Circadian regulation of hedonic appetite in mice by clocks in dopaminergic neurons of the VTA.

Unlimited access to calorie-dense, palatable food is a hallmark of Western societies and substantially contributes to the worldwide rise of metabolic disorders. In addition to promoting overconsumption, palatable diets dampen daily intake patterns, further augmenting metabolic disruption. We developed a paradigm to reveal differential timing in the regulation of food intake behavior in mice. While homeostatic intake peaks in the active phase, conditioned place preference and choice experiments show an increased sensitivity to overeating on palatable food during the rest phase. This hedonic appetite rhythm is driven by endogenous circadian clocks in dopaminergic neurons of the ventral tegmental area (VTA). Mice with disrupted clock function in the VTA lose their hedonic overconsumption rhythms without affecting homeostatic intake. These findings assign a functional role of VTA clocks in modulating palatable feeding behaviors and identify a potential therapeutic route to counteract hyperphagy in an obesogenic environment.

Interaction between circadian rhythms and stress.

Life on earth has adapted to the day-night cycle by evolution of internal, so-called circadian clocks that adjust behavior and physiology to the recurring changes in environmental conditions. In mammals, a master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus receives environmental light information and synchronizes peripheral tissues and central non-SCN clocks to geophysical time. Regulatory systems such as the hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS), both being important for the regulation of stress responses, receive strong circadian input. In this review, we summarize the interaction of circadian and stress systems and the resulting physiological and pathophysiological consequences. Finally, we critically discuss the relevance of rodent stress studies for humans, addressing complications of translational approaches and offering strategies to optimize animal studies from a chronobiological perspective.

Time-of-day-dependent adaptation of the HPA axis to predictable social defeat stress.

In modern societies, the risk of developing a whole array of affective and somatic disorders is associated with the prevalence of frequent psychosocial stress. Therefore, a better understanding of adaptive stress responses and their underlying molecular mechanisms is of high clinical interest. In response to an acute stressor, each organism can either show passive freezing or active fight-or-flight behaviour, with activation of sympathetic nervous system and the hypothalamus-pituitary-adrenal (HPA) axis providing the necessary energy for the latter by releasing catecholamines and glucocorticoids (GC). Recent data suggest that stress responses are also regulated by the endogenous circadian clock. In consequence, the timing of stress may critically affect adaptive responses to and/or pathological effects of repetitive stressor exposure. In this article, we characterize the impact of predictable social defeat stress during daytime versus nighttime on bodyweight development and HPA axis activity in mice. While 19 days of social daytime stress led to a transient reduction in bodyweight without altering HPA axis activity at the predicted time of stressor exposure, more detrimental effects were seen in anticipation of nighttime stress. Repeated nighttime stressor exposure led to alterations in food metabolization and reduced HPA axis activity with lower circulating adrenocorticotropic hormone (ACTH) and GC concentrations at the time of predicted stressor exposure. Our data reveal a circadian gating of stress adaptation to predictable social defeat stress at the level of the HPA axis with impact on metabolic homeostasis underpinning the importance of timing for the body's adaptability to repetitive stress.

High-fat diet induces leptin resistance in leptin-deficient mice.

