cord-injured rats: the effect of preventive interventions

Sedy, J., Zicha, J., Kunes, J., Hejcl, A., Sykova, E. — Fri, 25 Sep 2009 09:51:59 PDT

Neurogenic pulmonary edema (NPE) is an acute life-threatening complication following an injury of the spinal cord or brain, which is associated with sympathetic hyperactivity. The role of nitric oxide (NO) in NPE development in rats subjected to balloon compression of the spinal cord has not yet been examined. We, therefore, pretreated Wistar rats with the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) either acutely (just before the injury) or chronically (for 4 wk prior to the injury). Acute (but not chronic) l-NAME administration enhanced NPE severity in rats anesthetized with 1.5% isoflurane, leading to the death of 83% of the animals within 10 min after injury. Pretreatment with either the ganglionic blocker pentolinium (to reduce blood pressure rise) or the muscarinic receptor blocker atropine (to lessen heart rate decrease) prevented or attenuated NPE development in these rats. We did not observe any therapeutic effects of atropine administered 2 min after spinal cord compression. Our data indicate that NPE development is dependent upon a marked decrease of heart rate under the conditions of high blood pressure elicited by the activation of the sympathetic nervous system. These hemodynamic alterations are especially pronounced in rats subjected to acute NO synthase inhibition. In conclusion, nitric oxide has a partial protective effect on NPE development because it attenuates sympathetic vasoconstriction and consequent baroreflex-induced bradycardia following spinal cord injury.

Loss of vagal anti-inflammatory effect: in vivo visualization and adoptive transfer

O'Mahony, C., van der Kleij, H., Bienenstock, J., Shanahan, F., O'Mahony, L. — Fri, 25 Sep 2009 09:51:59 PDT

The vagus nerve is a conduit for bidirectional signaling between the brain and the viscera. Vagal signaling has been shown to downregulate gastrointestinal inflammation, and the mechanism is thought to involve acetylcholine binding to the alpha-7 subunit of the nicotinic acetylcholine receptor on macrophages. The aims of this study were to quantify the impact of vagotomy in vivo by visualizing nuclear factor (NF)-B activity and to determine if the proinflammatory impact of vagotomy could be transferred by lymphocytes. Real-time biophotonic imaging revealed that subdiaphragmatic vagotomy resulted in increased levels of NF-B in vivo. NF-B activation was further exaggerated in vivo following exposure to 4% DSS for 5 days. Vagotomized animals also exhibited higher disease activity scores and secreted more proinflammatory cytokines. Adoptive transfer of CD4⁺ T cells from vagotomized animals (but not CD4⁺ T cells from sham-operated controls) to naive dextran sulfate sodium (DSS)-treated recipients resulted in increased inflammatory scores. Further examination of the CD4⁺ T cells revealed that adoptive transfer of the CD25^– population alone from vagotomized donors (but not sham-operated donors) was sufficient to aggravate colitis in DSS-treated recipients. Increased DSS-induced inflammation was associated with reduced CD4⁺CD25⁺Foxp3⁺ regulatory T cell numbers in recipients. This study clearly demonstrates the ability of the vagus nerve to modulate activity of the proinflammatory transcription factor NF-B in vivo. The proinflammatory effect of vagotomy is transferable using splenic T cells and highlights a previously unappreciated cellular mechanism for linking central parasympathetic processes with mucosal inflammation and immune homeostasis.

Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells

Wang, Z.-Y., Wang, P., Bjorling, D. E. — Fri, 25 Sep 2009 09:51:59 PDT

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 µM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 µM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.

