Saline-infused Controls Research Articles

Central Thyrotropin-Releasing Hormone Infusion Opposes Cardiovascular and Metabolic Suppression during Caloric Restriction

Inhibition of hypothalamic thyrotropin-releasing hormone (TRH) neuronal activity is a well-established adaptation to caloric restriction (CR) that suppresses pituitary secretion of thyroid-stimulating hormone, but may also participate in modulation of autonomic function. Thus, we hypothesized that decreased hypothalamic TRH activity contributes to CR-induced bradycardia and decreased metabolic rate. To test this hypothesis, male Sprague-Dawley rats were instrumented with telemetry devices for measurement of heart rate (HR) and blood pressure (BP) and a lateral intracerebroventricular (i.c.v.) guide cannula for central infusions. After recovery, rats were housed in metabolic chambers and given either ad libitum(ad-lib) or CR treatments for 7 days; half of each diet group was then given continuous i.c.v. infusions of TRH (25 nmol/h) or saline (0.25 µl/h) for 7 days via osmotic pump. This dose of TRH did not significantly alter peripheral free T₄ levels. In ad-lib rats, TRH infusion produced small reductions in food intake and small increases in HR and BP over saline-infused controls. In CR rats, TRH infusion resulted in an increase in HR and also energy expenditure over saline-infused controls. These results support the hypothesis that suppression of central TRH activity contributes to the homeostatic suppression of energy expenditure and HR observed during CR.

Pediatric Research and Scholarship: Another Gordian Knot?

Peter J. Davis, MD Appearing in this issue of the journal are four clinical papers on the use of dexmedetomidine in children (1–4). Previous studies of the use of this drug in children have dealt with sedation in the intensive care unit (5), prevention of postoperative agitation associated with sevoflurane anesthesia (6,7), and sedation for noninvasive procedures (8–12). These articles also illustrate a number of subtle concerns that are relevant to drug development in children and the ethical principles that guide research in human subjects as proposed by the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, and summarized in the Belmont Report. In a randomized study of 30 pediatric cardiac surgical patients anesthetized with isoflurane and undergoing cardiopulmonary bypass (CPB), Mukhtar et al. (1) evaluated the effects of a continuous dexmedetomidine infusion on patients’ underlying hemodynamics and neuroendocrine system compared with patients receiving a saline control infusion. The authors infused dexmedetomidine after anesthetic induction until the termination of CPB. Dexmedetomidine attenuated the increase in the neuroendocrine markers of stress caused by sternotomy and CPB. In addition, dexmedetomidine provided better intraoperative hemodynamic stability in children with congenital heart disease. The report focused on hemodynamic outcome and did not address how this drug affected tracheal extubation time, recovery, length of hospital stay, or long-term outcome. Thus, the reader is left to ponder whether the reduction in the cortisol and norepinephrine, markers of neuroendocrine response, affect outcome. Moreover, we do not know how to titrate dexmedetomidine or if the optimum dose was administered. Are there dexmedetomidine doserelated adverse effects in this population? Should the dose be altered at the start of CPB? What is the effect of the fluctuation in the patient’s temperature (37° to 25° and return) on plasma levels achieved using a standard infusion rate? Why was dexmedetomidine not continued until the end of surgery? Why did the dexmedetomidine infusion not allow for less anesthetic administration, as had been shown in an adult study (13)? Did dexmedetomidine improve hemodynamic stability after bypass? Although this study measured biochemical endpoints to assess stress response, can we infer that the effect of this drug will be as salutary as the effect of opioids in the mortality of children having surgery for congenital heart disease? These questions were not answered because the study was not funded, limiting the number of subjects and the resources available to the investigators. Had this study been funded by the pharmaceutical manufacturer, the authors could have enrolled more patients, perhaps included other study centers, assayed blood for dexmedetomidine concentrations, developed pharmacokinetic models of dexmedetomidine in this population, and performed a pharmacodynamic concentration-versus-response analysis. In a study of 60 children undergoing magnetic resonance imaging, From the Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Anesthesia, Stanford University, Stanford, California; Department of Anesthesiology, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania. Accepted for publication March 22, 2006. Address correspondence and reprint requests to Peter J. Davis, MD, Professor of Anesthesia and Pediatrics Anesthesiologistin-Chief, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213. Address e-mail to davispj@anes.upmc.edu. Copyright © 2006 International Anesthesia Research Society DOI: 10.1213/01.ANE.0000220033.34889.CE

Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response

Application of ultrasound-mediated destruction of microbubbles (US + Bubble) to skeletal muscle creates capillary ruptures leading to leakage of the cell components. We studied whether US + Bubble combined with bone-marrow-derived mononuclear cells (BM-MNCs) infusion enables the targeted delivery of endothelial-lineage cells into the myocardium and improves cardiac function of the cardiomyopathy model due to the paucity of neocapillary formation. Pulsed US was applied to the anterior chest of BIOTO2 cardiomyopathy hamsters for 90 s after the intravenous injection of microbubble (Optison) followed by infusion of BM-MNCs. Cardiac samples from US + microbubble + BM-MNCs (US + Bubble + BM), US + Bubble, US + BM without Bubble, and saline infusion control groups were analyzed 12 weeks after treatment. Labeled BM-MNCs transplanted by US + Bubble were found to be mainly localized in the microvessels, but not by US stimulation without microbubble (121.2 +/- 24.5 vs. 2.80 +/- 1.30 cells/mm2, P < 0.001). Capillary densities in US + Bubble + BM group were increased 1.7-fold (P < 0.05) over the control, and neither US + Bubble nor US + BM enhanced neocapillary formation. 99mTc-Tetrofosmin scintigraphy revealed that blood perfusion area in the US + Bubble + BM group was 48% greater than the control (P < 0.01). US + Bubble stimulation induces the expression of adhesion molecules (VCAM-1 and ICAM-1) in capillaries, and the US + Bubble-mediated supply of BM-MNCs increased the myocardial content of VEGF and bFGF. The left ventricular wt/body wt, area of cardiac fibrosis, and apoptotic cell numbers in the US + Bubble + BM group significantly (P < 0.05) decreased by 82%, 73%, and 64% relative to the control, respectively. The cardiac function in myopathic hamsters (assessed by fractional shortening) was markedly improved 36% (P < 0.05) by US + Bubble + BM treatment. Targeted delivery of BM-MNCs by US + Bubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling, resulting in the prevention of heart failure. This non-invasive cell delivery system may be useful as a novel efficient approach for angiogenic cell therapy to the myocardium.

Reduced Ornithine Transcarbamylase Activity Does Not Impair Ureagenesis in Otcspf-ash Mice

Mouse models for urea cycle disorders have been available for the past 30 y; however, until now, no measurements of urea production in vivo have been conducted. Urea entry rate was determined in Otc(spf-ash) and littermate controls employing a primed-continuous infusion of 15N15N urea. A saline infusion control, a complete mixture of amino acids (AA), or a glycine-alanine (GA) mixture was infused at 86 (AA1 and GA1) and 172 mg N.kg(-1).h(-1) (AA2 and GA2) to impose a defined nitrogen load on the urea cycle. Urea entry rate and plasma urea concentration increased (P < 0.001) as a consequence of the increase in the infusion rate of the complete mixture of amino acids, but the 2 genotypes did not differ (P = 0.96 and P = 0.44, respectively). The infusion of the GA mixture, however, decreased (P < 0.001) the plasma urea concentration and urea entry rate in Otc(spf-ash) mice compared with controls. At the highest level (GA2), urea entry rate was further depressed (P < 0.001), Otc(spf-ash) mice became hyperammonemic (1701 +/- 150 micromol/L), and hyperammonemic symptoms were evident. An acute hepatic enlargement (P < 0.001) was also evident in Otc(spf-ash) mice infused with GA2. These results show that despite vestigial OTC activity, Otc(spf-ash) mice were able to maintain ureagenesis at the same rate of control animals when a complete mixture of amino acids was infused. This implies that Otc(spf-ash) mice are able to dispose of ammonia, without apparent adverse effects, when a balance mixture of amino acids is provided, despite reduced enzyme activity.