The occurrence of type II diabetes is highly correlated with obesity, although the mechanisms linking the two conditions are incompletely understood. Leptin is a potent insulin sensitiser and, in leptin-deficient, insulin insensitive, Lep(ob/ob) mice, leptin improves glucose tolerance, indicating that leptin resistance may link obesity to insulin insensitivity. Leptin resistance occurs in response to a high-fat diet (HFD) and both hyperleptinaemia and inflammation have been proposed as causative mechanisms. Scrutinising the role of hyperleptinaemia in this process, central hyperleptinaemia in Lep(ob/ob) mice was induced by chronic i.c.v. infusion of leptin (4.2 µg/day) over 10 days. This treatment led to a dramatic decline in body weight and food intake, as well as an improvement in glucose tolerance. Transfer to HFD for 4 days markedly arrested the beneficial effects of leptin on these parameters. Because Lep(ob/ob) mice are exquisitely sensitive to leptin, the possibility that leptin could reverse HFD-induced glucose intolerance in these animals was investigated. HFD led to increased body weight and glucose intolerance compared to a low-fat diet (LFD). Older and heavier Lep(ob/ob) mice were used as body weight-matched controls. Mice in each group received either i.p. leptin (1.25 mg/kg) or vehicle, and glucose tolerance, food intake and the number of phosphorylated signal transducer and activator of transcription (pSTAT)3 immunoreactive cells in the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) were analysed. Leptin improved glucose tolerance (P = 0. 019) and reduced food intake in Lep(ob/ob) mice on LFD (P ≤ 0.001) but was ineffective in mice on HFD. Furthermore, when leptin was administered centrally, the glucose tolerance of Lep(ob/ob) mice on HFD was significantly impaired (P = 0.007). Although leptin induced the number of pSTAT3 immunoreactive cells in the ARC and VMH of Lep(ob/ob) mice on LFD, HFD was associated with elevated pSTAT3 immunoreactivity in vehicle-treated Lep(ob/ob) mice that was unaffected by leptin treatment, suggesting central leptin resistance. Negating central inflammation by co-administering a c-Jun n-terminal kinase (JNK) inhibitor reinstated the glucose-lowering effects of leptin. These findings demonstrate that Lep(ob/ob) mice develop leptin resistance on a HFD independent of hyperleptinaemia and also indicate that the JNK inflammatory pathway plays a key role in the induction of diet-induced glucose intolerance.

Alterations in systemic and local colonic hemodynamic variables associated with intravenous infusion of ATP-MgCl2 in healthy anesthetized horses.

OBJECTIVE: To characterize alterations in systemic and local colonic hemodynamic variables associated with IV infusion of ATP-MgCl2 in healthy anesthetized horses. ANIMALS: 12 adult horses. PROCEDURE: Six horses were given ATP-MgCl2, IV, beginning at a rate of 0.1 mg of ATP/kg of body weight/min with incremental increases until a rate of 1.0 mg/kg/min was achieved. The remaining 6 horses were given an equivalent volume of saline (0.9% NaCl) solution over the same time period. Colonic and systemic hemodynamic variables and colonic plasma nitric oxide (NO) concentrations were determined before, during, and after infusion. RESULTS: Infusion of ATP-MgCl2 caused a rate-dependent decrease in systemic and colonic vascular resistance, principally via its vasodilatory effects. A rate of 0.3 mg of ATP/kg/min caused a significant decrease in systemic and colonic arterial pressure and colonic vascular resistance without a significant corresponding decrease in colonic arterial blood flow. Consistent alterations in NO concentrations of plasma obtained from colonic vasculature were not detected, despite profound vasodilatation of the colonic arterial vasculature. CONCLUSIONS AND CLINICAL RELEVANCE: Results revealed that IV infusion of ATP-MgCl2 may be beneficial in maintaining colonic perfusion in horses with ischemia of the gastrointestinal tract, provided a sufficient pressure gradient exists to maintain blood flow.

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins.

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.

Comparative responses of bronchial rings to mediators of airway hyperreactivity in healthy horses and those affected with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To compare responses of bronchial rings obtained from healthy horses and horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) to selected mediators of airway hyperreactivity in vitro. SAMPLE POPULATION: Bronchial rings from 6 healthy horses and 6 horses affected with SPAOPD. PROCEDURE: Bronchial rings obtained from each group of horses were mounted in organ baths and attached to force transducers interfaced with a polygraph. After applying 2g of tension, each ring was allowed to equilibrate for 45 minutes in Tyrode's solution at 37 C. Cumulative concentration-response relationships to graded concentrations of selected mediators (10(-8) to 10(-4) M) were determined and analyzed for significance at each concentration. RESULTS: Acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 induced concentration-dependent contractile responses in bronchial rings. Prostaglandin F2alpha induced weak and inconsistent contractile responses. The other 2 agents, norepinephrine and substance P, did not induce concentration-dependent responses. Considering the overall group-drug effect, acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 were effective in inducing consistent concentration-dependent contractile responses in both groups. Only 5-hydroxytryptamine and histamine induced significant responses in contractility between groups. The response of bronchial rings from horses with SPAOPD to 5-hydroxytryptamine was significantly greater than those from control horses, whereas the response to histamine was significantly lower. Significant responses were evident at concentrations ranging from 10(-6) to 10(-4) M for both drugs. CONCLUSIONS AND CLINICAL RELEVANCE: Because the airways of horses with SPAOPD had increased responsiveness to 5-hydroxytryptamine in vitro, treatment modalities using 5-hydroxytryptamine antagonists should be investigated to address this phenomenon.

