Caloric intake and aging: mechanisms in rodents and a study in nonhuman primates

Caloric restriction (CR) increases maximum life span in rodents while attenuating the development of age-associated pathological and biological changes. Although nearly all of the rodent studies have initiated CR early in life (1-3 months of age), CR, when started at 12 months of age, also extends maximum life span in mice. Two main questions face investigators of CR. One concerns the mechanisms by which CR retards aging and diseases in rodents. There is evidence that CR may act, at least in part, by reducing oxidative stress. A CR- induced decrease in oxidative stress appears to be most profound in post-mitotic tissues and may derive from lower mitochondrial production of free radicals. The second issue is whether CR will exert similar effects in primates. Studies on CR in rhesus monkeys (maximum life span approximately 40 years) support the notion of human translatability. We describe the University of Wisconsin Study of rhesus monkeys subjected to a 30% reduction of caloric intake starting at either 1989 or 1994 when they were approximately 10 years old. The data from our study and from other trials suggest that CR can be safely carried out in monkeys and that certain physiological effects of CR that occur in rodents (e.g., decreased blood glucose and insulin levels, improved insulin sensitivity, and lowering of body temperature) also occur in monkeys. Whether oxidative stress in monkeys is reduced by CR will be known by the year 2000, while effects on longevity and diseases should be clearly seen by, appropriately, 2020.      

Extrapyramidal syndromes in nonhuman primates: typical and atypical neuroleptics.

The traditional (typical) neuroleptic drugs produce acute extrapyramidal symptoms (EPS) in the majority of patients, whereas the atypical neuroleptics produce only minimal motor system side effects. Studies of acute dystonia in nonhuman primates with typical (haloperidol, fluphenazine), atypical (clozapine), and putative novel antipsychotic compounds with low EPS (remoxipride, melperone) were conducted across a wide dose range in double-blind, placebo-controlled trials. Haloperidol and fluphenazine caused dystonia, and clozapine did not. Remoxipride and melperone also produced dystonia, but remoxipride only did so at doses that were higher than needed for antipsychotic efficacy. Melperone produced dystonia in doses that are in the antipsychotic dose range. The clinical relevance of the findings is discussed.     

Fluoxetine: juvenile pharmacokinetics in a nonhuman primate model

Rationale

The selective serotonin reuptake inhibitor (SSRI) fluoxetine is the only psychopharmacological agent approved for use in children. While short-term studies of side effects have been performed, long-term consequences for brain development are not known. Such studies can be performed in appropriate animal models if doses modeling therapeutic use in children are known.

Objectives

The goal of this study was to identify a daily dose of fluoxetine in juvenile monkeys which would result in serum fluoxetine and norfluoxetine concentrations in the therapeutic range for children.

Methods

Juvenile (2.5-year-old rhesus monkeys, n?=?6) received single administration of doses of 1, 2, and 4 mg/kg day fluoxetine on a background of chronic dosing at an intermediate level to provide steady-state conditions to model therapeutic administration. Using nonlinear modeling, standard pharmacokinetic parameters were derived. Cerebrospinal fluid monoamine neurotransmitters were assayed to confirm the pharmacological effects.

Results

Dose-dependent area under the curve (AUC) and C _max values were seen, while T _max and absorption/elimination half-lives were minimally influenced by dose. A dosage of 2 mg/kg day given over a 14-week period led to steady-state serum concentrations of active agent (fluoxetine + norfluoxetine) similar to those recorded from drug monitoring studies in children. Cisternal cerebrospinal fluid concentrations of serotonin increased significantly over the 14-week period, while concentrations of the serotonin metabolite (5-HIAA) were lower but not significantly different.

Conclusions

A dose of 2 mg/kg day fluoxetine in juvenile rhesus monkeys provides an internal dose similar to therapeutic use in children and will help establish a valuable animal model for understanding fluoxetine’s therapeutic and potential adverse effects in children.     

