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Context Ovarian estradiol supports female sexual behavior and metabolic function. While ovariectomy (OVX) in rodents abolishes sexual behavior and enables obesity, OVX in nonhuman primates decreases, but does not abolish, sexual behavior, and inconsistently alters weight gain. Objective We hypothesize that extra-ovarian estradiol provides key support for both functions, and to test this idea, we employed aromatase inhibition to eliminate extra-ovarian estradiol biosynthesis and diet-induced obesity to enhance weight gain. Methods Thirteen adult female marmosets were OVX and received (1) estradiol-containing capsules and daily oral treatments of vehicle (E2; n = 5); empty capsules and daily oral treatments of either (2) vehicle (VEH, 1 mL/kg, n = 4), or (3) letrozole (LET, 1 mg/kg, n = 4). Results After 7 months, we observed robust sexual receptivity in E2, intermediate frequencies in VEH, and virtually none in LET females (P = .04). By contrast, few rejections of male mounts were observed in E2, intermediate frequencies in VEH, and high frequencies in LET females (P = .04). Receptive head turns were consistently observed in E2, but not in VEH and LET females. LET females, alone, exhibited robust aggressive rejection of males. VEH and LET females demonstrated increased % body weight gain (P = .01). Relative estradiol levels in peripheral serum were E2 >>> VEH > LET, while those in hypothalamus ranked E2 = VEH > LET, confirming inhibition of local hypothalamic estradiol synthesis by letrozole. Conclusion Our findings provide the first evidence for extra-ovarian estradiol contributing to female sexual behavior in a nonhuman primate, and prompt speculation that extra-ovarian estradiol, and in particular neuroestrogens, may similarly regulate sexual motivation in other primates, including humans.

The prevalence of advanced medical treatment has led to global population aging, resulting in increased numbers of dementia patients. One of the most intractable symptoms of dementia is apathy, or lack of interest and enthusiasm, which can accompany memory and cognitive deterioration. Development of a novel method to ameliorate apathy is desirable. In this feasibility trial, we propose a series of digital art installations as a candidate dementia intervention approach. Seven, three-minute scenes of digital images and sounds were presented to visitors either passively or in response to their reactions (motion and sound). We evaluated the potential of this application as an intervention against apathy in an elderly living home. We collected the dementia global standard Mini-Mental State Examination (MMSE) and questionnaire scores of sensory pleasure and motivation along with behavioral motion data in twenty participants. We further compared responses between the first and second experiences in the thirteen participants that were present for both days. Overall, we found a significant increase in participants&rsquo, motivation. In these subjects, head and right hand motion increased over different scenes and MMSE degrees, but was most significant during passive scenes. Despite a small number of subjects and limited evaluations, this new digital art technology holds promise as an apathy intervention in the elderly and can be improved with use of wearable motion sensors.

Aging is a multifactorial process characterized by progressive changes in gut physiology and the intestinal mucosal immune system. These changes, along with alterations in lifestyle, diet, nutrition, inflammation and immune function alter both composition and stability of the gut microbiota. Given the impact of environmental influences on the gut microbiota, animal models are particularly useful in this field. To understand the relationship between the gut microbiota and aging in nonhuman primates, we collected fecal samples from 20 male and 20 female rhesus macaques (Macaca mulatta), across the natural macaque age range, for 16S rRNA gene analyses. Operational taxonomic units were then grouped together to summarize taxon abundance at different hierarchical levels of classification and alpha- and beta-diversity were calculated. There were no age or sex differences in alpha diversity. At the phylum level, relative abundance of Proteobacteria and Firmicutes and Firmicutes to Bacteriodetes ratio were different between age groups though significance disappeared after correction for multiple comparisons. At the class level, relative abundance of Firmicutes_Bacilli decreased and Proteobacteria_Alphaproteobacteria and Proteobacteria_Betaproteobacteria increased with each successively older group. Only differences in Firmicutes_Bacilli remained significant after correction for multiple comparisons. No sex differences were identified in relative abundances after correction for multiple comparisons. Our results are not surprising given the known impact of environmental factors on the gut microbiota.

The use of marmosets as nonhuman primate models of human disease has undergone rapid expansion in the United States in the last decade, with an emphasis in the field of neuroscience. With this expanding need, there has been an increase in the formation of small marmoset colonies. The standardization in care and husbandry techniques for marmosets has historically lagged behind other established nonhuman primate models, resulting in a great deal of variation in practices between colonies. There remains a lack of consensus and evidence-based recommendations regarding best standards for nutrition, enrichment, weight management, and diagnostics for clinical metabolic disease. Marmoset base diets vary broadly in their nutritional value, and therefore the physiological responses to these diets also vary broadly. In this review, we briefly outline what is known about nutrition for captive marmosets and highlight what is known regarding metabolic dysfunction and obesity.