Sex-specific differences in cardiac control and hematology of sockeye salmon (Oncorhynchus nerka) approaching their spawning grounds

Sandblom, E., Clark, T. D., Hinch, S. G., Farrell, A. P. — Fri, 25 Sep 2009 09:51:59 PDT

Some male salmonids (e.g., rainbow trout) display profound cardiovascular adjustments during sexual maturation, including cardiac growth and hypertension, and tachycardia has been observed in free-ranging male salmonids near their spawning grounds. In the present study, we investigated cardiac control, dorsal aortic blood pressure, cardiac morphometrics, and hematological variables in wild, sexually maturing sockeye salmon (Oncorhynchus nerka) with a particular aim to decipher any sex-specific differences. Routine heart rate (f_H) was significantly higher in females (52 vs. 43 beats/min), which was due to significantly lower cholinergic tone (28 vs. 46%), because there were no differences in adrenergic tone or intrinsic heart rate between sexes. No differences in blood pressure were observed despite males possessing an 11% greater relative ventricular mass. Concomitant with higher routine heart rates, female sockeye had significantly higher levels of cortisol, testosterone, and 17β-estradiol, whereas the level of 11-ketotestosterone was higher in males. There were no differences in hematocrit or hemoglobin concentration between the sexes. The findings of this study highlight the importance of considering sex as a variable in research fields such as conservation biology and when modeling the consequences of local and global climate change. Indeed, this study helps to provide a mechanistic basis for the significantly higher rates of female mortality observed in previous studies of wild-caught sockeye salmon.

Impaired exercise tolerance and skeletal muscle myopathy in sulfonylurea receptor-2 mutant mice

Fri, 25 Sep 2009 09:51:59 PDT

By sensing intracellular energy levels, ATP-sensitive potassium (K_ATP) channels help regulate vascular tone, glucose metabolism, and cardioprotection. SUR2 mutant mice lack full-length K_ATP channels in striated and smooth muscle and display a complex phenotype of hypertension and coronary vasospasm. SUR2 mutant mice also display baseline cardioprotection and can withstand acute sympathetic stress better than normal mice. We now studied response to a form of chronic stress, namely that induced by 4 wk of daily exercise on SUR2 mutant mice. Control mice increased exercise capacity by 400% over the training period, while SUR2 mutant mice showed little increase in exercise capacity. Unexercised SUR2 mutant showed necrotic and regenerating fibers in multiple muscle skeletal muscles, including quadriceps, tibialis anterior, and diaphragm muscles. Unlike exercised control animals, SUR2 mutant mice did not lose weight, presumably due to less overall exertion. Unexercised SUR2 mutant mice showed a trend of mildly reduced cardiac function, measured by fractional shortening, (46 ± 4% vs. 57 ± 7% for SUR2 mutant and control, respectively), and this decrease was not exacerbated by chronic exercise exposure. Despite an improved response to acute sympathetic stress and baseline cardioprotection, exercise intolerance results from lack of SUR2 K_ATP channels in mice.

Opioid growth factor-opioid growth factor receptor axis is a physiological determinant of cell proliferation in diverse human cancers

Zagon, I. S., Donahue, R. N., McLaughlin, P. J. — Fri, 25 Sep 2009 09:51:59 PDT

The opioid growth factor (OGF) regulates cell proliferation of human cancer cells through the cyclin-dependent kinase inhibitory pathway, with mediation of this action by the OGF receptor (OGFr). The ubiquity of the OGF-OGFr axis in human cancer is unknown. We used 31 human cancer cell lines, representative of more than 90% of neoplasias occurring in humans, and found that OGF and OGFr were detected in the cytoplasm and nucleus by immunohistochemistry. The addition of OGF to cultures depressed cell number up to 41%, whereas naltrexone (NTX) increased cell proliferation by up to 44%, a total of 85% in the modulating capacity for the OGF-OGFr axis. Neutralization of OGF by specific antibodies led to a marked increase in cell number. Knockdown of OGFr by OGFr-siRNA resulted in a significant increase in the number of cells, even in the face of the addition of exogenous OGF. The cultures to which NTX was added and subjected to OGFr-siRNA were similar to those with OGF-siRNA alone. The OGF-OGFr axis, a physiological determinant of cell-proliferative activity, is a ubiquitous feature of human cancer cells. The identification of this native biological system in neoplasia may be important in understanding the pathophysiology of neoplasia, and in designing treatment modalities that utilize the body's own chemistry.