In vitro responses of equine colonic arterial and venous rings to adenosine triphosphate.

OBJECTIVE: To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. SAMPLE POPULATION: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. PROCEDURES: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine triphosphate (10(-8) to 10(-3) M) was added in a noncumulative manner, and relaxation percentage versus time curves were generated. Areas under the curves (ie, percentage of relaxation time) were calculated. RESULTS: Relaxation response of arterial and venous rings to ATP was dose-dependent. Percentage of relaxation time in response to 10(-4) and 10(-3) MATP was significantly greater, compared with that for rings not treated with ATP Removal of endothelium attenuated but did not eliminate the relaxation response. Addition of L-NAME did not attenuate the relaxation response in arteries. At higher concentrations, the vascular response to ATP was biphasic. CONCLUSIONS AND CLINICAL RELEVANCE: ATP applied to equine colonic arterial and venous rings with and without intact endothelium induced a biphasic response characterized by transient contraction followed by slow, substantial, and sustained relaxation. This ATP-induced response is possibly mediated by a mechanism other than NO. Adenosine triphosphate may be a useful treatment to modulate colonic vasomotor tone in horses with strangulating volvulus of the ascending colon.

Cimetidine inhibits nitric oxide associated nitrate production in a soft-tissue inflammation model in the horse.

Cimetidine (CIM) is an H2-receptor antagonist that has been used in racehorses in an attempt to reduce the occurrence of stress-related gastric ulceration. It has also been shown to produce several useful effects other than its gastric acid suppression properties. Further, it is a well documented antagonist of cytochrome P-450 (CYP) mediated oxygenation reactions. Nitric oxide (NO), a recently discovered mediator or modifier of numerous physiological functions, is generated by several forms of nitric oxide synthase (NOS), one of which is inducible (iNOS). Inducible NOS, expressed in neutrophils and macrophages as part of the inflammatory response to noxious stimuli, contains both a CYP and a CYP reductase domain. Because of the similarity of structure of iNOS and CYP, it was decided to determine whether CIM could reduce NO production, using a carrageenan inflammation model in the horse. Two experiments were conducted. In Trial 1, six female Thoroughbred horses each had three tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into three treatments: 0.9% NaCl (NaCI), CIM (3 mg/kg), and aminoguanidine (AG; 25 mg/kg), an inhibitor of iNOS. Each mare received three i.v. injections 12 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and tissue chamber fluid (TCF) were collected serially. Concentrations of NO3- (the major metabolite of NO), albumin, total protein, CIM and AG were measured and complete cell counts and differentials were conducted. Trial 2 also used six female Thoroughbred horses implanted with at least two tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into two treatments: NaCl (0.9%) and CIM (6 mg/kg). Each mare received seven i.v. injections of either NaCl or CIM 8 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and TCF were collected serially as before, and analysed for NO3- and CIM content. Areas under the curve (AUC) of the different parameters were calculated for the periods of -1-1, -1-3 and -1-7 days (Trial 1) and -2-1 for Trial 2. Absolute values were also compared at 4, 8 and 12 h postcarrageenan. Saline treatment did not reduce the elevated concentrations of NO3- in either plasma or TCF. Plasma, test chamber and control chamber NO3-concentrations rose from 0 to 12 h, and were very similar in all three sampled fluids. Cimetidine significantly (P< or =0.05) decreased NO3- production in plasma over the periods of -1-1, -1-3, and -1-7 days post inflammation when compared to NaCl treatment in Trial 1. Aminoguanidine and CIM decreased NO3-production in TCF for the periods -1-1, 1-3, and -1-7 days post inflammation in Trial 1 and -2-1 for Trial 2. Both CIM and AG also significantly reduced NO3-concentrations in plasma and TCF at 12 h postinitiation (Trials 1 and 2). Thus CIM, at the doses studied, was capable of reducing NO3- concentrations in this model as effectively as AG, a relatively specific inhibitor of iNOS activity.