Metabolic syndrome increases risk of complicating co-morbidities. Current clinical indicators reflect established metabolic impairment, preventing earlier intervention strategies. Here we show that circulating sphingolipids are altered in the very early stages of insulin resistance development. The study involved 16 paired overweight but healthy monkeys, one-half of which spontaneously developed metabolic syndrome over the course of 2 years. Importantly, animals did not differ in adiposity and were euglycemic throughout the study period. Using mass spectrometry, circulating sphingolipids, including ceramides and sphingomyelins, were detected and quantified for healthy and impaired animals at both time points. At time of diagnosis, several ceramides were significantly different between healthy and impaired animals. Correlation analysis revealed differences in the interactions among ceramides in impaired animals at diagnosis and pre-diagnosis when animals were clinically indistinguishable from controls. Furthermore, correlations between ceramides and early-stage markers of insulin resistance, diacylglycerols and non-esterified fatty acids, were distinct for healthy and impaired states. Regression analysis identifies coordinated changes in lipid handling across lipid classes as animals progress from healthy to insulin resistant. Correlations between ceramides and the adipose-derived adipokine adiponectin were apparent in healthy animals but not in the metabolically impaired animals, even in advance of loss in insulin sensitivity. These data suggest that circulating ceramides are clinically relevant in identifying disease risk independent of differences in adiposity, and may be important in devising preventative strategies.

Nonhuman primates (NHPs) are a critical component of translational/preclinical biomedical research due to the strong similarities between NHP and human physiology and disease pathology. In some cases, NHPs represent the most appropriate, or even the only, animal model for complex metabolic, neurological, and infectious diseases. The increased demand for and limited availability of these valuable research subjects requires that rigor and reproducibility be a prime consideration to ensure the maximal utility of this scarce resource. Here, we discuss a number of approaches that collectively can contribute to enhanced rigor and reproducibility in NHP research.

IntroductionInsulin is a peptide hormone that is secreted by the β cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS).MethodsMarmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin.Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled (13C, 15N) synthetic marmoset insulin tryptic peptide was used as internal standard.ResultsThe assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 4.28 pmol/L and the limit of quantitation was 8.62 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L.ConclusionsThis paper presents a simple, reproducible method to measure marmoset insulin using LC-MS/MS.

Age is a major risk factor for late‐onset Alzheimer's disease (AD) but seldom features in laboratory models of the disease. Furthermore, heterogeneity in size and density of AD plaques observed in individuals are not recapitulated in transgenic mouse models, presenting an incomplete picture. We show that the amyloid plaque microenvironment is not equivalent between rodent and primate species, and that differences in the impact of AD pathology on local metabolism and inflammation might explain established differences in neurodegeneration and functional decline. Using brain tissue from transgenic APP/PSEN1 mice, rhesus monkeys with age‐related amyloid plaques, and human subjects with confirmed AD, we report altered energetics in the plaque microenvironment. Metabolic features included changes in mitochondrial distribution and enzymatic activity, and changes in redox cofactors NAD(P)H that were shared among species. A greater burden of lipofuscin was detected in the brains from monkeys and humans of advanced age compared to transgenic mice. Local inflammatory signatures indexed by astrogliosis and microglial activation were detected in each species; however, the inflamed zone was considerably larger for monkeys and humans. These data demonstrate the advantage of nonhuman primates in modeling the plaque microenvironment, and provide a new framework to investigate how AD pathology might contribute to functional loss., Souder et al. report the outcome of a cross‐species comparison of the local impact of amyloid plaques in the brains of APP/PSEN mouse model, aged monkeys, and human subjects diagnosed with Alzheimer's disease. Microenvironment mitochondrial clustering and altered redox status were common to each species, but lipofuscin accumulation and elevated inflammation were more pronounced in monkeys and humans, indicating that age contributes to amyloid pathology.

BACKGROUND: A survey was developed to characterize disease incidence, common pathology lesions, environmental characteristics, and nutrition programs within captive research marmoset colonies. METHODS: 17 research facilities completed the electronic survey. RESULTS: Nutritional management programs varied amongst research institutions housing marmosets; eight primary base diets were reported. The most common clinical syndromes reported were gastrointestinal (GI) disease (i.e. inflammatory bowel disease (IBD) like disease, chronic lymphocytic enteritis (CLE), chronic malabsorption, chronic diarrhea), metabolic bone disease or fracture, infectious diarrhea, and oral disease (tooth root abscesses, gingivitis, tooth root resorption). The five most common pathology morphologic diagnoses were colitis, nephropathy/ nephritis, enteritis, chronic lymphoplasmacytic enteritis, and cholecystitis. Obesity was more common (average 20% of a reporting institution’s population) than thin body condition (average 5%). CONCLUSIONS: Through review of current practices, we aim to inspire development of evidence based practices to standardize husbandry and nutrition practices for marmoset research colonies.