Saliva and other taste stimuli are important for gustatory processing of linoleic acid

Stratford, J. M., Contreras, R. J. — Fri, 25 Sep 2009 09:51:59 PDT

Paradoxically, bilateral transection of the chorda tympani nerve (CTX) raises the taste discrimination threshold for the free fatty acid, linoleic acid (LA), yet the chorda tympani nerve (CT) is unresponsive to lingual application of LA alone. LA may require a background of saliva to activate taste cells, since CTX decreases saliva production through denervation of the submaxillary and sublingual salivary glands. To assess the role of saliva, we measured LA taste discrimination thresholds for animals whose submaxillary and sublingual salivary glands were removed and also recorded CT responses to LA mixed in artificial saliva. Partial desalivation shifted LA discrimination thresholds from between 5.5 and 11 µM to between 11 and 22 µM. However, this effect was not as pronounced as previously seen with CTX animals. Surprisingly, the CT was unresponsive to LA mixed with artificial saliva, suggesting that artificial saliva may lack components necessary for LA taste. Additionally, fats may primarily enhance other tastes. We previously reported that LA increases CT responses to monosodium glutamate (MSG). Thus we also recorded CT whole nerve responses to taste mixtures of LA and sodium chloride (NaCl), sucrose (SUC), citric acid (CA), or quinine hydrochloride (QHCl) in anesthetized rats. We found that LA increased CT responses to NaCl but did not alter CT responses to SUC, CA, and QHCl. Thus CT recordings either lack the sensitivity to detect small changes to SUC, CA, and QHCl or LA may affect CT responses to MSG and NaCl only, perhaps by specifically modulating gustatory processing of Na⁺.

Ductus arteriosus wave intensity analysis in fetal lambs: midsystolic ductal flow augmentation is due to antegrade pulmonary arterial wave transmission

Smolich, J. J., Mynard, J. P., Penny, D. J. — Fri, 25 Sep 2009 09:51:59 PDT

In midsystole, fetal pulmonary trunk (PT) and arterial (PA) blood flows characteristically fall, despite pulmonary blood pressure increasing, while ductus arteriosus (DA) flow continues to rise to a delayed peak. Wave intensity (WI) analysis indicates that midsystolic fetal PT and PA flow reductions are related to a very large midsystolic PA backward-running compression wave (BCW_ms), which originates in the pulmonary microvasculature and is partially transmitted into the PT. This study tested the hypothesis that midsystolic augmentation of DA blood flow was related to transmission of the PA BCW_ms into the DA. DA, PT, and PA WI analysis was performed in eight anesthetized late-gestation fetal sheep instrumented with DA, PT, and left PA micromanometer catheters to measure pressure (P) and transit-time flow probes to obtain blood velocity (U). In a subgroup (n = 5), the main PA was briefly occluded to abolish wave transmission from the lungs. WI was calculated as the product of P and U rates of change. PA and PT WI profiles both contained a prominent BCW_ms, ~5-fold larger in the PA (P < 0.005), which increased P but decreased U. By contrast, the DA WI profile demonstrated a large midsystolic forward-running compression wave (FCW_ms), which increased DA P and U, and occurred 5 ms after PA BCW_ms. Furthermore, both DA FCW_ms and PT BCW_ms were abolished by main PA occlusion. These results suggest that the fetal PA BCW_ms undergoes retrograde transmission into the PT as a BCW_ms, but antegrade transmission into the DA as a FCW_ms that augments midsystolic DA flow.