In vitro response of large colon arterial and venous rings to vasodilating drugs in horses.

OBJECTIVE: To determine in vitro vasomotor response of equine large colon arterial and venous rings with and without endothelium to vasodilator drugs, including dopamine (DOP), dopexamine (DPX), acepromazine (ACE), isoxsuprine (ISX), and nifedipine (NFP). ANIMALS: 7 adult horses. PROCEDURE: Relaxation of large colon arteries and veins in response to vasodilating drugs was determined by measuring the change in tension of vessel rings when exposed to a cumulative concentration range (10(-8) to 10(-4)M) of each drug. Vessel rings, with and without endothelium, were mounted in organ baths, attached to a transducer, and contracted with norepinephrine (NE). Cumulative concentration-response relationships, percentage maximal relaxation, and EC50 (concentration of drug required to relax the NE-induced contracted tissue to 50% of its contracted state) values were calculated. RESULTS: There were significant differences among drugs for EC50 (ACE = ISX < NFP) and percentage maximal relaxation (ACE = ISX > NFP = DPX > DOP) values in veins. Endothelium removal from veins had no significant effect. There were no differences in EC50 values for arteries; however, percentage maximal relaxation was significantly different among drugs (ACE = ISX = NFP > DPX = DOP). Endothelial removal resulted in higher EC50 and lower percentage maximal relaxation values, compared with endothelium-intact arteries. CONCLUSION AND CLINICAL RELEVANCE: ACE and ISX were the most potent and efficacious drugs evaluated and could potentially be used to improve blood flow after correction of large-colon volvulus. Dopamine cannot be recommended because of its biphasic response and potential to further decrease blood flow. Endothelium removal altered the vasodilatory responses of colonic arterial rings, but did not affect venous rings.

Biphasic responses of equine colonic vessel rings to vasoactive inflammatory mediators.

1. The role of endothelium in modulating equine colonic vessel responses to histamine (HST), 5-hydroxytryptamine (5-HT), bradykinin (BK) and acetylcholine (ACh) was evaluated in vitro. 2. Segments of mesenteric arteries and veins were collected from the left ventral colon of six adult horses destined for euthanasia for reasons unrelated to cardiovascular or gastrointestinal systems. Vessels were gently cleansed and cut into 4 mm wide rings. 3. Three vessel conditions namely endothelium intact, endothelium removed and N omega-nitro-L-arginine methyl ester (L-NAME)-treated were used for both arterial and venous rings. Each ring was placed in an organ bath with oxygenated Tyrode's solution. One side of the ring was fixed to the floor of the bath and the other side to a force-displacement transducer interfaced with a polygraph. 4. An initial tension of 2 g was applied to rings which were allowed to equilibrate for 45 min. The bath solution was gently replaced every 15 min and tension was readjusted to 2 g each time except following the last wash. 5. Rings were precontracted with a single EC25 dose of noradrenaline and after the response plateaued, cumulative concentration (10(-12)-10(-4) M) response curves were determined for each agent on separate rings. The relaxation from the precontracted level to the baseline was considered as 100% relaxation. Maximal relaxation and maximal contractions were statistically analyzed. 6. All agents induced a relaxation response initially, followed by a contractile phase as the concentrations increased in both arteries and veins, thus, making a biphasic concentration-response curve. In arteries, relaxation produced by ACh was significantly greater than 5-HT. Endothelium removal and L-NAME treatment significantly reduced relaxation in arteries. Only endothelium removal produced a significant reduction of relaxation in veins. 7. In both arteries and veins, HST and 5-HT produced significantly greater contraction than ACh or BK. No significant change in contraction was observed in arteries either by endothelium removal or L-NAME treatment, however, contraction was significantly reduced in veins by endothelium removal. 8. These findings suggest that the endothelium plays a major role in modulating equine colonic arterial relaxation via nitric oxide and venous contraction via endothelium-derived contractile mediators, probably endothelin and/or arachidonates.

