Nucleus accumbens cocaine-amphetamine regulated transcript mediates food intake during novelty conflict.

Obesity is a persistent and pervasive problem, particularly in industrialized nations. It has come to be appreciated that the metabolic health of an individual can influence brain function and subsequent behavioral patterns. To examine the relationship between metabolic phenotype and central systems that regulate behavior, we tested rats with divergent metabolic phenotypes (Low Capacity Runner: LCR vs. High Capacity Runner: HCR) for behavioral responses to the conflict between hunger and environmental novelty using the novelty suppressed feeding (NSF) paradigm. Additionally, we measured expression of mRNA, for peptides involved in energy management, in response to fasting. Following a 24-h fast, LCR rats showed lower latencies to begin eating in a novel environment compared to HCR rats. A 48-h fast equilibrated the latency to begin eating in the novel environment. A 24-h fast differentially affected expression of cocaine-amphetamine regulated transcript (CART) mRNA in the nucleus accumbens (NAc), where 24-h of fasting reduced CART mRNA in LCR rats. Bilateral microinjections of CART 55-102 peptide into the NAc increased the latency to begin eating in the NSF paradigm following a 24-h fast in LCR rats. These results indicate that metabolic phenotype influences how animals cope with the conflict between hunger and novelty, and that these differences are at least partially mediated by CART signaling in the NAc. For individuals with poor metabolic health who have to navigate food-rich and stressful environments, changes in central systems that mediate conflicting drives may feed into the rates of obesity and exacerbate the difficulty individuals have in maintaining weight loss.

Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats.

Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of MC peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how variance in MC signaling may underlie individual differences in physical activity levels, we examined behavioral response to MC receptor agonists and antagonists in rats that show high and low levels of physical activity and NEAT, that is, high- and low-capacity runners (HCR, LCR), developed by artificial selection for differential intrinsic aerobic running capacity. Focusing on the hypothalamus, we identified brain region-specific elevations in expression of MCR 3, 4, and also MC5R, in the highly active, lean HCR relative to the less active and obesity-prone LCR. Further, the differences in activity and associated EE as a result of MCR activation or suppression using specific agonists and antagonists were similarly region-specific and directly corresponded to the differential MCR expression patterns. The agonists and antagonists investigated here did not significantly impact food intake at the doses used, suggesting that the differential pattern of receptor expression may by more meaningful to physical activity than to other aspects of energy balance regulation. Thus, MCR-mediated physical activity may be a key neural mechanism in distinguishing the lean phenotype and a target for enhancing physical activity and NEAT.

High inborn aerobic capacity does not protect the heart following myocardial infarction.

Maximal oxygen uptake (Vo2max) is a strong prognostic marker for morbidity and mortality, but the cardio-protective effect of high inborn Vo2max remains unresolved. We aimed to investigate whether rats with high inborn Vo2max yield cardio-protection after myocardial infarction (MI) compared with rats with low inborn Vo2max. Rats breed for high capacity of running (HCR) or low capacity of running (LCR) were randomized into HCR-SH (sham), HCR-MI, LCR-SH, and LCR-MI. Vo2max was lower in HCR-MI and LCR-MI compared with respective sham (P < 0.01), supported by a loss in global cardiac function, assessed by echocardiography. Fura 2-AM loaded cardiomyocyte experiments revealed that HCR-MI and LCR-MI decreased cardiomyocyte shortening (39%, and 34% reduction, respectively, both P < 0.01), lowered Ca(2+) transient amplitude (37%, P < 0.01, and 20% reduction, respectively), and reduced sarcoplasmic reticulum (SR) Ca(2+) content (both; 20%, P < 0.01) compared with respective sham. Diastolic Ca(2+) cycling was impaired in HCR-MI and LCR-MI evidenced by prolonged time to 50% Ca(2+) decay that was partly explained by the 47% (P < 0.01) and 44% (P < 0.05) decrease in SR Ca(2+)-ATPase Ca(2+) removal, respectively. SR Ca(2+) leak increased by 177% in HCR-MI (P < 0.01) and 67% in LCR-MI (P < 0.01), which was abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase II. This study demonstrates that the effect of MI in HCR rats was similar or even more pronounced on cardiac- and cardiomyocyte contractile function, as well as on Ca(2+) handling properties compared with observations in LCR. Thus our data do not support a cardio-protective effect of higher inborn aerobic capacity.

Osteoblast response to ovariectomy is enhanced in intrinsically high aerobic-capacity rats.

