Repeated measurements of motor activity in rats in long-term toxicity studies.

INTRODUCTION: In the light of 3R (replace, reduce, refine) principles in animal experimentation and increased focus on delayed effects of treatment on central nervous system, the incorporation of behavioural tests into standard toxicology studies as a complement or substitution of a stand-alone safety pharmacology study appears very attractive, but poses some challenges. In the present study, we evaluated the results of an open field test (standard part of the behavioural test batte- ries) incorporated into the 3-month regulatory toxicology study. METHODS: The study was performed in two rat strains most commonly used in toxicology studies (Wistar and Sprague Dawley (SprD)). Open field test was performed according to the standard protocol for stand-alone behavioural test (modified Irwin test) before the start of treat- ment (Day-7, "naïve" animals), on Day 2, inWeek 6 and inWeek 13 of treatment with saline. RESULTS AND DISCUSSION: There was no overall difference between strains, and only minor differences were detected at the individual time points. With regard to time effect, the average values for most of the parameters were comparable throughout the study but individual variability in the performance in the arena was increased at repeated measurements compared to the start. In conclusion, performance in the open field arena did not differ principally between Wistar and SprD rats of both genders. However, individual variability in the behaviour in the open field arena increased with time. This has clear implications for deciding the appropriate group size for this type of study and has to be taken into account in the design of a toxicology study with integrated safety pharmacology endpoints.

Evalution of the castrated male Sprague-Dawley rat as a model of the metabolic syndrome and type 2 diabetes.

OBJECTIVE: Low testosterone levels have been shown to be predictive for the development of the metabolic syndrome in men. The aim of this study was to describe effects of testosterone deficiency on metabolic syndrome-related parameters in male rats in order to evaluate the rat as a model for the human metabolic syndrome related to low testosterone levels. METHODS: Male Sprague-Dawley rats were castrated or sham operated at 16 weeks of age and fed either a standard or a high energy diet. Measured parameters were: food intake, body weight, fat distribution, energy expenditure, physical activity and blood/plasma parameters related to glucose and lipid metabolism. RESULTS: Castration led to an increase in the amount of subcutaneous fat, but did not result in any changes in the visceral fat. Fasting blood glucose levels were increased and free fatty acids concentration decreased in the castrated rats from 2 weeks after castration and throughout the study, whereas no significant differences between the groups were found in any of the other parameters measured. A high-energy diet did not change the response to castration in male Sprague-Dawley rats. CONCLUSION: Compared to humans rats respond differently to testosterone deficiency. Only few of the features typical for the human metabolic syndrome were observed in castrated male Sprague-Dawley rats. Therefore, we conclude that with the present experimental setup the castrated rat is not an optimal model for studies on the influence of testosterone deficiency on body fat distribution and the development of other central components of the metabolic syndrome.

Influence of a selective histamine H3 receptor antagonist on hypothalamic neural activity, food intake and body weight.

OBJECTIVE: This study was conducted to elucidate whether antagonistic targeting of the histamine H3 receptor increases hypothalamic histamine levels, in parallel with decreases in food intake and body weight. METHODS: The competitive antagonist potency of a recently synthesized histamine H3 receptor antagonist, NNC 38-1049, was studied in intact HEK293 cells expressing human or rat histamine H3 receptor, in which NNC 38-1049 was allowed to antagonize the effect of the H3 receptor agonist R-alpha-methylhistamine on isoprenaline-induced accumulation of cAMP. The affinity of NNC 38-1049 for a number of variants of the histamine receptor was also determined. Following single dosing of normal rats with NNC 38-1049, hypothalamic histamine levels were assessed by means of microdialysis. Plasma and brain levels of NNC 38-1049 and acute effects on food intake and energy expenditure were followed after oral doses of 3-60 mg/kg. Potential side effects were examined with rat models of behaviour satiety sequence (BSS), pica behaviour and conditioned taste aversion (CTA). Intakes of food and water together with body weight were recorded for 15 days during daily dosing of dietary obese rats. RESULTS: NNC 38-1049 was found to be a highly specific and competitive antagonist towards both human and rat histamine H3 receptors, and measurable amounts of NNC 38-1049 were found in the plasma of rats following single oral doses of 3-60 mg/kg and in the brain after 15-60 mg/kg. Following single intraperitoneal injections of NNC 38-1049 (20 mg/kg), significant increases in extracellular histamine concentrations were observed. The same dose did not change BSS or pica behaviour acutely, nor did it induce CTA following repeated administration for 7 days. Reductions in food intake were seen very soon after administration, and occurred in a dose-dependent fashion. Energy expenditure was unchanged, but the respiratory quotient (RQ) tended to decrease at higher doses, indicating an increase in lipid oxidation. Twice daily administration of 20 mg/kg of NNC 38-1049 in old and dietary obese rats resulted in sustained reduction of food intake throughout a 2-week study, and was associated with a highly significant (P<0.01) decrease in body weight compared with controls (-18.4+/-3.4 vs +0.4+/-2.7 g). The same dose of NNC 38-1049 produced an acute decrease of water intake, but 24 h intakes were not significantly changed. CONCLUSIONS: The results of this study strongly support the idea that an increase in the hypothalamic concentration of histamine produces a specific reduction of food intake and that this effect can be translated into a decrease in body weight.