Insulin is a peptide hormone that is secreted by the β cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). Marmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin. Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled ((13)C, (15)N) synthetic marmoset insulin tryptic peptide was used as internal standard. The assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 15.49 pmol/L and the limit of quantitation was 140.78 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L. In summary, this paper presents a simple, reproducible method to measure marmoset insulin in serum using LC-MS/MS.

The use of home-based image sensors for biological and environmental monitoring provides novel insight into health and development but it is difficult to evaluate people during their normal activities in their home. Therefore, we developed a low-cost infrared (IR) technology-based motion, location, temperature and thermal environment detection system that can be used non-invasively for long-term studies in the home environment. We tested this technology along with the associated analysis algorithm to visualize the effects of parental care and thermal environment on developmental state change in a non-human primate model, the common marmoset (Callithrix jacchus). To validate this system, we first compared it to a manual analysis technique and we then assessed the development of circadian rhythms in common marmosets from postnatal day 15&ndash, 45. The semi-automatically tracked biological indices of locomotion velocity (BV) and body surface temperature (BT) and the potential psychological index of place preference toward the door (BD), showed age-dependent shifts in circadian phase patterns. Although environmental variables appeared to affect circadian rhythm development, principal component analysis and signal superimposing imaging methods revealed a novel phasic pattern of BD-BT correlation day/night switching in animals older than postnatal day 38 (approximately equivalent to one year of age in humans). The origin of this switch was related to earlier development of body temperature (BT) rhythms and alteration of psychological behavior rhythms (BD) around earlier feeding times. We propose that this cost-effective, inclusive sensing and analytic technique has value for understanding developmental care conditions for which continual home non-invasive monitoring would be beneficial and further suggest the potential to adapt this technique for use in humans.

The impact of chronic caloric restriction (CR) on health and survival is complex with poorly understood underlying molecular mechanisms. A recent study in mice addressing the diets used in nonhuman primate CR studies found that while diet composition did not impact longevity, fasting time and total calorie intake were determinant for increased survival. Here, integrated analysis of physiological and multi-omics data (transcriptomics-metabolomics) from ad libitum, meal-fed, or CR animals was used to gain insight into core and specific pathways associated with improved health and survival. We uncover a potential pivotal role played by the glycine-serine-threonine metabolism in longevity and related life-sustaining mechanisms. Direct comparison of the different feeding strategies unveiled a pattern of shared pathways of improved health that included short-chain fatty acids and essential PUFA metabolism. These findings were recapitulated in the serum metabolome from nonhuman primates. We propose that the pathways identified might be targeted for their potential role in healthy aging.

Declining serum estradiol (E2) levels during the menopausal transition are associated with increased central adiposity and heightened risk for metabolic disease. Estrogenic effects on adiposity and metabolism in female rodents are primarily mediated by estrogen receptor alpha (ESR1) activation in ventromedial (VMN) and arcuate (ARC) nuclei within the mediobasal hypothalamus (MBH). The role of hypothalamic ESR1 in the menopausal transition, and in regulating body weight, body composition and energy homeostasis in female primates, however, remains unclear. To investigate the involvement of ESR1 in regulating female primate body weight, we employed RNAi technology to assess ESR1 gene knockdown throughout the MBH of adult, full-grown, ovary intact female rhesus macaques. Using MRI-guided stereotaxic targeting, adeno-associated viral vector 8 (AAV8) expressing shRNA-ESR1 (ERαKD) (n=6), or a scrambled control sequence (n=4), were infused bilaterally into the MBH to knockdown ESR1 expression. Results: ERαKD females exhibited a ~22% (+2.0 ± 0.1 kg) increase in body weight to attain 10.4±0.9 kg after ~12-24 months (mo) (p