The role of exercise in promoting bone health is typically attributed to increased mechanical loading, which induces functional adaptation. Recent evidence suggests that habitual aerobic exercise has influence at the cellular level as well. The effect of aerobic capacity on osteoblast-lineage cell differentiation and function as well as skeletal phenotype is unknown. Using a rat model of high-capacity and low-capacity runners (HCRs and LCRs, respectively), in which an intrinsic functional genomic difference in aerobic capacity exists between nontrained animals, this study evaluated the effects of aerobic capacity on measures of bone mass and strength as well as osteoblast activity following ovariectomy. The ovariectomized rat emulates the clinical features of the estrogen-depleted human skeleton and represents a valuable model for studying short-term upregulation of osteoblast activity. We hypothesized that intrinsically high aerobic capacity would augment osteoblast response, which would mitigate the deleterious effects of hormone withdrawal. Femora and tibiae were assessed by micro-computed tomography, mechanical testing, and dynamic histomorphometry. HCRs had enhanced femoral tissue mineral density and estimated elastic modulus relative to LCRs. At 4 weeks postovariectomy, HCRs demonstrated a more robust osteoblast response. Markers of bone formation were upregulated to a greater extent in HCRs than LCRs, suggesting a role for aerobic capacity in governing osteoblast activity. Results from this and future studies will help to identify the influence of cellular aerobic metabolism on bone health, which may lead to new strategies for targeting diseases of the skeleton.

Phenotypic and evolutionary plasticity of body composition in rats selectively bred for high endurance capacity.

We investigated the effects of genetic selection and prolonged wheel access (8 wk) on food consumption and body composition in lines of rats selected for high and low intrinsic (untrained) endurance running capacity (HCR and LCR, respectively) to test the generality of phenotypic correlations between physical activity levels, aerobic capacity, and body composition. HCR rats ran more minutes per day on activity wheels than LCR rats, supporting the hypothesis that voluntary activity and physiological capacity are genetically correlated (self-induced adaptive plasticity). Both treatments (selection and wheel access) significantly affected food consumption. HCR rats consumed and digested more food than LCR rats. Access to running wheels did not result in changes in overall body mass, but lean body mass increased and percent body fat decreased in both lines. Selection for high endurance capacity resulted in hypertrophy of the heart and kidneys and decreased long intestine length. We found significant phenotypic flexibility in a number of organ masses after wheel running. Specifically, access to running wheels resulted in hypertrophy of the heart, liver, kidney, stomach, and small and large intestines in LCR and HCR rats. The selected line×wheel access interaction was significantly greater in HCR rats in relative mass for the heart and lung. Compared with LCR rats, HCR rats fortify wheel running with increased food consumption along with greater hypertrophy of key organs for O2 transport.

Enhanced mitochondrial sensitivity to creatine in rats bred for high aerobic capacity.

Qualitative and quantitative measures of mitochondrial function were performed in rats selectively bred 15 generations for intrinsic aerobic high running capacity (HCR; n = 8) or low running capacity (LCR; n=8). As estimated from a speed-ramped treadmill exercise test to exhaustion (15 degrees slope; initial velocity of 10 m/min, increased 1 m/min every 2 min), HCR rats ran 10 times further (2,375+/-80 m) compared with LCR rats (238+/-12 m). Fiber bundles were obtained from the soleus and chemically permeabilized. Respiration was measured 1) in the absence of ADP, 2) in the presence of a submaximally stimulating concentration of ADP (0.1 mM ADP, with and without 20 mM creatine), and 3) in the presence of a maximally stimulating concentration of ADP (2 mM). Although non-ADP-stimulated and maximally ADP-stimulated rates of respiration were 13% higher in HCR compared with LCR, the difference was not statistically significant (P>0.05). Despite a similar rate of respiration in the presence of 0.1 mM ADP, HCR rats demonstrated a higher rate of respiration in the presence of 0.1 mM ADP+20 mM creatine (HCR 33% higher vs. LCR, P<0.05). Thus mitochondria from HCR rats exhibit enhanced mitochondrial sensitivity to creatine (i.e., the ability of creatine to decrease the Km for ADP). We propose that increased respiratory sensitivity to ADP in the presence of creatine can effectively increase muscle sensitivity to ADP during exercise (when creatine is increased) and may be, in part, a contributing factor for the increased running capacity in HCR rats.

Cardiac performance in inbred rat genetic models of low and high running capacity.

