Ibipinabant attenuates ß-cell loss in male Zucker diabetic fatty rats independently of its effects on body weight.

AIM: To test the antidiabetic efficacy of ibipinabant, this new cannabinoid receptor 1 (CB1) antagonist was compared with food-restriction-induced weight loss, rosiglitazone (4 mg/kg) and rimonabant (3 and 10 mg/kg), using parameters of glycaemic control in male Zucker diabetic fatty (ZDF) rats. METHODS: Body weight, food and water intake, fasted and non-fasted glucose and insulin, glucose tolerance and glycosylated haemoglobin (HbA1c) were all assessed over the course of the 9-week study. Pancreatic insulin content and islet area were also evaluated. RESULTS: At the end of the study, vehicle-treated ZDF rats were severely hyperglycaemic and showed signs of ß-cell decline, including dramatic reductions in unfasted insulin levels. Ibipinanbant (10 mg/kg) reduced the following relative to vehicle controls: fasting glucose (-61%), glucose excursion area under the curve (AUC) in an oral glucose tolerance test (OGTT, -44%) and HbA1c (-50%). Furthermore, non-fasting insulin, islet area and islet insulin content were all increased (71, 40 and 76%, respectively) relative to vehicle controls by the end of the study. All of these effects were similar to those of rimonabant and rosiglitazone, where ibipinabant was slightly more effective than rimonabant at the lowest dose and somewhat less effective than rosiglitazone at all doses. These antidiabetic effects appear independent of weight loss because none of the parameters above were consistently improved by the comparable weight loss induced by food restriction. CONCLUSIONS: Ibipinabant may have weight loss-independent antidiabetic effects and may have the potential to attenuate ß-cell loss in a model of progressive ß-cell dysfunction.

Effects of the obese gene product on body weight regulation in ob/ob mice.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

Urocortin, corticotropin releasing factor-2 receptors and energy balance.

Although there is considerable information regarding the role of brain CRF in energy balance, relatively little is known about the role of urocortin (UCN), which is an equally potent anorexic agent. Therefore, the effects of intracerebroventricular (icv) administration of UCN (0.01-1 nmol/day) on food intake and body weight were assessed over a period of 13 days and compared with data from CRF-infused counterparts. Although both peptides dose dependently reduced food intake and weight gain, the effects of CRF were much greater in magnitude than those of UCN, particularly on body weight. Pair-feeding studies suggested that, while the effects of CRF on body weight could not be completely explained by appetite suppression, the effects of UCN appeared to be due to its initial impact on food intake. CRF increased brown adipose fat pad and adrenal weights, whereas it reduced thymus and spleen weights. CRF also increased serum corticosterone, triglyceride, FFA, and cholesterol levels, whereas it reduced glucose. UCN did not produce any consistent changes in any of these indices of sympathetic nervous system activation. Concurrent administration of the CRF(2)-selective antagonist, antisauvagine-30 (ASV-30) (30 nmol/day) completely reversed or attenuated the effects of UCN and CRF (1 nmol/day) on food intake and body weight. ASV-30 did not significantly attenuate any of the above CRF-induced changes in tissue weights or serum chemistry. These data suggest that the central CRF(2) receptor may primarily mediate the anorexic, but not the metabolic effects of CRF.

A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse.

The central melanocortin system is critical for the long term regulation of energy homeostasis. Null mutations of the melanocortin-4 receptor (MC4-R) are associated with hyperphagia, obesity, and accelerated longitudinal growth in mice and humans. However, little is known about the function of another central melanocortin receptor, the MC3-R. To assess the role of the MC3-R in energy homeostasis, the majority of the mc3r coding sequence was deleted from the mouse genome. In contrast to the MC4-R knockout, which exhibits increased food intake, increased somatic growth, and defects in metabolism, mc3r-/- mice exhibit an exclusively metabolic syndrome. Homozygous null mc3r mice, while not significantly overweight, exhibit an approximately 50% to 60% increase in adipose mass. Mc3r-/- mice also exhibit an unusual increase in respiratory quotient when transferred onto high fat chow, suggesting a reduced ratio of fat/carbohydrate oxidation. Furthermore, male mc3r-/- mice also exhibit an approximately 50% reduction in locomotory behavior on the running wheel, suggesting reduced energy expenditure.

Systemically and topically administered leptin both accelerate wound healing in diabetic ob/ob mice.