SERT and NET occupancy by venlafaxine and milnacipran in nonhuman primates: a PET study

Introduction

Serotonin and norepinephrine reuptake inhibitors (SNRIs) are antidepressants which have high affinity to both serotonin transporter (SERT) and norepinephrine transporter (NET). In studies in vitro, SNRIs have been reported to show a large variability in the affinity ratio between SERT and NET. For instance, the reported affinity ratio is about 30 for venlafaxine and 1.6 for milnacipran. In this study in nonhuman primates, we aimed to investigate the relationship between SERT and NET affinity by measuring the in vivo occupancy at both transporters of venlafaxine and milnacipran.

Methods

PET measurements with [¹¹C]MADAM and [¹⁸F]FMeNER-D₂ were performed in two female cynomolgus monkeys at baseline and after pretreatment with venlafaxine and milnacipran, respectively. Relationships between dose, plasma concentration, and transporter occupancy were evaluated by saturation analysis using a hyperbolic function. Binding affinity (Kd^plasma) was expressed by the dose or plasma concentration at which 50 % of the transporter was occupied.

Results

SERT and NET occupancy by venlafaxine and milnacipran increased in a dose and plasma concentration-dependent manner. The Kd^plasma ratio of SERT to NET was 1.9 for venlafaxine and 0.6 for milnacipran.

Conclusions

In this nonhuman primate PET study, the affinity in vivo for SERT and NET, respectively, was shown to be at a similar level for venlafaxine and milnacipran. Both drugs were found to produce balanced inhibition of SERT and NET binding. This observation is not consistent with previous in vitro binding data and illustrates the need to characterize antidepressants at in vivo condition.     

Calorie restriction in nonhuman primates: mechanisms of reduced morbidity and mortality

Long term chronic calorie restriction (CR) of adult nonhuman primates significantly reduces morbidity and increases median age of death. The present review is focused upon an ongoing study of sustained adult- onset calorie restriction, which has been underway for 15 years. Monkeys, initially calorie restricted at about 10 years of age, are now approximately 25 years old. The median life span of these restricted monkeys is increasing, now exceeding that of ad libitum (AL)-fed monkeys. In our laboratory, maximum life span for AL-fed monkeys appears to be about 40 years. Thus, whether CR can also increase maximal life span, as it does in rodents, cannot be determined for at least another 15 years. The earliest detectable positive benefit on morbidity in these monkeys was previously reported as the prevention of obesity. Current evidence, as reviewed here, suggests that much obesity- associated morbidity is also mitigated by sustained calorie restraint in nonhuman primates. Furthermore, probably because of the prevention of obesity, diabetes has also been prevented. Recent findings include the identification of extraordinary changes in the glycogen synthesis pathway, and on the phosphorylation of glycogen synthase in response to insulin. This calorie restriction-induced prevention of morbidity does not require excessive leanness, but is clearly present when body fat is within the normal range of 10 to 22%, and this is likely to be true in humans as well.      

Measuring anxiety in nonhuman primates: effect of lorazepam on macaque scratching

Lorazepam (0.2 mg/kg IM) was given to group-living female macaques to assess the effect of anxiolytic treatment on scratching, a behavior pattern referred to as a displacement activity in the primate literature. Lorazepam selectively diminished scratching behavior. The drug effect was status-dependent: especially low-ranking animals showed a marked reduction in scratching. Lorazepam exerted a direct effect on scratching, that is the effect was not due to sedation or mediated by the influence of the drug on other behaviors. These results provide pharmacological validation to the ethological finding that scratching may be a manifestation of anxiety in monkeys. In addition, they suggest to use scratching as a behavioral measure in studies investigating nonhuman primate models of anxiety.     