Life without UCP1: mitochondrial, cellular and organismal characteristics of the UCP1-ablated mice.

Mice devoid of the original uncoupling protein UCP1 have provided opportunities to delineate UCP1 function in a series of biochemical and physiological contexts. The isolated brown-fat mitochondria from such mice are fully coupled (without the addition of GDP), but still exhibit a depressed capacity for ATP synthesis. However, they only show a 2-fold decrease in sensitivity to the de-energizing effect of free fatty acids, compared with UCP1-containing mitochondria, whereas they possess a (UCP1-independent) 50-fold higher sensitivity than liver mitochondria; the fatty acid sensitivities in wild-type and UCP1-deficient mitochondria may, however, be of different natures. Despite the fact that brown-fat cells from UCP1-ablated mice cannot produce heat when stimulated by noradrenaline ('norepinephrine') or fatty acids, UCP1-ablated mice can be induced to tolerate extended cold exposure, but the heat then fully results from shivering thermogenesis. Recruitable or adaptive (by cold acclimation or adaptation to a cafeteria diet) adrenergically-stimulated thermogenesis does not exist in the UCP1-ablated animals, demonstrating the unique ability of UCP1 to mediate recruitable non-shivering thermogenesis. In addition to information on the function of UCP1, the UCP1-ablated mice can be used to gain information concerning the function of the UCP1 homologues. Thus whereas an uncoupling function of the UCP1 homologues cannot be excluded, UCP1-ablated animals clearly lack any ability to recruit any UCP1 homologue to functionally replace the loss of thermogenesis resulting from UCP1. UCP1 (thermogenin) thus remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis.

Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold.

Adaptive nonshivering thermogenesis may have profound effects on energy balance and is therefore therefore is a potential mechanism for counteracting the development of obesity. The molecular basis for adaptive nonshivering thermogenesis has remained a challenge that sparked acute interest with the identification of proteins (UCP2, UCP3, etc.) with high-sequence similarity to the original uncoupling protein-1 (UCP1), which is localized only in brown adipose tissue. Using UCP1-ablated mice, we examined whether any adaptive nonshivering thermogenesis could be recruited by acclimation to cold. Remarkably, by successive acclimation, the UCP1-ablated mice could be made to subsist for several weeks at 4C during which they had to constantly produce heat at four times their resting levels. Despite these extreme requirements for adaptive nonshivering thermogenesis, however, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, although the existence of, for example, muscular mechanisms for adaptive nonshivering thermogenesis has recurrently been implied, we did not find any indication of such thermogenesis. Not even during prolonged and enhanced demand for extra heat production was any endogenous hormone or neurotransmitter able to recruit any UCP1-independent adaptive nonshivering thermogenic process in muscle or in any other organ, and no proteins other than UCP1-not even UCP2 or UCP3-therefore have the ability to mediate adaptive nonshivering thermogenesis in the cold.

Arotinolol is a weak partial agonist on beta 3-adrenergic receptors in brown adipocytes.