Comparison of sarcoplasmic reticulum calcium content in atrial and ventricular myocytes of three fish species

Haverinen, J., Vornanen, M. — Fri, 25 Sep 2009 09:51:59 PDT

Ryanodine (Ry) sensitivity of cardiac contraction differs between teleost species, between atrium and ventricle, and according to the thermal history of the fish. The hypothesis that variability in Ry sensitivity of contraction is due to species-specific, chamber-specific, and temperature-related differences in the sarcoplasmic reticulum (SR) Ca²⁺ content, was tested by comparing steady-state (SS) and maximal (Max) Ca²⁺ loads of the SR in three teleost fish, rainbow trout (Oncorhynchus mykiss), burbot (Lota lota), and crucian carp (Carassius carassius), which differ in the extent of SR contribution to excitation-contraction coupling. Fish were acclimated at 4°C (cold-acclimation, CA) or 18°C (warm-acclimation, WA), and SR Ca²⁺ content was released by a rapid application of 10 mM caffeine to single cardiac myocytes; its amount was determined from the Na⁺-Ca²⁺ exchange current at 18°C. SS Ca²⁺ load was larger in atrial (304–915 µmol/l) than ventricular (224–540 µmol/l) myocytes in all fish species (P < 0.05), and the same was true for Max SR Ca²⁺ content: 550–1,522 µmol/l and 438–840 µmol/l for atrial and ventricular myocytes, respectively (P < 0.05). Consistent with the hypothesis, acclimation to cold increased Ca²⁺ load of the cardiac SR in the burbot heart, but contrary to the hypothesis, temperature acclimation did not affect SR Ca²⁺ content in rainbow trout and crucian carp hearts. Furthermore, there was an inverse relation between SR Ca²⁺ content and Ry sensitivity of contraction force: the species with the smallest SR Ca²⁺ content (burbot) is most sensitive to Ry. Collectively, these findings show that SR Ca²⁺ content of fish cardiac myocytes is several times larger than that in mammalian cardiac SR.

Benefits of oat {beta}-glucan and sucrose feedings on infection and macrophage antiviral resistance following exercise stress

Murphy, E. A., Davis, J. M., Carmichael, M. D., Mayer, E. P., Ghaffar, A. — Fri, 25 Sep 2009 09:51:59 PDT

Oat β-glucan can counteract the exercise-induced increased risk for upper respiratory tract infection (URTI) in mice, which is at least partly mediated by its effects on lung macrophages. Substantial evidence in humans indicates that carbohydrate-containing sports drinks can offset the decreased immune function associated with stressful exercise. However, no studies in animals or humans have directly examined their effects on URTI using a controlled virus-challenge model. We examined the effects of sucrose feedings alone and in combination with oat β-glucan on susceptibility to infection and on macrophage antiviral resistance in mice following stressful exercise. These effects were also examined in rested, nonimmunocompromised control mice. Mice were assigned to one of four groups: H₂O (water), sucrose (S), oat β-glucan (OβG), and sucrose + oat β-glucan (S+OβG). OβG and S treatments consisted of a solution of 50% OβG and 6% sucrose, respectively, and were administered in drinking water for 10 consecutive days. Exercise consisted of a treadmill run to fatigue performed on three consecutive days. Mice were then intranasally inoculated with a standardized dose of herpes simplex virus 1 (HSV-1) and monitored for morbidity and mortality for 21 days. Additional mice were used to determine macrophage antiviral resistance. In the exercise experiment, S, OβG, and S+OβG all reduced morbidity (P < 0.05), while only S+OβG reduced mortality (P < 0.05). Macrophage antiviral resistance was also increased in S, OβG, and S+OβG treatments (P < 0.05). In resting controls, S and S+OβG reduced morbidity and mortality (P < 0.05) and showed a trend toward increased macrophage antiviral resistance. There was no significant additive effect of S and OβG in either control or exercised animals. These data extend our previous work on the benefits of oat β-glucan to show that sucrose feedings have similar effects on susceptibility to respiratory infection and macrophage antiviral resistance in both resting controls and following exercise stress.