Calorie restriction in nonhuman primates: effects on diabetes and cardiovascular disease risk

The effects of calorie restriction (CR) on life span, disease, and aging in physiological systems have been documented extensively in rodent models. However, whether CR has similar effects in longer-lived species more closely related to humans remains unknown. Studies of CR and aging using nonhuman primates (rhesus monkeys) have been ongoing for several years at the National Institute on Aging and the University of Wisconsin-Madison. The majority of data published from these studies are consistent with the extensive findings reported in rodents. For example, monkeys on CR weigh less and have less body fat. Monkeys on CR also exhibit lower body temperature, fasting blood glucose and insulin, and serum lipids. In addition, insulin sensitivity is increased in monkeys on CR. Recent efforts in the NIA study have focused on the effect of this intervention on risk factors for various age-related diseases, in particular for diabetes and cardiovascular disease. We have shown that monkeys on CR have lower blood pressure, reduced body fat, and a reduced trunk:leg fat ratio. Also, monkeys on CR have reduced triglycerides and cholesterol and have increased levels of HDL2B. Low levels of this HDL subfraction have been associated with increased cardiovascular disease in humans. In short-term studies, older (> 18 years) monkeys on CR exhibit reductions in insulin and triglycerides before changes in body composition and fat distribution became evident. These and other findings have suggested that CR might have beneficial effects on certain disease risk factors independent of reductions in body weight or prevention of obesity.      

Serotonin and aggressive behavior in rodents and nonhuman primates: predispositions and plasticity

This review analyzes psychosocial and genetic determinants of aggressive behavior in rodents and nonhuman primates and the role of the serotonin (5-HT) system on aggressive behaviors in order to trace possible evolutionary common origins between psychopathological and adaptive forms of aggression. Studies in primates suggest that deficit in serotonin activity, as indicated by the levels of the cerebrospinal fluid (CSF) serotonin major metabolite 5-hydroxyindoleacetic acid (5-HIAA) correlates with impulsive and aggressive behavior. It is possible that CSF 5-HIAA reflects the prevailing serotonergic tone and may be related to an aggressive trait. Superimposed on this tone are phasic serotonin changes that may be related to the inhibition of aggressive acts. Genetic factors determine aggressive behaviors as demonstrated by classic selection and strain comparison studies. Manipulations of genes targeting 5-HT receptors, transporters and enzymes can influence aggression. Some of these genes related to the serotonin transporter (5-HTT) and the monoamine oxidase A (MAO-A) show a polymorphism that may predispose, under specific environmental conditions, certain individuals to display pathological forms of aggression.     

Effect of response-independent candy on responding maintained by candy using a novel model of commodity acquisition and consumption in nonhuman primates

Ingestive behavior consists of appetitive or foraging behavior, i.e., "acquisition," followed by consummatory behavior. Responding of six adult rhesus monkeys, living in three-chambered enclosures, was studied under an operant chain schedule that simulated commodity acquisition and commodity consumption. Responding during the initial acquisition component was reinforced by stimuli paired with that commodity, while responding during the following consumption component was reinforced with that commodity. Throughout the 10-h experimental day, monkeys experienced multiple candy (plain M & Ms) and fruit-drink (Kool-Aid) sessions in different end chambers. The effects of response-independent candy reinforcement, in the context of extinction, were examined when monkeys received a daily food ration of 8 or 20 chow. Response-independent candy increased responding during the acquisition components of candy sessions when monkeys received a daily food ration of 8 chow but not when the food ration was 20 chow. Furthermore, response-independent candy increased candy choice over fruit-drink during choice opportunities and increased the length of time spent in the candy chamber when the candy stimulus lights were illuminated under both food ration conditions, i.e., location preference. The present procedure, which combines operant and place preference measures of commodity acquisition, when used in combination with methods of studying reinstatement of responding, may prove useful in analyzing factors affecting relapse.     