Arotinolol, a clinically used alpha/beta-adrenergic blocker, has been demonstrated to be an anti-obesity agent. The anti-obesity effect of arotinolol was suggested to be the result of direct activation of thermogenesis in brown-fat cells. We tested the ability of arotinolol to stimulate thermogenesis (oxygen consumption) in isolated brown-fat cells and in intact animals. Arotinolol stimulated thermogenesis in brown-fat cells isolated from mouse and hamster. A relatively low sensitivity to the beta-adrenergic antagonist propranolol (pK(B) approximately 6) indicated that arotinolol interacted with the beta3-adrenergic receptor. On the beta3-receptor, arotinolol was a very weak (EC50 approximately 20 microM) and only partial (approximately 50%) agonist, but arotinolol also demonstrated the properties of being a beta3-receptor antagonist with a pK(B) of 5.7. In intact animals, only the antagonistic action of arotinolol could be observed. Because arotinolol is only a very weak and partial agonist on the beta3-receptors, direct stimulation of thermogenesis in brown adipose tissue is unlikely to be sufficient to cause significant weight loss. It may be necessary to invoke additional pathways to explain the anti-obesity effects of chronic treatment with arotinolol.

UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency.

The uniqueness of UCP1 (as compared to UCP2/UCP3) is evident from expression analysis and ablation studies. UCP1 expression is positively correlated with metabolic inefficiency, being increased by cold acclimation (in adults or perinatally) and overfeeding, and reduced in fasting and genetic obesity. Such a simple relationship is not observable for UCP2/UCP3. Studies with UCP1-ablated animals substantiate the unique role of UCP1: the phenomenon of adaptive adrenergic non-shivering thermogenesis in the intact animal is fully dependent on the presence of UCP1, and so is any kind of cold acclimation-recruited non-shivering thermogenesis; thus UCP2/UCP3 (or any other proteins or metabolic processes) cannot substitute for UCP1 physiologically, irrespective of their demonstrated ability to show uncoupling in reconstituted systems or when ectopically expressed. Norepinephrine-induced thermogenesis in brown-fat cells is absolutely dependent on UCP1, as is the uncoupled state and the recoupling by purine nucleotides in isolated brown-fat mitochondria. Although very high UCP2/UCP3 mRNA levels are observed in brown adipose tissue of UCP1-ablated mice, there is no indication that the isolated brown-fat mitochondria are uncoupled; thus, high expression of UCP2/UCP3 does not necessarily confer to the mitochondria of a tissue a propensity for being innately uncoupled. Whereas the thermogenic effect of fatty acids in brown-fat cells is fully UCP1-dependent, this is not the case in brown-fat mitochondria; this adds complexity to the issues concerning the mechanisms of UCP1 function and the pathway from beta(3)-adrenoceptor stimulation to UCP1 activation and thermogenesis. In addition to amino acid sequences conserved in all UCPs as part of the tripartite structure, all UCPs contain certain residues associated with nucleotide binding. However, conserved amongst all UCP1s so far sequenced, and without parallel in all UCP2/UCP3, are two sequences: 144SHLHGIKP and the C-terminal sequence RQTVDC(A/T)T; these sequences may therefore be essential for the unique thermogenic function of UCP1. The level of UCP1 in the organism is basically regulated at the transcriptional level (physiologically probably mainly through the beta(3)-adrenoceptor/CREB pathway), with influences from UCP1 mRNA stability and from the delay caused by translation. It is concluded that UCP1 is unique amongst the uncoupling proteins and is the only protein able to mediate adaptive non-shivering thermogenesis and the ensuing metabolic inefficiency.

beta1 to beta3 switch in control of cyclic adenosine monophosphate during brown adipocyte development explains distinct beta-adrenoceptor subtype mediation of proliferation and differentiation.