Vestigial ornithine transcarbamylase activity does not impair ureagenesis in Otc spf‐ash mice

Mouse models for urea cycle (UC) disorders have been available for the past 30 years; however, until now no measurements of urea entry rate (UER) in vivo have been conducted. The purpose of this research was the study of urea metabolism in conscious Otcspf-ash mice, a model of ornithine transcarbamylase deficiency, under defined nitrogen load conditions. UER was determined in mutant and littermate controls by a primed-continuous infusion of 15N15N urea. A saline infusion control, a complete mixture of amino acids (AA) or a Glycine-Alanine (GA) mixture were infused at 0 (Saline), 86 (AA1, GA1) and 172 mg N kg−1 h−1 (AA2, GA2) to impose a defined nitrogen load on the UC. UER and plasma urea concentration (PU) increased (P < 0.001) as a consequence of the increase in level of infusion of the complete mixture of amino acids, but no difference (P = 0.96, P = 0.44, respectively) between the two genotypes was detected. The infusion of the GA mixture, however, decreased the PU (P < 0.001) and UER in Otcspf-ash compared to controls. At the higher level (GA2) UER was further depressed (P < 0.001) and Otcspf-ash mice became hyperammonemic (1701±150 μM). An acute hepatic enlargement (P < 0.001) was also evident in Otcspf-ash mice infused with GA2. The present results show that despite very low OTC activity, Otcspf-ash mice were able to maintain UER at the same rate as control animals, if arginine or UC intermediates were provided. This implies that there is a hepatic requirement for these UC intermediates to sustain ammonia detoxification and urea production in Otcspf-as mice.

A Novel Gene (Cmya3) Induced in the Heart by Angiotensin II-Dependent but not Salt-Dependent Hypertension in Mice

In previous studies using serial analysis of gene expression for elucidation of the molecular pathways of angiotensin II (Ang II)-induced hypertensive/ischemic cardiomyopathy in mice, we found that a hitherto unknown transcript, designated initially as 2310008C07Rik, an unknown expressed sequence tag (EST), was highly significantly upregulated in myocardial tissue. The current experiments were designed to further characterize this gene and to evaluate its expression in various types of hypertension. Mice rendered hypertensive by Ang II infused intravenously at 30 ng/min for 6 h or by osmotic minipump at 0.9 mug/h for 7 or 14 days, were compared to saline-infused normotensive controls and to mice with hypertension induced by subtotal nephrectomy and 1% saline as drinking water. At end point, mice were euthanized, their tissues processed for gene expression analysis, and results were confirmed by ribonuclease protection assay. The Ang II-infused mice developed systolic blood pressure (BP) of 134 +/- 7, 158 +/- 13, and 149 +/- 15 mm Hg at 6 h, 7 days, and 14 days, respectively, compared to 102 +/- 9, 110 +/- 8, and 114 +/- 7 mm Hg in their respective controls and subtotally nephrectomized salt-fed mice had end point blood pressure of 153 +/- 5 v 112 +/- 7 mm Hg in controls. Through sequencing and expression analysis we found that the unknown transcript is part of the cardiomyopathy associated 3 (Cmya3) gene, being overexpressed in Ang II-induced but not salt-induced hypertension. The highly expressed 2310008C07Rik EST was found to be part of Cmya3 and its upregulation is due to Ang II-induced myocardial damage and not to BP elevation per se.

Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness.

The Effect of Insulin Infusion Upon Protein Metabolism in Neonates on Extracorporeal Life Support