1. Previous work demonstrating that DA inbred rats are superior to COP inbred rats in aerobic treadmill running capacity has indicated their utility as genetic models to explore this trait. We tested the general hypothesis that intermediate phenotypes of cardiac function and calcium metabolism are responsible for the difference in capacity between these strains. 2. Logical cardiac trait differences were estimated at a tissue (isolated papillary muscle), cellular (isolated left ventricular cells), and biochemical level of organization. 3. DA hearts were found to give significantly higher values than COP hearts for: (1) maximal developed tension (38.3 % greater), and rates of tension change in contraction (61 %) or relaxation (59 %) of isolated papillary muscle, (2) fractional shortening (50 %), amplitude of the Ca(2+) transient (78.6 %), and caffeine-induced release of Ca(2+) from the sarcoplasmic reticulum (SR; 260 %) in isolated ventricular myocytes, and (3) Na(+),K(+)-ATPase activity of isolated myocytes (17.3 %). 4. Our results suggest that these trait differences may prove useful for further studies into the genes responsible for natural variations in both ventricular function and aerobic endurance capacity. Understanding the genetic basis of aerobic capacity will help define the continuum between health and disease.

Hepatorenal reflex in the rat.

The possible existence of a hepatorenal reflex was evaluated in male Sprague-Dawley rats. Sodium excretion was measured in two groups of six rats each, during the first 4 h following acute ingestion of a known amount of high salt chow (2.0-2.5 mequiv. NaCl). Hourly excretion rates for sodium before surgery were compared with results following 7 days of recovery from either hepatic denervation (n = 6) or sham denervation (n = 6). Before denervation, hourly sodium excretion between the groups was not different. Following surgery for hepatic denervation, sodium excretion was 91% lower than presurgery values for the 1st h (p < 0.02) and 44% lower in the 2nd h (p < 0.04). Sham denervation caused no significant change in sodium excretion when compared with presurgery results. A test for completeness of denervation showed that norepinephrine concentration in liver tissue taken from denervated rats was 5.1 +/- 8 ng/g and that taken from sham rats was 22.8 +/- 1 ng/g (p < 0.001). These data demonstrate that the liver is essential for the normal postprandial excretion of sodium following ingestion of a high salt meal in rats.

Autoregulatory capacity in renal and mesenteric vasculatures of mineralocorticoid hypertensive dogs.

Experiments were performed in seven conscious dogs to quantitate the renal and mesenteric autoregulatory capacity in deoxycorticosterone acetate (DOCA)-salt hypertensive dogs. Each dog was chronically instrumented for the measurement of aortic blood pressure and blood flows through the superior mesenteric and left renal arteries (Doppler measurements); a right nephrectomy was also performed. To induce hypertension, animals were administered 5 mg/kg DOCA each day and given 1% saline as a drinking solution. After arterial pressure had increased by 25% (8-14 days of DOCA), a large-bore catheter was positioned retrograde in the left common carotid artery for subsequent connection to a gravity reservoir system for acute control of arterial pressure. Autoregulatory capacity was evaluated by controlling arterial pressure via the gravity reservoir at hypertensive and then normotensive values for 15-min periods. In neurally intact animals the renal, but not the mesenteric, circulation displayed autoregulatory capacity; the closed-loop gain of renal flow control averaged 0.92 +/- 0.06. Inhibition of autonomic ganglionic transmission (hexamethonium) increased the gain of flow regulation in the mesenteric circulation, but mesenteric autoregulation was not manifest. Ganglionic blockade, inhibition of prostaglandin formation, or blockade of histamine or adenosine receptors did not significantly influence renal autoregulatory capacity. These data demonstrate that the kidney has a high capacity to autoregulate in DOCA hypertension and suggest that autoregulatory-mediated renal vasoconstriction contributes to the increased total peripheral resistance.

Reflex versus autoregulatory control of hindlimb blood flow during treadmill exercise in dogs.