Developmental toxicity testing of biopharmaceuticals in nonhuman primates: previous experience and future directions

Developmental toxicity studies for pharmaceutical safety testing are designed to evaluate potential adverse effects of drug treatment on pregnancy and on the developing embryo/fetus. Biopharmaceuticals present specific challenges for developmental toxicity testing because the pharmacology of these molecules, which are frequently human-specific proteins, is often restricted to humans and nonhuman primates (NHPs). For those species-restricted molecules, the only option for the evaluation of potential effects on development of the human biopharmaceutical is to use NHPs. This article reviews each of the stages of development in cynomolgus macaques (the most frequently used NHP) and the potential exposure of the embryo, fetus, and infant following administration of a biopharmaceutical during pregnancy and lactation. Because the purpose of the NHP developmental studies is to identify potential human risks, a comparison between macaque and human development and potential exposure has been made when possible. Understanding the potential exposure of the conceptus relative to critical periods in development is essential to designing a scientifically based study that adequately addresses human risks. Some options for NHP study designs, including the option of combining end points into a single study, and the pros and cons of each of the study options have been reviewed. Developmental studies for biopharmaceuticals in NHPs need to be optimally designed on a case-by-case basis taking into consideration the pharmacology of the molecule, the type of molecule (antibody or non-antibody), the potential exposure relative to the development of potential target organs, the clinical use, and the ethical considerations associated with the use of NHPs.     

The behavioral pharmacology of anorexigenic drugs in nonhuman primates: 30 years of progress

Comparatively few studies over the past 30 years have used pharmacological manipulations as a means of understanding processes underlying feeding behavior of nonhuman primates. In the 1970s and early 1980s, four laboratories provided data on the anorexigenic effects of a range of drugs on rhesus monkeys and baboons, and a fifth laboratory studied the effects of neuropeptides on feeding behavior of baboons. There were differences in the way anorexigenic drugs altered eating topography, and those that increased dopamine levels had greater abuse liability than those that increased serotonin levels. Studies in the 1980s and 1990s used foraging models and principles of behavioral economics to understand food-drug interactions. Experimenter-given anorexigenic drugs did not function as economic substitutes for food. Recent studies have examined the effects of a range of drugs on consumption of highly palatable food and model diet-induced obesity. Although some drugs, including stimulants, N-methyl-D-aspartate antagonists, and a cannabinoid antagonist increased the latency to standard food consumption, there was little evidence for a selective effect of any drug on highly palatable food consumption. Results obtained in nonhuman primates did not always confirm those observed in rodents. Future studies looking at sex differences and social factors may provide insight into factors related to human obesity.     

The automated bioaerosol exposure system: preclinical platform development and a respiratory dosimetry application with nonhuman primates

INTRODUCTION: Well-characterized inhalation exposure systems are critical for preclinical testing and pathogenesis studies. The automated bioaerosol exposure system (ABES) provides a microprocessor-driven inhalation platform that provides exquisite data acquisition and control over all aspects of inhalation exposures. Because this represents a new technology, the development and characteristics of the ABES are thoroughly discussed. In addition to control over homeostatic and aerosol conditions, the ABES incorporates a dosimetry function based on respiratory performance of the test animal during inhalation. METHODS: To test the system, rhesus macaques were initially sham-exposed using the ABES in a head-only inhalation configuration. The ABES was subsequently used under biosafety level (BSL)-III conditions in a vaccine efficacy challenge using aerosolized staphylococcal enterotoxin B (SEB) toxin, again using the real-time dosimetry function of the system. RESULTS: Sham exposure results indicated significant departures from corresponding whole-body plethysmography (WBP) respiratory function estimates taken before the inhalation procedure. The results of the SEB exposure demonstrated the utility of using the ABES to generate consistently accurate and precise inhalation dose. DISCUSSION: Taken together, the results of the sham and toxin challenge experiments demonstrate that the dosimetry function of the ABES improves the precision and accuracy of inhaled dose delivery and calculation as compared to predictive WBP conducted before the exposure. The ABES represents a highly adaptable platform for the design of inhalation systems to suit the requirements of a variety of animal models.     

Methylmercury: exposure duration and regional distribution as determinants of neurotoxicity in nonhuman primates.