Critically ill neonates on extracorporeal life support (ECLS) demonstrate elevated rates of protein breakdown that, in turn, are associated with increased morbidity and mortality. This study sought to determine if the administration of the anabolic hormone insulin improved net protein balance in neonates on ECLS. Twelve parenterally fed neonates, on ECLS, were enrolled in a randomized, prospective, crossover trial. Subjects were administered a hyperinsulinemic euglycemic clamp and a control saline infusion. Protein metabolism was quantified using ring-D5-phenylyalanine and ring-D2-tyrosine stable isotopic infusions. Statistical comparisons were made by paired sample t tests (significance at P < 0.05). Serum insulin concentration increased 20-fold during insulin infusion compared with saline infusion control (P < 0.0001). Protein breakdown was significantly decreased during insulin infusion compared with controls (7.98 +/- 1.82 vs. 6.89 +/- 1.03 g/kg per day; P < 0.05). Serum amino acid concentrations were significantly decreased by insulin infusion (28,450 +/- 9270 vs. 20,830 +/- 8110 micromol/L; P < 0.02). Insulin administration tended to decrease protein synthesis (9.58 +/- 2.10 g/kg per day vs. 8.60 +/- 1.20; P = 0.05). For the whole cohort, insulin only slightly improved net protein balance (protein synthesis minus protein breakdown) (1.60 +/- 0.80 vs. 1.71 +/- 0.89 g/kg per day; P = 0.08). In neonates receiving > or =2 g/kg per day of dietary amino acids insulin significantly improved net protein balance (2.17 +/- 0.34 vs. 2.40 +/- 0.26 g/kg per day; P < 0.01). Insulin effectively decreases protein breakdown in critically ill neonates on ECLS. However, this is associated with a significant reduction in plasma amino acids and a trend toward decreased protein synthesis. Insulin administration significantly improves net protein balance only in those ECLS neonates in whom adequate dietary protein is provided.

Pioglitazone increases non-esterified fatty acid clearance in upper body obesity

Plasma NEFA concentrations are largely determined by adipose tissue lipolysis. Insulin suppression of lipolysis is commonly impaired with insulin resistance and improves with thiazolidinedione treatment of type 2 diabetes. The present studies were designed to assess the effects of thiazolidinedione on NEFA (oleate) metabolism that are independent of improved glycaemic control. We measured plasma oleate concentration and flux ([(3)H]oleate), glucose kinetics ([6-(2)H(2)]glucose) and substrate oxidation (indirect calorimetry) before and after pioglitazone (30 mg/day for approximately 20 weeks) in 20 non-diabetic adults with upper body obesity. To assess the effects of improved insulin sensitivity per se we performed the same measurements in a matched group of volunteers treated with diet/exercise. Half of the two groups underwent these measurements during a hyperinsulinaemic-euglycaemic clamp, and the other half had their measurements taken during a (control) saline infusion before and after the intervention. Both interventions increased insulin-stimulated glucose disposal and reduced plasma oleate concentrations during the insulin clamp. After diet/exercise, oleate flux decreased (p=0.03) during the insulin clamp and oleate clearance remained unchanged (p=0.55), whereas in the pioglitazone group, oleate flux during the clamp was unchanged (p=0.97) and oleate clearance increased (p=0.003). Oleate clearance in the saline control condition was increased in the pioglitazone group compared with the diet/exercise group (p=0.02). In insulin-resistant, non-diabetic adults, pioglitazone increases NEFA clearance during physiological hyperinsulinaemia, whereas improved insulin sensitivity achieved by diet/exercise does not alter NEFA clearance but enhances insulin suppression of NEFA release. This action of pioglitazone may contribute to improved glucose metabolism in type 2 diabetes.

Cell-Based Cardiac Repair

Received February 28, 2005; revision received April 25, 2005; accepted April 29, 2005. It has now been more than a decade since the first experiments were performed using cell transplantation for the prevention and treatment of heart failure.1–3 Although the biomedical community was initially somewhat skeptical of this approach, a large body of experimental evidence was amassed showing that injected cells could create new tissue and improve function of the failing heart. This evidence, coupled with the recognized limitations of heart failure treatments and the intuitively appealing concept of “regenerative medicine,” has contributed to a crescendo of activity in cell-based cardiac repair. Given the flurry of clinical trials that are currently under way, we think it is timely to review progress over the past 10 years and provide a critical assessment of where the field stands and where it appears to be headed. Cell-based cardiac repair began with studies of skeletal myoblasts derived from skeletal muscle satellite cells.1–3 Myoblasts were the initial choice because of their availability from autologous or syngeneic sources, their ability to proliferate, and their ability to withstand ischemia better than many cell types. Although it was originally hoped that these cells would transdifferentiate into cardiomyocytes, it is now clear that myoblasts remain stubbornly committed to form mature skeletal muscle in the heart3–5 (with the exception of rare cell fusion events at the graft–host interface6). Skeletal muscle is one of the few cell types in the body that does not normally express gap junction proteins, and hence, structural and physiological studies indicate that skeletal muscle cells do not form electromechanical junctions with cardiomyocytes when engrafted into the heart.7,8 Despite this, numerous studies have shown beneficial effects of skeletal myoblast grafting into the infarcted heart in rodents and large animals.8–13 Cardiomyocytes would …