To evaluate the competition between local autoregulation and reflex neurohumoral control of hindlimb blood flow (HLBF), the hindlimb vascular pressure-flow relationship was determined in nine dogs in response to a 10% decrease in mean arterial pressure (AP) imposed during both low (3.0 km/h, 0% grade) and high (5.5 km/h, 14% grade) intensities of treadmill exercise. HLBF was measured with a Doppler flow probe on the left external iliac artery, and AP was controlled with a gravity reservoir connected to the left carotid artery. A 10 +/- 2% reduction in AP for 25 min caused HLBF to decrease 25 +/- 2% during low-exercise intensity but only 10 +/- 2% during high-exercise intensity. The corresponding closed-loop gains (Gc) of HLBF regulation [Gc = 1 - (% delta hindlimb blood flow/% delta hindlimb perfusion pressure) were -1.6 +/- 0.4 and -0.06 +/- 0.2 during low- and high-exercise intensity, respectively. Autonomic ganglionic blockade (hexamethonium) increased the Gc during low-intensity exercise to 0.07 +/- 0.2. Antagonism of adenosine receptors (aminophylline) decreased the Gc of HLBF regulation during high-intensity exercise to -0.57 +/- 0.3. These data demonstrate that in response to an imposed decrease in AP, autonomic vasoconstriction overrides autoregulatory vasodilatory mechanisms during low-intensity exercise. HLBF regulation increases at a higher exercise intensity, in part due to adenosine, but autoregulation does not predominate over arterial pressure regulating mechanisms.

Adenosine is not essential for exercise hyperaemia in the hindlimb in conscious dogs.

1. The contribution of endogenous adenosine to the increase in hindlimb blood flow that occurs during treadmill exercise was evaluated in conscious dogs. We postulated that if adenosine is essential for the hindlimb hyperaemic response, then pharmacological treatment of the animals with adenosine receptor antagonists should decrease hindlimb blood flow during treadmill exercise. 2. A total of twenty-three dogs were chronically instrumented for measurement of aortic blood pressure and hindlimb blood flow using electromagnetic or Doppler flow probes on the left external iliac artery. Measurements of arterial blood pressure, hindlimb blood flow and heart rate were made during steady-state treadmill exercise in both the presence and the absence of adenosine receptor antagonists. Four different protocols were performed using different routes of administration of two adenosine receptor antagonists. Aminophylline was used in most of the experiments, and the effects of the more potent antagonist, 8-phenyltheophylline, were also evaluated. In addition, the dogs exercised at varying intensities ranging from a low level of 5.5 km h-1 at 0% gradient to a high intensity of 5.5 km h-1 at 21% gradient. 3. Aminophylline given as a single intravenous dose, or as a constant infusion either intravenously or directly into the hindlimb artery, did not decrease hindlimb blood flow at low, moderate or high intensities of exercise. Likewise, the blockade of adenosine receptors with 8-phenyltheophylline, given systemically or as a bolus injection administered directly into the hindlimb circulation during moderate exercise, did not attenuate the hindlimb blood flow response. 4. Our data demonstrate that exercise hyperaemia of the hindlimb is not reduced by antagonism of adenosine receptors. These findings are consistent with the hypothesis that adenosine is not an essential mediator of hindlimb vasodilatation during exercise.

Effect of aminophylline on hindlimb blood flow autoregulation during increased metabolism in dogs.

The contribution of adenosine to hindlimb blood flow autoregulation during treadmill exercise or the administration of 2,4-dinitrophenol (DNP) was evaluated in 9 conscious dogs by determining hindlimb vascular bed pressure-flow relationships in the presence and absence of the adenosine receptor site antagonist, aminophylline. Hindlimb pressure-flow relationships were obtained by measuring blood flow during stepwise reductions in perfusion pressure produced with an occlusion cuff located distal to a flow probe on the external iliac artery. The efficiency of autoregulation was quantitated by calculating the closed-loop gain of flow regulation (Gc) at each pressure decrement utilizing the equation Gc = 1 - (% delta flow/% delta pressure). A Gc of one represents perfect autoregulation of flow, and a Gc of zero is indicative of a rigid system. During exercise, Gc averaged 0.44 +/- 0.07. Aminophylline reduced the Gc during exercise to -0.07 +/- 0.06 (P less than 0.05). During DNP administration, Gc averaged 0.54 +/- 0.09 and declined to -0.09 +/- 0.10 in the presence of aminophylline (P less than 0.05). These results support the hypothesis that adenosine is a primary mediator of hindlimb blood flow autoregulation during conditions that increase hindlimb metabolism.

Jejunal blood flow after exposure to light in rats injected with hematoporphyrin derivative.