Exposure duration appears to be an important determinant, along with dosage, blood, and brain Hg concentration, of sublethal methylmercury toxicity in macaque and squirrel monkeys. Animals were evaluated for objective neural signs while maintained with blood and brain Hg near equilibrium. Chronic methylmercury, po, produced similar bodily distributions of Hg and neural signs for both species. The characteristic brain regional distribution of Hg is established well in advance of signs, emphasizing the importance of exposure duration and arguing against a major redistribution of Hg as the critical event preceding toxic signs. The calcarine cortex, lateral geniculate, and corpus striatum are candidates for critical brain regions. When near equilibrium, the primate brain Hg concentration is 1.8 to 4.9 times that of whole blood, depending upon region; higher brain/blood ratios are obtained 1 week or more following the end of exposure. Clearance of Hg from most regions of the macaque brain appeared to be similar to the rate of clearance from whole blood ($\text{t}\text{1}\text{2}\text{= 21}\text{days}$) and thus is considerably shorter than half-times reported for primate hair and whole body. The latent period preceding neural signs was inversely related to whole blood Hg concentration within the range associated with neurological signs in humans (0.5–4.5 ppm). Evidence of a “no-effect” dose in the primate will require lower level exposures in excess of 1000 days. This experimental model can suggest critical indices of human longterm, low-level exposures and it provides a basis for evaluating results from nonprimate species.     

Butyrylcholinesterase accelerates cocaine metabolism: in vitro and in vivo effects in nonhuman primates and humans.

Butyrylcholinesterase (BChE) is known to metabolize cocaine in humans. In the present study, three different experiments were performed to determine whether the addition of horse serum-derived BChE would accelerate the metabolism of cocaine. In the first experiment, the addition of BChE to squirrel monkey plasma in vitro reduced the half-life of cocaine by over 80%, decreased the production of the metabolic product benzoylecgonine, and increased ecgonine methyl ester formation. The effect of BChE on cocaine metabolism was reversed by a specific BChE inhibitor. In the second, in vivo, experiment, exogenously administered BChE reduced peak cocaine concentrations when given to anesthetized squirrel monkeys. Finally, incubation of cocaine with added BChE in human plasma in vitro resulted in a decrease in cocaine half-life similar to that observed with squirrel monkey plasma. The magnitude of the decrease in cocaine half-life was proportional to the amount of added BChE. Together, these results indicate that exogenously administered BChE can accelerate cocaine metabolism in such a way as to potentially lessen the behavioral and toxic effects of cocaine. Therefore, BChE may be useful as a treatment for cocaine addiction and toxicity.     

Influence of caloric restriction on the development of atherosclerosis in nonhuman primates: progress to date

Caloric restriction (CR) has been observed to retard aging processes and extend the maximum life span in rodents. In an effort to evaluate the effect of this nutritional intervention on physiologic variables in higher species, several nonhuman primate trials are ongoing. In particular, a study evaluating the independent effect of CR on the extent of atherosclerosis was initiated in 1993 in 32 adult cynomolgus monkeys. Therefore, the trial was designed to achieve identical cholesterol intake after animals were randomized to a control group or a calorie-restricted group (30% reduction from baseline caloric intake). The animals were routinely evaluated for glycated proteins, plasma insulin and glucose levels, insulin sensitivity, and specific measures for abdominal fat distribution by CT scans over a 4-year interval. The results from 4 years of intervention demonstrate that CR improves cardiovascular risk factors (such as visceral fat accumulation) and improves insulin sensitivity. In contrast to other primate studies with normolipidemic animals, CR had no independent effects on plasma lipid levels and composition in the presence of equivalent amounts of dietary cholesterol intake. Preliminary analysis of atherosclerotic lesion extent in the abdominal aorta has failed to demonstrate differences between control animals and CR animals. Follow- up studies are being conducted to determine the effect of CR on atherosclerosis extent in coronary and carotid arteries.      