Mitochondrial-dependent apoptosis in experimental rodent abdominal aortic aneurysms

While extrinsic mechanisms of apoptosis in abdominal aortic aneurysms (AAAs) are recognized, this project hypothesizes that an intrinsic, mitochondrial-dependent, mechanism of apoptosis also contributes to experimental AAA formation. Rat aortas were perfused with either saline or elastase (N = 5 per group) and harvested 7 days postperfusion. The aortas were placed in gluteraldehyde for subsequent transmission electron microscopy, Bouin's solution for TUNEL, or paraformaldehyde for immunohistochemical staining for caspase-9, caspase-3, and Bid. Abdominal aortic diameters increased 168 +/- 25% (mean +/- SEM) after elastase perfusion. compared with 30 +/- 5% after saline perfusion (P < .001). Apoptosis of aortic smooth muscle cells, macrophages, and neutrophils was evidenced by transmission electron microscopy and TUNEL in the elastase-perfused aneurysmal aortas. Quantitative analysis of the apoptotic cells revealed a significant (P < .01) increase in the number of total apoptotic cells in the elastase-perfused aortas (12 +/- 3 cells per high-power field), compared with that of saline-infused controls (1.3 +/- 0.2). Caspase-9, the key initiator in the mitochondrial-dependent apoptotic pathway, stained positively in only elastase-perfused aortas. Bid staining was not detected in either the elastase-perfused aortas or the saline controls. Apoptosis is evident in multiple cell lines in elastase-perfused aneurysmal aortas, but rarely observed in control aortas. Caspase-9, the key initiator of intrinsic apoptosis, was documented only in elastase-perfused aortas. These results suggest that mitochondrial-dependent apoptosis is associated with abdominal aortic aneurysm formation.

Diet-induced obesity and acute hyperlipidemia reduce IκBα levels in rat skeletal muscle in a fiber-type dependent manner

Increased activity of proinflammatory/stress pathways has been implicated in the pathogenesis of insulin resistance in obesity. However, the effects of obesity on the activity of these pathways in skeletal muscle, the major insulin-sensitive tissue by mass, are poorly understood. Furthermore, the mechanisms that activate proinflammatory/stress pathways in obesity are unknown. The present study addressed the effects of diet-induced obesity (DIO; 6 wk of high-fat feeding) and acute (6-h) hyperlipidemia (HL) in rats on activity of IKK/IkappaB/NF-kappaB c-Jun NH(2)-terminal kinase, and p38 MAPK in three skeletal muscles differing in fiber type [superficial vastus (Vas; fast twitch-glycolytic), soleus (Sol; slow twitch-oxidative), and gastrocnemius (Gas; mixed)]. DIO decreased the levels of the IkappaBalpha in Vas (24 +/- 3%, P = 0.001, n = 8) but not in Sol or Gas compared with standard chow-fed controls. Similar to DIO, HL decreased IkappaBalpha levels in Vas (26 +/- 5%, P = 0.006, n = 6) and in Gas (15 +/- 4%, P = 0.01, n = 7) but not in Sol compared with saline-infused controls. Importantly, the fiber-type-dependent effects on IkappaBalpha levels could not be explained by differential accumulation of triglyceride in Sol and Vas. HL, but not DIO, decreased phospho-p38 MAPK levels in Vas (41 +/- 7% P = 0.004, n = 6) but not in Sol or Gas. Finally, skeletal muscle c-Jun NH(2)-terminal kinase activity was unchanged by DIO or HL. We conclude that diet-induced obesity and acute HL reduce IkappaBalpha levels in rat skeletal muscle in a fiber-type-dependent manner.