To explain the distinctive pharmacological profiles observed for adrenergic stimulation of cell proliferation (beta1) and cell differentiation (beta3), the adrenergic control of cAMP accumulation was investigated during brown adipocyte development. In preadipocytes, norepinephrine (NE) increased cAMP levels but the beta3-agonists BRL-37344 and CGP-12177 did not; in contrast, when the cells had differentiated into mature brown adipocytes, a large cAMP response to the beta3-agonists had emerged and was now double that to NE (although the affinity of NE had increased 10-fold). Beta1-messenger RNA (mRNA) levels were high in both pre- and mature brown adipocytes; beta3-mRNA did not appear until maturation but then abruptly. Although beta1-receptors remained detectable by [3H]CGP-12177 binding in the mature brown adipocytes, the cAMP response to NE (based on propranolol inhibitory potency) switched from beta1 to beta3. Even the established beta1-agonist dobutamine acted through beta3-receptors in the mature brown adipocytes. The increases in cAMP levels could adequately explain the increased cell proliferation in NE-stimulated preadipocytes and the NE-induced UCP1 gene expression in mature brown adipocytes. The distinctive adrenergic profiles for stimulation of proliferation and of differentiation were thus not due to the existence of additional pathways but to a switch in the type of beta-receptor mediating the NE response, coordinated with an alteration in the nuclear response to increased cAMP levels. The study implies that full recruitment of brown adipose tissue cannot be induced by exclusive beta3-stimulation.

Benidipine induces thermogenesis in brown adipose tissue by releasing endogenous noradrenaline: a possible mechanism for the anti-obesity effect of calcium antagonists.

BACKGROUND: Anti-obesity effects of calcium antagonists such as benidipine and nifedipine have been described in rodent obesity models, but the mode of action of the calcium antagonists as anti-obesity agents has not been established. OBJECTIVE: To examine whether the anti-obesity effects of calcium antagonists (here benidipine) could be ascribed to a direct stimulation of brown adipose tissue (BAT) thermogenesis. METHODS: Examination of the ability of benidipine to induce thermogenesis (increased rate of oxygen consumption) in isolated brown-fat cells from rats, mice and hamsters--and in intact cold-acclimated rats. RESULTS: Benidipine itself, or in combination with any dose of noradrenaline (NA), was totally unable to induce or augment thermogenesis in isolated brown-fat cells of any species tested. However, it markedly induced thermogenesis in intact animals (approx 60% increase over resting metabolic rate). This effect could be fully inhibited by propranolol. CONCLUSION: Benidipine is itself without thermogenic effect. The thermogenic response in-vivo (and thus presumably the anti-obesity effect) is probably secondary to a previously described general side-effect of calcium antagonists: a release of NA from sympathetic nerves, here most likely directly from nerves in the BAT. The anti-obesity effect of benedipine is thus probably not due to its calcium channel blocking effect. PERSPECTIVES: It is probable that the anti-obesity effects of calcium antagonists reported in several models of genetically obese rodents (MSG-obese and agouti mice, SHHF/Mcc-fa(cp) and JCR:LA-corpulent rats) are mediated via an indirect stimulation of BAT. To what extent calcium antagonists may induce similar effects in a clinical situation, is currently unknown.

UCP1: the original uncoupling protein--and perhaps the only one? New perspectives on UCP1, UCP2, and UCP3 in the light of the bioenergetics of the UCP1-ablated mice.

The availability of a UCP1-ablated mouse has enabled critical studies of the function of UCP1, UCP2, and UCP3. Concerning UCP1, its presence in brown-fat mitochondria is associated with innate uncoupling, high GDP-binding capacity, and GDP-inhibitable Cl- permeability and uncoupling--but the high fatty acid sensitivity found in these mitochondria is observed even in the absence of UCP1. The absence of UCP1 leads to low cold tolerance but not to obesity. UCP1 ablation also leads to an augmented expression of UCP2 and UCP3 in brown adipose tissue, making this tissue probably the one that boasts the highest expression of these UCPs. However, these very high expression levels are not associated with any inherent uncoupling, or with a specific GDP-binding capacity, or with a GDP-sensitive Cl- permeability, or with any effect of GDP on mitochondrial membrane potential, or with an increased basal metabolism of cells, or with the presence of norepinephrine- or fatty acid-induced thermogenesis in cells, and not with a cold-acclimation recruited, norepinephrine-induced thermogenic response in the intact animal. Therefore, it can be discussed whether any uncoupling effect is associated with UCP2 or UCP3 when they are endogenously expressed and, consequently, whether (loss of) uncoupling (thermogenic) effects of UCP2 or UCP3 can be invoked to explain metabolic phenomena, such as obesity.