[Hygienic significance of sterigmatocystin in vegetable foods. 1. Analytical detection of sterigmatocystin]. / Zur hygienischen Bedeutung von Sterigmatocystin in pflanzlichen Lebensmitteln. 1. Mitt. Analytischer Nachweis von Sterigmatocystin.

The present paper gives a survey of the chemical, physical and biological properties of the mycotoxin sterigmatocystin. It must be regarded as toxic to warm-blooded animals and as cancerogenic. It is likely to occur in foods; therefore, its analytical detection is necessary. With reference to known confirmatory reactions, a method is described for the semi-quantitative determination of sterigmatocystin in fruit and vegetables. This method permits to detect 20 microgram/kg of food by means of thin-layer chromatography, after column-chromatographic purification on silica gel. The identity is confirmed by derivatization to the semi-acetal by treatment with trifluoro-acetic anhydride. The method is suited especially for the routine control of foods.

[Hygienic significance of sterigmatocystin in vegetable foods. 3. Occurrence of sterigmatocystin]. / Zur hygienischen Bedeutung von Sterigmatocystin in pflanzlichen Lebensmitteln. 3. Mitt. Zum Vorkommen von Sterigmatocystin.

A total of 142 samples of vegetable foods was examined for the occurrence of sterigmatocystin. The samples examined were fruits and vegetables which had spontaneously gone mouldy or begun to rot under natural conditions on the one hand, and organoleptically impeccable fruit juices and maize specimens on the other hand. The samples were taken at the manufacturing plant or procured on the market in the framework of operative controls. Sterigmatocystin was detected in none of the samples under investigation. From this it may be concluded that the risk of its occurrence in vegetable foods is not very great in our country. Nevertheless, due to its cancerogenic and toxic properties, sterigmatocystin should remain included in the examination for mycotoxins in the framework of food control.

[Hygienic significance of patulin in foods. 1. Analytical detection of patulin]. / Zur hygienischen Bedeutung von Patulin in Lebensmitteln. 1. Mitt. Zum analytischen Nachweis von Patulin.

The authors describe a thin-layer chromatographic method for determining patulin in fruit and vegetable products which is suited for routine work in hygiene practice. The samples are extracted with ethyl acetate, and the extracts are purified on a Florisil column. Separation is performed by means of a one-dimensional technique, using toluene/ehtyl acetate/formic acid (5 + 4 + 1), or, in the presence of interfering contaminants, by means of a two-dimensional technique, using benzene/methanol/glacial acetic acid (90 + 5 + 5) for the first run, and toluene/ethyl acetate/formic acid (5 + 4 + 1) for the second run. Patulin is detected by spraying with a benzidine solution, after chlorination. The limits of detection are 5 microgram/l of juice and 5 microgram/kg of fruit or vegetable. Derivatization with acetic anhydride/pyridine is used for corroborating the results obtained. The significance of 5-hydroxymethylfurfural as an interfering substance in apple juices is discussed.

[Hygienic significance of patulin in food. 2. Occurrence of patulin in fruit and vegetables]. / Zur hygienischen Bedeutung von Patulin in Lebensmitteln. 2. Mitt. Zum Vorkommen von Patulin in Obst und Gemüse.

The analyses of more than 200 samples of various foods of plant origin showed that patulin was contained in 36% of the fresh and canned fruits infested with mould, and in 7% of the vegetables. Besides apples, pears, plums, peaches and tomatoes contained also patulin. In organoleptically impeccable fruit juices, the contamination rates were 40% (for apple juice) and 16% (for the other juices, such as sour cherry, currant, sea buckthorn juices). The patulin content varied from 20 to 200 microgram/l, the mean value being 80 microgram/l. It ranged from 0.1 to 5 microgram/g in apples and sterile apple preserves. The authors discuss the hygienic-toxicologic significance of these findings, and suggest to include patulin in the examination of foods for mycotoxins, stipulating a permissible value.