Resting and Evoked Spinal Substance P Release during Chronic Intrathecal Morphine Infusion: Parallels with Tolerance and Dependence

Spinal opiate analgesia is associated with presynaptic inhibition of release of excitatory neurotransmitters/neuromodulators, e.g., substance P (SP), from primary afferent terminals. Chronic intrathecal (i.t.) administration of opiates such as morphine results in an initial analgesia followed by tolerance and a state of dependence. In this study, we examined the resting and evoked neurokinin 1 receptor (NK1r) internalization, indicative of endogenous SP release, in dorsal horn neurons of the lumbar spinal cord by immunocytochemistry during chronic i.t. infusion of morphine in rats. Noxious mechanical stimulation (compression) applied to unilateral hind paw evoked a significant increase in NK1r internalization in lamina I neurons in the ipsilateral dorsal horn. Intrathecal morphine infusion (40 nmol/microl/h) for 1 day possessed similar analgesic efficacy as acute morphine and blocked compression-induced spinal NK1r internalization. After 5 days of morphine infusion, thermal escape latencies were the same as in preinfusion animals or saline-infused controls, and compression-evoked NK1r internalization was no longer suppressed. Systemic administration of naloxone to rats on day 6 of morphine infusion resulted in prominent withdrawal behaviors and a concomitant increase in NK1r internalization in dorsal horn. The naloxone-induced internalization was blocked by NK1r antagonist L-703,606 [cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1 azabicyclo[2.2.2]octan-3-amine] or pretreatment with capsaicin, confirming that the internalization is due to the endogenous SP release from the primary afferents. We conclude that inability to suppress release of excitatory neurotransmitters/neuromodulators from primary afferents by morphine after chronic exposure is an important component in spinal morphine tolerance, and excessive release from these afferents contributes to the spinal morphine withdrawal syndrome.

CHRONIC EFFECTS OF ANGIOTENSIN II and AT1 RECEPTOR ANTAGONISTS IN SUBFORNICAL ORGAN‐LESIONED RATS

1. Angiotensin (Ang) II is known to exert some of its effects centrally via circumventricular organs. These unique central nervous system areas lack the normal blood-brain barrier and, therefore, allow peptide hormones access to the brain. Of these, the subfornical organ (SFO) has been shown to be involved in many of the acute dipsogenic and pressor effects of AngII, but much less is known about the role of the SFO in the chronic effects of AngII. We hypothesized that the SFO is a central site involved in the chronic hypotensive effects of endogenous AT(1) receptor blockade, as well as the chronic hypertensive effects of exogenously administered AngII. 2. In order to test these hypotheses, SFO-lesioned (SFOx) or sham Sprague-Dawley rats were instrumented with venous catheters and radiotelemetric pressure transducers for intravenous administration of losartan or AngII and continuous measurement of blood pressure and heart rate. Rats were given 3 days of saline control infusion (7 mL/day of 0.9% NaCl) and were then infused with either losartan (10 mg/kg per day) or AngII (10 ng/kg per min) for 10 days. 3. By day 4 of losartan treatment, arterial pressure had decreased 24 +/- 2 and 18 +/- 2 mmHg in sham (n = 9) and SFOx (n = 10) rats, respectively. Furthermore, by day 5 of AngII infusion, arterial pressure had increased 12 +/- 3 mmHg in sham rats (n = 9), but only by 4 +/- 1 mmHg in SFOx rats (n = 9). In each treatment group, these attenuated pressure responses in SFOx rats continued through day 10 of treatment. 4. These results support the hypotheses that the SFO plays a role in both the hypotensive effects of chronic AT(1) receptor blockade and the chronic hypertensive phase of exogenously administered AngII.

Chronically Administered Islet Amyloid Polypeptide in Rats Serves as an Adiposity Inhibitor and Regulates Energy Homeostasis

Aims/Hypothesis: Islet amyloid polypeptide (IAPP) reduces food intake and body weight in laboratory animals. In addition, IAPP appears to regulate nutrient metabolism. In the present studies, we investigated the effect of chronic IAPP treatment on different aspects of energy homeostasis. Methods: IAPP was infused (25 pmol/kg/min) from subcutaneous osmotic pumps for 2–7 days. Rats in 2 saline-infused control groups were fed ad libitum (AF) or pair-fed (PF) against the IAPP-treated rats. Results: As expected, the IAPP infusion reduced food intake and body weight gain. In addition, the IAPP treatment decreased the epididymal fat pad (vs. PF rats, p < 0.05) and lowered circulating levels of triglycerides (vs. PF rats, p < 0.05), free fatty acids (vs. PF rats, p < 0.05), leptin (vs. both AF and PF rats, p < 0.05) and insulin (vs. AF rats, p < 0.05). In contrast, glucose and protein metabolism in the IAPP-treated rats was largely unchanged, as shown in results regarding serum glucose, glucose transport in skeletal muscle, blood urea nitrogen, and glycogen and protein content in the liver and in skeletal muscle. Conclusion/Interpretation: In summary, chronic IAPP exposure led to a changed lipid metabolism, which was characterized by decreased adiposity, hypolipidemia and hypoleptinemia, and to unchanged glucose and protein homeostasis. These results were similar to those seen in rodents during chronic exposure to another satiety/adiposity regulator, leptin. In conclusion, chronically administered IAPP plays a role as a satiety and adiposity signal in rats, and helps regulate energy homeostasis.