Leptin is a 16 kD protein that is produced by adipocytes and induces weight loss in both normal and genetically obese ob/ob mice. ob/ob mice are obese, have multiple metabolic abnormalities, and exhibit impaired wound healing. Exogenous administration of leptin to these animals induces weight loss and corrects their metabolic defects. Leptin's effect on wound repair, however, has not been studied. Systemic administration of leptin at doses ranging from 0.1 to 10 mg/kg/day induced a highly significant acceleration in wound repair in ob/ob mice (p<0.0001), but not in db/db mice, indicating that leptin's effects on wound repair were mediated through the leptin receptor. We then investigated the possibility that leptin was acting directly at the wound site by administering leptin topically, and found that topical leptin also induced a dose dependent acceleration in wound repair (p<0.0001). In addition, we found that all forms of leptin receptor, including the signal transducing long form, were present in skin by RNase protection assay, and that leptin receptor localized to subcutaneous vessels of wounded skin by in situ hybridization. Finally, we investigated the possibility that leptin stimulated angiogenesis in wounds by analyzing wound hemoglobin and wound vessel density. Neither systemic nor topical leptin induced any significant changes in either parameter, suggesting that leptin accelerates wound repair by a mechanism other than stimulation of angiogenesis. In summary, both systemic and topical leptin accelerate wound repair in diabetic ob/ob mice, possibly via the direct interaction of leptin with its receptors in wounded skin, but do not appear to significantly stimulate wound angiogenesis. Further studies to better elucidate the mechanisms of leptin's effects on wound repair are warranted.

An age-related spatial learning deficit: choline uptake distinguishes "impaired" and "unimpaired" rats.

A functional decline in the hippocampal formation may underlie the emergence of spatial learning deficits in aged rodents. In this study, sodium-dependent high-affinity choline uptake (HACU) was used to monitor hippocampal function in response to training on a spatial task. The subjects were male Long-Evans rats at either 4 months or 22-24 months of age. Animals were trained to locate a camouflaged escape platform in the Morris water maze. Each animal that received place training had a yoked counterpart that was exposed to swimming in the maze but was not required to learn the task. Animals, both young and aged, were sacrificed after attaining a criterion performance. Relative to animals in the yoked condition, place training significantly reduced HACU in both the young rats and in a subpopulation of the aged animals that learned the task rapidly. In contrast, for aged rats that had an impaired rate of acquisition, no effect of place training on HACU was observed. These results provide evidence for a relationship between the behavioral capacities of aged rats and changes in the status of hippocampal function.

Spatial learning deficits in old rats: a model for memory decline in the aged.

Spatial learning tasks are sensitive to functional decline in aged laboratory rodents. This is a review of recent work that has examined both the nature of age-related impairments on spatial tasks, and the relation of such deficits to underlying neurobiological mechanisms. The review supports the notion that hippocampal dysfunction underlies the mild/moderate cognitive decline that often accompanies normal aging. Thus the spatial learning deficit in aged rodents is a promising model for understanding the effect of age on brain systems that serve a memory function in humans.

Differential effects of nucleus basalis lesions in young adult and aging rats.

To characterize age-related changes in frontal cortical plasticity, we assessed maze learning and frontal cortical pharmacology in young adult, middle-aged, and aged rats. Rats received either ibotenic acid or sham lesions of the nucleus basalis magnocellularis (NBM) and were then trained on a radial maze task. After training, we assessed [3H]desmethylimipramine (DMI), [3H]muscimol, [3H]AMPA, and [3H]QNB binding using quantitative autoradiography. Both middle-aged and aged rats were impaired on the radial maze task. DMI binding was increased in both middle-aged and aged rats, while QNB binding was decreased in aged rats. While lesions impaired maze performance at all ages, middle-aged and aged rats showed more profound lesion-induced deficits. Lesions increased GABA, and AMPA receptor binding in young adult rats only. These lesion-induced changes may reflect a compensatory response that is lost with advancing age.

Y1 and Y2 receptor selective neuropeptide Y analogues: evidence for a Y1 receptor subclass.

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.

Feeding, activity, and body temperature following 6-hydroxydopamine lesions in diabetes (db/db) mice.

Reductions in central catecholamines produced by intraventricular injections of 6-hydroxydopamine (6-OHDA) cause weight loss and decreased plasma glucose in diabetes (db/db) mice. The effects of this treatment were examined in short-term (64-day) and long-term (120-day) survival groups. Diabetes mice treated with 6-OHDA decreased food intake, lost weight, and maintained a lower weight than vehicle-treated controls until vehicle-treated animals began to enter the terminal stages of the syndrome, indicated by a loss of body weight. Diabetes mice given 6-OHDA lost weight despite reduced body temperatures and activity levels. Blood glucose levels were always lower in 6-OHDA than in ad lib fed vehicle-treated db/db mice. The 6-OHDA treatment also improved pancreatic islet granulation. Pair feeding vehicle-treated with 6-OHDA-treated db/db mice did not halt weight gain in the vehicle-treated group. However, measurement of carcass fat indicated similar losses in db/db-6-OHDA mice and vehicle-treated mice when the vehicle group was pair-fed with lean controls. Treatment with 6-OHDA produced long-term improvement in the diabetes syndrome, but the decreased body weight of the 6-OHDA-treated diabetes mice could not be completely accounted for by changes in food intake or measured indices of energy expenditure.