Noradrenergic correlates of chronic cocaine craving: neuromelanin and functional brain imaging

Preclinical studies have implicated noradrenergic (NA) dysfunction in cocaine addiction. In particular, the NA system plays a central role in motivated behavior and may partake in the regulation of craving and drug use. Yet, human studies of the NA system are scarce, likely hampered by the difficulty in precisely localizing the locus coeruleus (LC). Here, we used neuromelanin imaging to localize the LC and quantified LC neuromelanin signal (NMS) intensity in 44 current cocaine users (CU; 37 men) and 59 nondrug users (NU; 44 men). We also employed fMRI to investigate cue-induced regional responses and LC functional connectivities, as quantified by generalized psychophysiological interaction (gPPI), in CU. Imaging data were processed by published routines and the findings were evaluated with a corrected threshold. We examined how these neural measures were associated with chronic cocaine craving, as assessed by the Cocaine Craving Questionnaire (CCQ). Compared to NU, CU demonstrated higher LC NMS for all probabilistic thresholds defined of 50–90% of the peak. In contrast, NMS of the ventral tegmental area/substantia nigra (VTA/SN) did not show significant group differences. Drug as compared to neutral cues elicited higher activations of many cortical and subcortical regions, none of which were significantly correlated with CCQ score. Drug vs. neutral cues also elicited “deactivation” of bilateral parahippocampal gyri (PHG) and PHG gPPI with a wide array of cortical and subcortical regions, including the ventral striatum and, with small volume correction, the LC. Less deactivation of the PHG (r = 0.40, p = 0.008) and higher PHG-LC gPPI (r = 0.44, p = 0.003) were positively correlated with the CCQ score. In contrast, PHG-VTA/SN connectivity did not correlate with the CCQ score. Together, chronic cocaine exposure may induce higher NMS intensity, suggesting neurotoxic effects on the LC. The correlation of cue-elicited PHG LC connectivity with CCQ score suggests a noradrenergic correlate of chronic cocaine craving. Potentially compensating for memory functions as in neurodegenerative conditions, cue-elicited PHG LC circuit connectivity plays an ill-adaptive role in supporting cocaine craving.Subject terms: Predictive markers, Motivation     

Chronic maternal methanol inhalation in nonhuman primates (Macaca fascicularis): reproductive performance and birth outcome

The present study was designed to characterize maternal reproductive performance and early offspring effects following exposure to methanol (MeOH) vapor in a nonhuman primate model. The two-cohort study design used 48 adult female Macaca fascicularis (24/cohort) monkeys exposed to 0, 200, 600, or 1800 ppm MeOH vapor for approximately 2.5 h/day, 7 days/week prior to breeding and throughout pregnancy. Maternal body weight measurement, clinical observations and health assessments were conducted routinely throughout the study. Menstrual cyclicity was monitored during the pre-breeding and breeding periods and timed matings were conducted with nonexposed males. Females were monitored closely during the last month of pregnancy. At birth, infant physical characteristics were measured and a newborn health assessment was conducted. Methanol exposure did not alter menstrual cycles, the number of breedings to conception or conception rate. A total of 34 live-born infants were delivered (control=8, 200 ppm=9, 600 ppm=8, 1800 ppm=9). One female each in the control and 600-ppm group delivered a stillborn infant and a cesarean section (C-section) was required to deliver a hydrocephalic infant who died in utero in the maternal 1800-ppm group. Although not statistically significant, five MeOH-exposed females were C-sectioned due to pregnancy complications such as uterine bleeding and prolonged unproductive labor. These complications were not observed in the control group. The mean length of pregnancy in the MeOH-exposed groups was significantly decreased by 6 to 8 days when compared to controls. There were no MeOH-related effects on offspring birthweight or newborn health status. The consistent reduction in length of pregnancy observed in the MeOH females may reflect a treatment effect on the fetal neuroendocrine system. Given that the fetal hypothalamic--pituitary-adrenal axis controls pregnancy length in most species, these results suggest a modest but significant effect of MeOH on the biochemical events that control the timing of birth.