Neuropeptide W as a Stress Mediator in the Hypothalamus

Neuropeptide W (NPW) was isolated and found to be an endogenous peptide ligand for the orphan receptors GPR7 and GPR8. Centrally administered NPW caused a dose-dependent increase in corticosterone levels in rats. This observation indicates that NPW may play an important role in the hypothalamic organization of the endocrine response to stress. We examined the effects of immobilization stress and cold exposure on NPW-containing neurons in the hypothalamus of the rat, using dual immunostaining for NPW and Fos. In addition, to analyze the function of NPW, we studied the effect of intracerebroventricular (icv) NPW administration on Fos protein accumulation in the brain. Double immunohistochemistry for NPW and Fos showed that the percentage of Fos expression in the NPW-immunoreactive cells of the perifornical nucleus was significantly increased by immobilization stress compared with that in nonstressed rats. Similarly, the results indicated that cold exposure activates NPW-immunoreactive neurons in the perifornical nucleus. An icv administration of NPW resulted in significant Fos expression in the paraventricular nucleus, as compared with saline-infused controls. These results suggest that NPW is related to stress-responsive signal transduction, and that NPW may modulate the hypothalamus-pituitary-adrenal axis.

Contribution of the subfornical organ to angiotensin II-induced hypertension.

Previous studies clearly demonstrated acute actions of angiotensin II (ANG II) at one of the central circumventricular organs, the subfornical organ (SFO), but studies demonstrating a role for the SFO in the chronic actions of ANG II remain uncertain. The purpose of this study was to examine the role of the SFO in the chronic hypertensive phase of ANG II-induced hypertension. We hypothesized that the SFO is necessary for the full hypertensive response observed during the chronic phase of ANG II-induced hypertension. To test this hypothesis, male Sprague-Dawley rats were subjected to sham operation (sham rats) or electrolytic lesion of the SFO (SFOx rats). After 1 wk, the rats were instrumented with venous catheters and radiotelemetric transducers for intravenous administration of ANG II and measurement of blood pressure and heart rate, respectively. Rats were then allowed 1 wk for recovery. After 3 days of saline control infusion (7 ml of 0.9% NaCl/day), sham and SFOx rats were infused with ANG II at 10 ng.kg(-1).min(-1) i.v. for 10 consecutive days and then allowed to recover for 3 days. A 0.4% NaCl diet and distilled water were provided ad libitum. At day 5 of ANG II infusion, mean arterial pressure increased 11.7 +/- 3.0 mmHg in sham rats (n = 9) but increased only 3.7 +/- 1.4 mmHg in SFOx rats (n = 9). This trend continued through day 10 of ANG II treatment. These results support the hypothesis that the SFO is necessary for the full hypertensive response to chronic ANG II administration.