Evaluation of different methods for assessment of insulin sensitivity in Gottingen minipigs: introduction of a new, simpler method

Christoffersen, B., Ribel, U., Raun, K., Golozoubova, V., Pacini, G. — Fri, 25 Sep 2009 09:51:59 PDT

The use of animal models in diabetes research requires reliable tests for evaluation of insulin sensitivity and β-cell function. Minipigs are being increasingly used in metabolic research, and the aim of this study was to compare different tests and indexes for evaluation of insulin sensitivity and β-cell function in Göttingen minipigs. Hyperinsulinemic, isoglycemic clamp, intravenous (IVGTT) and oral glucose tolerance tests (OGTT), and a modified insulin tolerance test were performed in minipigs fed either low- or high-energy diet. Furthermore, the reproducibility of IVGTT-derived parameters was assessed. Previously described insulin sensitivity indexes [steady-state glucose infusion rate/glucose concentration/insulin concentration from clamp (M/G/I); oral glucose insulin sensitivity (OGIS) and ISI_comp from OGTT; S_I from minimal model analysis of IVGTT; and quantitative insulin sensitivity check index from fasting values] were calculated together with an insulin sensitivity index from the modified insulin tolerance test (ISI_ITT) and a new simple index (S2) derived from the first 30 min of the IVGTT. β-Cell function was assessed from the IVGTT and the OGTT. Reproducibility of the IVGTT-derived parameters was calculated as median intraindividual coefficient of variation (CV%).M/G/I correlated significantly only with S2 (P < 0.05, r = 0.54). S2 furthermore correlated with S_I (P < 0.001, r = 0.81), ISI_ITT (P < 0.001, r = 0.57), and the two indexes from OGTT, ISI_comp (P < 0.001, r = 0.78) and OGIS (p < 0.05, r = 0.48). No correlation was found between β-cell function indexes from OGTT and IVGTT. The median CV% of the new S2 index was 13. In conclusion, the new simple index of insulin sensitivity, S2, was revealed to be useful for evaluation of insulin sensitivity in pigs.

Cardiac and skeletal muscle fatty acid transport and transporters and triacylglycerol and fatty acid oxidation in lean and Zucker diabetic fatty rats

Fri, 25 Sep 2009 09:51:59 PDT

We examined fatty acid transporters, transport, and metabolism in hearts and red and white muscles of lean and insulin-resistant (week 6) and type 2 diabetic (week 24) Zucker diabetic fatty (ZDF) rats. Cardiac fatty acid transport was similar in lean and ZDF hearts at week 6 but was reduced at week 24 (–40%) in lean but not ZDF hearts. Red muscle of ZDF rats exhibited an early susceptibility to upregulation (+66%) of fatty acid transport at week 6 that was increased by 50% in lean and ZDF rats at week 24 but remained 44% greater in red muscle of ZDF rats. In white muscle, no differences were observed in fatty acid transport between groups or from week 6 to week 24. In all tissues (heart and red and white muscle), FAT/CD36 protein and plasmalemmal content paralleled the changes in fatty acid transport. Triacylglycerol content in red and white muscles, but not heart, in lean and ZDF rats correlated with fatty acid transport (r = 0.91) and sarcolemmal FAT/CD36 (r = 0.98). Red and white muscle fatty acid oxidation by isolated mitochondria was not impaired in ZDF rats but was reduced by 18–24% in red muscle of lean rats at week 24. Thus, in red, but not white, muscle of insulin-resistant and type 2 diabetic animals, a marked upregulation in fatty acid transport and intramuscular triacylglycerol was associated with increased levels of FAT/CD36 expression and plasmalemmal content. In heart, greater rates of fatty acid transport and FAT/CD36 in ZDF rats (week 24) were attributable to the inhibition of age-related reductions in these parameters. However, intramuscular triacylglycerol did not accumulate in hearts of ZDF rats. Thus insulin resistance and type 2 diabetes are accompanied by tissue-specific differences in FAT/CD36 and fatty acid transport and metabolism. Upregulation of fatty acid transport increased red muscle, but not cardiac, triacylglycerol accumulation. White muscle lipid metabolism dysregulation was not observed.

Corrigendum

Fri, 25 Sep 2009 09:51:59 PDT