Nigral 5-HT and substance P-induced enhancement of passive avoidance retention.

Peripheral, posttraining injection of substance P (SP) has been shown to facilitate the retention of aversive and appetitive learning tasks, suggesting that SP may play a role in information processing. In addition, SP may modulate the release of nigrostriatal monoamines, which have also been linked with avoidance learning. This paper examines the interaction between SP and nigrostriatal monoamines by observing the behavioral effects of neurochemical lesions on SP-induced avoidance retention, and by measuring changes in nigrostriatal monoamine activity and receptor regulation following avoidance training and SP injection. In Expt. 1, 5,7-dihydroxytryptamine lesions of the substantia nigra, but not the caudate, attenuated the retention-enhancing effects of posttraining SP injection. Further, 6-hydroxydopamine lesions of the substantia nigra produced a deficit in avoidance conditioning that was reversed by posttraining SP injection. Expts. 2 and 3 demonstrated that although passive avoidance training and posttraining SP injections did not significantly alter nigral 5-hydroxytryptamine (5-HT) activity, SP increased 5-HT1 receptor density. It was concluded that SP may affect avoidance retention by modulating nigral 5-HT activity.

The effect of substance P and its fragments on passive avoidance retention and brain monoamine activity.

It has been shown that substance P(SP), as well as its carboxy and amino terminal fragments, affects a wide range of behaviors. In order to test the CNS activity of these fragments, we measured their effects on passive avoidance learning and monoamine activity. Following one-trial passive avoidance training, mice were injected intraventricularly with either a carboxy or amino terminal SP fragment (SP-C or SP-N), SP itself or phosphate-buffered saline (PBS). SP-N enhanced avoidance retention, which was tested 24 h after training. In a second experiment, monoamine activity was measured one hour after intraventricular injection of SP, PBS or SP fragments. SP-C decreased both nigral 5-hydroxyindoleacetic acid/5-hydroxytryptamine (5-HIAA/5-HT) and, to a lesser extent, 3,4-dihydroxyphenylacetic acid/dopamine, while SP-N increased nigral 5-HIAA/5-HT. It was concluded that SP-N and SP-C can exert behavioral and neurochemical effects that may be independent of the parent SP molecule.

D2 dopamine receptors in the rat prefrontal cortex: characterization and alteration by stress.

D2 dopamine (DA) receptors were characterized in the medial prefrontal cortex (MPFC) of the rat by employing radioligand binding techniques which greatly reduced [3H]spiperone binding to filters, S2 serotonin receptors and spirodecanone sites. Competition studies suggested that the MPFC contains a higher proportion of D2 [high] receptors than does the striatum. The IC50 values of DA receptor antagonists in assays of MPFC tissue were correlated with their antipsychotic potencies. Stress (footshock or tailshock) increased the density of MPFC D2 dopamine receptors and decreased their affinity for [3H]spiperone. These changes were delayed in onset, being present at 27 h but not at 3 h after exposure to stress. The binding assay detected the changes when it was performed at pH 7.9 but not when the pH was reduced to 6.2. D2 dopamine receptors in the striatum and nucleus accumbens were not affected by stress.

An age-related decline in striatal taurine is correlated with a loss of dopaminergic markers.

Taurine is present in high concentration in the mammalian brain and is known to decline with aging. The present studies examined the relationship between the loss of striatal neurotransmitters and spatial learning ability in aged male Long-Evans rats. The effects of intrahippocampal infusions of neurotrophic factors-nerve growth factor (NGF) and brain-derived neurotrophic factor-were also examined for their ability to ameliorate the age-related decline in brain amino acid content. Taurine content was found to be significantly reduced in the striatum of aged rats (26 months old) that were impaired in spatial learning performance when compared to young unimpaired rats (5 months old). Aged rats that were behaviorally unimpaired had more modest reductions in taurine. Striatal dopamine content was also significantly reduced in aged learning-impaired rats. There was a significant (p < 0.001) correlation (r=0.61) between the striatal content of taurine and dopamine, but no such correlation was found for other striatal transmitters (glutamate, serotonin, norepinephrine). Treatment with neurotrophins had little effect on the age-related decline in striatal amino acids, although NGF treatment did improve spatial learning. These studies suggest (1) a link between age-related declines in striatal dopamine and taurine and (2) that NGF-induced improvement in spatial learning is not related to mechanisms involving changes in taurine or glutamate content.

Effects of idebenone on information processing in aged Long-Evans rats.

Idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) is a benzoquinone that has been shown to improve cognitive function in animals subjected to cerebral ischemia and in rats with lesions of the basal forebrain cholinergic system. Because the cognitive deficits observed in aged rats have been associated with decreased cerebral blood flow and basal forebrain cholinergic dysfunction, it was hypothesized that IDE might improve cognition in aged animals. In the present study, the effects of idebenone on cognitive function in aged Long-Evans rats were assessed using a battery of tests that evaluated attention, habituation, and spatial learning. Selective attention was assessed using an overshadowing paradigm, where IDE (30 mg/kg, IP) was injected 30 min prior to compound cue exposure. IDE enhanced the overshadowing effect in aged rats. The Morris water maze was used to assess spatial learning, where IDE (3 mg/kg, IP) was injected daily throughout the course of training. IDE did not improve the impaired performance of aged rats in the Morris task. Habituation was tested by measuring recovery from gustatory neophobia. IDE (30 mg/kg, IP) was injected 30 min prior to the first exposure to the novel taste. IDE normalized habituation rate in aged rats. It was concluded that IDE improves some forms of acquisition in aged rats, and may do so by decreasing general reactivity to novel stimuli.

Substance P enhancement of passive and active avoidance conditioning in mice.

In a series of seven experiments we explored the effects of peripherally administered substance P on passive and active avoidance conditioning in mice of two genotypes. The peripheral post-trial administration of substance P significantly enhanced the retention of a single-trial passive avoidance task. This effect was dose dependent; 1 ng/g of substance P enhanced the retention of this habit, whereas higher and lower doses were either less effective or ineffective. In heterogeneous strain (HS) mice, substance P administered before training on an active avoidance task did not alter the rate at which these animals learned this habit. However, animals that had been trained with substance P were significantly more resistant to extinction than were animals that had been injected with vehicle. Similarly, C57Bl/6J mice that had been treated with substance P immediately after active avoidance training were more resistant to extinction than were mice that had been given control injections. The enhancement of retention of the passive avoidance habit with substance P was reversed in animals that had been pretreated with naltrexone. Substance P enhancement of the retention of the passive avoidance habit, and its reversal with naltrexone, was observed in both sham operated and adrenalectomized mice.

Leptin prevents posthibernation weight gain but does not reduce energy expenditure in arctic ground squirrels.

In mammals, leptin reduces energy intake and may increase energy expenditure as a means to maintain body weight and/or adiposity at an appropriate level. Hibernating mammals seasonally alter body mass, food intake, and body composition and, therefore, represent an attractive model for investigating the physiological regulation of changing body mass and adiposity. Previous experiments in our laboratory demonstrated that administration of mouse recombinant leptin reduces food intake and body weight in arctic ground squirrels during prehibernation fattening. In addition, leptin appeared to reduce metabolic efficiency (weight gain per unit of energy intake). This result suggests that reduced food intake alone may not account for the observed weight loss. Here, we describe the effect of a 3-week constant infusion of leptin given to posthibernation arctic ground squirrels on food consumption and energy expenditure. Mouse recombinant leptin (1 mg/ml) was administered through subcutaneously implanted mini-osmotic pumps (10 microliters/hr flow rate). Resting metabolic rate was monitored before and during the 3-week leptin administration period by indirect calorimetry. Body temperature and locomotory activity were monitored continuously by abdominal radiotransmitters. At the end of the leptin administration period, thermogenic capacity was evaluated by measuring brown fat uncoupling protein-1 mRNA and protein levels. Leptin administration resulted in reduced food intake and prevented posthibernation weight gain, but it did not alter any of the measured parameters of energy expenditure.

Effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats.

The relation of forebrain cholinergic function to learning and memory was explored by identification and characterization of a training-induced change in high-affinity choline uptake (HACU), an index of cholinergic activity. Young adult rats were trained to find an invisible escape platform in a water tank using environmental cues. After 4 d of this place-training (16 trials), hippocampal HACU was significantly reduced relative to that observed in rats trained to find a visible platform (cue-training), even when cue- and place-trained rats were yoked for swim time. These place- but not cue-trained rats showed significantly lower hippocampal HACU than did naive rats, and no effect of training was noted after only 1 d of training. Similar results were obtained in parietal cortex. These differential training effects on HACU correspond to previous reports that muscarinic blockade impairs place, but not cue, learning. A further experiment revealed that the decrease in HACU in hippocampus, but not in parietal cortex, occurred only during the acquisition phase of learning and was related to the rate of acquisition for individual animals. Hippocampal HACU in naive young and aged (24-27 months) rats did not differ, but the response of the septohippocampal cholinergic system to training was diminished in the aged rats. Old rats displayed impaired place learning and a corresponding dampening of the training-induced change in HACU. These results suggest that there is a task-specific engagement of cholinergic function in young animals that does not occur in behaviorally impaired aged animals, a finding that is consistent with a role for cholinergic dysfunction in memory impairments associated with aging.