OUTCOME OF THE BLADDER COOLING TEST IN CHILDREN WITH NONNEUROGENIC BLADDER PROBLEMS

The bladder cooling test (BCT) engages a primitive neonatal spinal reflex that becomes suppressed by descending signals in older children and may reappear with suprasacral lesions. We assessed the outcome of the BCT in a large group of children with nonneurogenic bladder problems. The BCT was evaluated in a consecutive series of 178 girls and 106 boys, 1 month to 18 years old with bladder problems without overt neurology. The test was performed at the end of routine cystometry by a rapid control infusion of body warm saline followed, after fluid evacuation, by the same volume of cold saline (3 to 10C). The test was considered positive if a detrusor contraction greater than 30 cm H2O was evoked by the cold but not the warm fluid. Most children younger than 4 years had a history of pyelonephritis (29 of 34) and/or had vesicoureteral reflux (grade IV to V in 26 of 34). For those younger than 2 years 87% of the BCTs were positive while only 21% of the tests were positive in 2 to 3-year-old children. Most children older than 4 years had idiopathic urge incontinence, and greater than 50% of the BCTs were positive in the youngest (less than 6 years) with a gradual decline to 0% at age 13 years. Conversion of positive to negative BCTs at about age 2 years presumably represents normal maturation while positive tests in older incontinent children suggest delayed maturation of the central neuronal control of the bladder.

Effects of PYY[3-36] in rodent models of diabetes and obesity.

Peptide YY (PYY) is a 36 amino-acid peptide secreted from ileal L cells following meals. The cleaved subpeptide PYY[3-36] is biologically active and may constitute the majority of circulating PYY-like immunoreactivity. The peptide family that includes PYY, pancreatic peptide and neuropeptide Y is noted for its orexigenic effect following intracerebroventricular administration. To investigate the effects of peripheral (intraperitoneal and chronic subcutaneous) infusions of PYY[3-36] on food intake, body weight and glycemic indices. Food intake was measured in normal mice and in several rodent models of obesity and type II diabetes. In marked contrast to the reported central orexigenic effects, in the present study, PYY[3-36] acutely inhibited food intake by up to 45%, with an ED(50) of 12.5 microg/kg in fasted female NIH/Swiss mice. A 4-week infusion reduced weight gain in female ob/ob mice, without affecting the cumulative food intake. In diet-induced obese male mice, PYY[3-36] infusion reduced cumulative food intake, weight gain and epididymal fat weight (as a fraction of carcass) with similar ED(50)'s (466, 297 and 201 microg/kg/day, respectively) and prevented a diet-induced increase in HbA1c. Infusion at 100 microg/kg/day for 8 weeks in male fa/fa rats reduced the weight gain (288+/-11 vs 326+/-12 g in saline-infused controls; P<0.05), similar to effects in a pair-fed group. In female ob/ob and db/db mice, there was no acute effect of PYY[3-36] on plasma glucose concentrations. In male diabetic fatty Zucker rats, PYY[3-36] infused for 4 weeks reduced HbA1c and fructosamine (ED(50)'s 30 and 44 microg/kg/day). Peripheral PYY[3-36] administration reduced the food intake, body weight gain and glycemic indices in diverse rodent models of metabolic disease of both sexes. These findings justify further exploration of the potential physiologic and therapeutic roles of PYY[3-36].

Intravenous insulin decreases protein breakdown in infants on extracorporeal membrane oxygenation

Background/Purpose Infants requiring extracorporeal membrane oxygenation (ECMO) have the highest rates of protein catabolism ever reported. Recent investigations have found that such extreme protein breakdown is refractory to conventional nutritional management. In this pilot study, the authors sought to use the anabolic hormone insulin to reduce the profound protein degradation in this cohort. Methods Four parenterally fed infants on ECMO were enrolled in a prospective, randomized, crossover trial. Subjects were administered an insulin infusion using a 4-hour hyperinsulinemic euglycemic clamp followed by a control saline infusion on consecutive days in random order. Whole-body protein flux and breakdown were quantified using a primed continuous infusion of the stable isotope l-[1- 13C]leucine. Statistical analyses were performed using paired t tests. Results Serum insulin levels were increased 15-fold during the insulin clamp compared with the saline control (407 ± 103 v 26 ± 12 μU/mL; P < .05). During the insulin infusion, infants had decreased rates of total leucine flux (214 ± 25 v 298 ± 38 μmol/kg/h; P < .05) and leucine flux derived from protein breakdown (156 ± 40 v 227 ± 54 μmol/kg/h; P < .05) when compared with saline control. Overall, insulin administration produced a 32% reduction in protein breakdown ( P < .05). Conclusions In this pilot study, the anabolic hormone insulin markedly reduced protein breakdown in critically ill infants on ECMO. Because elevated protein breakdown correlates with mortality and morbidity, the administration of intravenous insulin may ultimately have broad applicability to the metabolic management of critically ill infants.