Vitamin D(3) (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultra violet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D(3) due to lack of sunlight exposure. The minimum dietary vitamin D(3) requirement and the maximum amount of vitamin D(3) that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D(3) provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D(3) (25(OH)D(3)), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D(3) in captive marmosets, but 25(OH)D(3) is not the final product of vitamin D(3) metabolism. In addition to serum 25(OH)D(3), we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and the less well understood metabolite, 24,25-dihydroxyvitamin D(3) (24,25(OH)(2)D(3)) to characterize the marmoset’s ability to metabolize dietary vitamin D(3). We present vitamin D(3) metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D(3) per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D(3) (426ng/ml (SD 200) versus 215ng/ml (SD 113)) and 24,25(OH)(2)D(3) (53ng/ml (SD 35) versus 7ng/ml (SD 5)). There was no difference in serum 1,25(OH)(2)D(3) between the colonies. Serum 1,25(OH)(2)D(3) increased and 25(OH)D(3) decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D(3) into the active metabolite 1,25(OH)(2)D(3;) excess 25(OH)D(3) is metabolized into 24,25(OH)(2)D(3). This ability explains the tolerance of high levels of dietary vitamin D(3) by marmosets, however, our data suggest that these high dietary levels are not required.

Summary Aging leads to profound changes in glucose homeostasis, weight, and adiposity, which are considered good predictors of health and survival in humans. Direct evidence that these age-associated metabolic alterations are recapitulated in animal models is lacking, impeding progress to develop and test interventions that delay the onset of metabolic dysfunction and promote healthy aging and longevity. We compared longitudinal trajectories, rates of change, and mortality risks of fasting blood glucose, body weight, and fat mass in mice, nonhuman primates, and humans throughout their lifespans and found similar trajectories of body weight and fat in the three species. In contrast, fasting blood glucose decreased late in life in mice but increased over the lifespan of nonhuman primates and humans. Higher glucose was associated with lower mortality in mice but higher mortality in nonhuman primates and humans, providing a cautionary tale for translating age-associated metabolic changes from mice to humans.

Aging is the biggest risk factor for the most serious chronic diseases and disabilities. Cellular senescence, a state in which cells stop dividing but release factors that damage other cells, may contribute to both age-related and chronic diseases. Removal of senescent cells from aged mice has been shown to delay aging and age–related disabilities. Our goal was to determine the ability of potential senolytic agents to remove senescent cells in a primate model. Several agents and combinations were tested including Fisetin, Navitoclax, combined Dasatinib and Quercetin, and combined Dasatinib and Fisetin. Here we describe the Dasatinib and Fisetin trial. Dasatinib is an FDA approved oral anticancer drug that has been used to treat chronic myelogenous leukemia in humans. Fisetin is a flavonoid that can be found in many plants, particularly strawberries, and acts as a coloring agent. After baseline measurements, six older (mean age=21 years) female rhesus monkeys (Macaca mulatta) were given a combined oral dose of Dasatinib (5 mg/kg) and Fisetin (100 mg/kg) on two consecutive days. Animals were additionally assessed at 1- and 7-weeks following dosing. At 7 weeks post dosing, there were fewer (p

In mammals, caloric restriction consistently results in extended lifespan. Epigenetic information encoded by DNA methylation is tightly regulated, but shows a striking drift associated with age that includes both gains and losses of DNA methylation at various sites. Here, we report that epigenetic drift is conserved across species and the rate of drift correlates with lifespan when comparing mice, rhesus monkeys, and humans. Twenty-two to 30-year-old rhesus monkeys exposed to 30% caloric restriction since 7–14 years of age showed attenuation of age-related methylation drift compared to ad libitum-fed controls such that their blood methylation age appeared 7 years younger than their chronologic age. Even more pronounced effects were seen in 2.7–3.2-year-old mice exposed to 40% caloric restriction starting at 0.3 years of age. The effects of caloric restriction on DNA methylation were detectable across different tissues and correlated with gene expression. We propose that epigenetic drift is a determinant of lifespan in mammals., Caloric restriction has been shown to increase lifespan in mammals. Here, the authors provide evidence that age-related methylation drift correlates with lifespan and that caloric restriction in mice and rhesus monkeys results in attenuation of age-related methylation drift.

Summary Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR-regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR-125a-5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR-associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR-induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA-based homeostatic mechanisms to coordinate a program of delayed aging.

Caloric restriction (CR) without malnutrition extends lifespan and delays the onset of age-related disorders in most species but its impact in nonhuman primates has been controversial. In the late 1980s two parallel studies were initiated to determine the effect of CR in rhesus monkeys. The University of Wisconsin study reported a significant positive impact of CR on survival, but the National Institute on Aging study detected no significant survival effect. Here we present a direct comparison of longitudinal data from both studies including survival, bodyweight, food intake, fasting glucose levels and age-related morbidity. We describe differences in study design that could contribute to differences in outcomes, and we report species specificity in the impact of CR in terms of optimal onset and diet. Taken together these data confirm that health benefits of CR are conserved in monkeys and suggest that CR mechanisms are likely translatable to human health., Caloric restriction (CR) delays ageing of model organisms, but whether it works in nonhuman primates has been controversial. Here, the authors pool and reanalyse data from two long-running CR primate studies, concluding that moderate CR indeed improves health and survival of rhesus monkeys.