Experiments were performed to determine the effect of hematoporphyrin derivative (HPD) photodynamic therapy on blood flow to normal rat intestine. A segment of rat jejunum was exposed to red (greater than 590 nm) light (200 mW/cm2) 24 h after the i.v. administration of HPD. Blood flow to the light exposed segment was determined using the radioactive microsphere technique while blood flow to an adjacent light shielded segment of intestine served as an internal control. Animals were divided into six groups of six each: Group I, no HPD, no light; Group II, light, no HPD; Group III, HPD (20 micrograms/g body weight), no light; and Group IV, HPD (20 micrograms/g body weight), light. Blood flow in these four groups was determined 10 min after completion of a 30-min exposure to light. Only in Group IV was there a statistically significant decrease (P less than 0.005) in blood flow to the segment treated with HPD and light. In Groups V [HPD (20 micrograms/g body weight), light] and VI [HPD (10 micrograms/g body weight), light] blood flows were determined 24 h after exposure to light. In both of these groups there was also a significant (P less than 0.05) decrease in blood flow in the segment treated with HPD and light. This study demonstrates that normal intestinal blood flow can be disrupted by HPD photodynamic therapy.

Morphologic studies of bladder tumors treated with hematoporphyrin derivative photochemotherapy.

The morphologic changes that occurred in transplanted rat bladder tumors after treatment with hematoporphyrin derivative (HPD) and/or phototherapy were investigated. Transitional cell bladder tumors were initiated subcutaneously in male F344 rats by injection of AY27 cells. When tumors reached 1 cm in diameter, the rats received either HPD (10 mg/kg body weight) photochemotherapy, HPD only, phototherapy only, or no treatment. Tumors were sampled immediately (0 time), 1/2, 1, 2, 4, and 24 hours after phototreatment for light and electron microscopy. Tumors receiving HPD-photochemotherapy displayed progressive injury to both tumor cells and endothelial cells. Early changes (0-2 hours) included focal tumor and endothelial cell vacuolation and swelling as well as sloughing of tumor cells into papillary spaces. Tumor cells and endothelial cells displayed vacuolization and damage to cell mitochondria immediately after phototreatment. Intercellular spaces also increased in size. Lethally injured cells were apparent in papillary spaces. At 4 hours after phototherapy, tumor cells and endothelial cells exhibited extensive cell damage, including mitochondrial destruction, endoplasmic reticulum swelling, polyribosome disaggregation, and plasma membrane blebbing. By 24 hours after phototherapy, the majority of cells within the tumor were necrotic. Untreated tumors and those treated with phototherapy-only did not exhibit these changes. Tumors that received HPD only exhibited focal areas of cell swelling and focal mitochondrial vacuolization in both tumor and endothelial cells. These changes, unlike the HPD-light-treated group did not progress and were reversible.

Hematoporphyrin derivative photochemotherapy of experimental bladder tumors.

Recent studies have shown that disruption of tumor blood flow is a major consequence of hematoporphyrin derivative photochemotherapy. A series of experiments was undertaken on the transplantable N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide induced urothelial tumor in Fischer 344 rats to determine a dose response for both hematoporphyrin derivative and light. Tumor blood flow was used as the biologic criteria of response. Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to cause a significant decrease in tumor blood flow when the tumors were illuminated with 360 joules/cm.2 of noncoherent red light (greater than 590 nm.). With a constant hematoporphyrin derivative dose of 20 micrograms./gm. body weight, significantly lower tumor blood flows were observed with fluences of 240 joules/cm.2 and above. In order to correlate dose response to tumor regression, experiments were done in which tumor dry weights were determined 3 weeks after completion of photochemotherapy (360 joules/cm.2). Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to induce tumor regression. These studies support the hypothesis that disruption of tumor blood flow is a tumoricidal mechanism of hematoporphyrin derivative photochemotherapy.

Blood flow in transplantable bladder tumors treated with hematoporphyrin derivative and light.

Following hematoporphyrin derivative (HPD) photochemotherapy, blood flow to transplantable N-[4-(5-nitro-2-furyl)-2-thia-zolyl] formamide-induced urothelial tumors was determined by a radioactive microsphere technique using either 103Ru or 141Ce. Two tumors were implanted s.c. on the abdominal wall of Fischer 344 weanling rats. HPD (10 mg/kg body weight) was administered 24 hr prior to phototherapy (red light, greater than 590 nm; 360 J/sq cm). One of the two tumors was shielded from light exposure and served as an internal control. Blood flows were determined in control animals that received no treatment (Group 1), HPD only (Group 2), or light only (Group 3). In Groups 4 and 5, animals received the combination of HPD and light but differed in the time interval between treatment and blood flow determinations (10 min and 24 hr, respectively). Only blood flow to tumors treated with HPD and light showed a significant decrease (p less than 0.05) when compared with their internal controls both at 10 min (Group 4) and 24 hr (Group 5) after completion of phototherapy. These studies suggest that disruption of tumor blood flow may be an important mechanism of action of this method of cancer therapy.