Calorie restriction (CR) improved health span in 2 longitudinal studies in nonhuman primates (NHPs), yet only the University of Wisconsin (UW) study demonstrated an increase in survival in CR monkeys relative to controls; the National Institute on Aging (NIA) study did not. Here, analysis of left ventricle samples showed that CR did not reduce cardiac fibrosis relative to controls. However, there was a 5.9-fold increase of total fibrosis in UW hearts, compared with NIA hearts. Diet composition was a prominent difference between the studies; therefore, we used the NHP diets to characterize diet-associated molecular and functional changes in the hearts of mice. Consistent with the findings from the NHP samples, mice fed a UW or a modified NIA diet with increased sucrose and fat developed greater cardiac fibrosis compared with mice fed the NIA diet, and transcriptomics analysis revealed diet-induced activation of myocardial oxidative phosphorylation and cardiac muscle contraction pathways.

Common marmosets (Callithrix jacchus) are susceptible to intestinal inflammation which leads to chronic diarrhea, weight loss, and vitamin D deficiency. We examined food intake and digestion in three mixed-sex groups of adult marmosets maintained on three commercial base diets. Animals underwent two consecutive 4-day digestion trials. Body mass stayed constant. Feces and diet were assayed for Mn, fat, and gross energy (GE). Apparent digestibility of dry matter (ADDM) was calculated by the total collection method and from dietary and fecal Mn; the methods produced correlated results (r = 0.658, p 10%) was associated with ADfat of zero, consistent with lipid malabsorption. Mean digestible energy intake (DEI) was equal to 1.5 the estimated metabolic rate, but varied widely between individuals. The diet with the fewest animals with high fecal fat had the highest mean DEI and most animals above 450 g, suggesting it may be obesogenic.

Caloric restriction (CR) improves survival in nonhuman primates and delays the onset of age-related morbidities including sarcopenia, which is characterized by the age-related loss of muscle mass and function. A shift in metabolism anticipates the onset of muscle-aging phenotypes in nonhuman primates, suggesting a potential role for metabolism in the protective effects of CR. Here, we show that CR induced profound changes in muscle composition and the cellular metabolic environment. Bioinformatic analysis linked these adaptations to proteostasis, RNA processing, and lipid synthetic pathways. At the tissue level, CR maintained contractile content and attenuated age-related metabolic shifts among individual fiber types with higher mitochondrial activity, altered redox metabolism, and smaller lipid droplet size. Biometric and metabolic rate data confirm preserved metabolic efficiency in CR animals that correlated with the attenuation of age-related muscle mass and physical activity. These data suggest that CR-induced reprogramming of metabolism plays a role in delayed aging of skeletal muscle in rhesus monkeys.

Sarcomeric proteins, including myofilament and Z-disk proteins, play critical roles in regulating muscle contractile properties. A variety of isoforms and post-translational modifications (PTMs) of sarcomeric proteins have been shown to be associated with modulation of muscle functions and the occurrence of muscle diseases. Non-human primates (NHPs) are excellent research models for sarcopenia, a disease associated with alterations in sarcomeric proteins, due to their marked similarities to humans. However, the sarcomeric proteins in NHP skeletal muscle have not been well characterized. To gain a deeper understanding of sarcomeric proteins in NHP skeletal muscle, we employed top-down mass spectrometry (MS) to conduct a comprehensive analysis on isoforms and PTMs of sarcomeric proteins in rhesus macaque skeletal muscle. We identified 23 protein isoforms with 46 proteoforms of sarcomeric proteins, including 6 isoforms with 18 proteoforms from fast skeletal troponin T. Particularly, for the first time, a novel PDZ/LIM domain protein isoform, PDLIM7, was characterized with a newly identified protein sequence. Moreover, we also identified multiple PTMs on these proteins, including deamidation, methylation, acetylation, tri-methylation, phosphorylation, and S-glutathionylation. Most PTM sites were localized, including Asn13 deamidation on MLC-2S; His73 methylation on αactin; N-terminal acetylation on most identified proteins; N-terminal tri-methylation on MLC-1S, MLC-1F, MLC-2S, and MLC-2F; Ser14 phosphorylation on MLC-2S; and Ser15 and Ser16 phosphorylation on MLC-2F. In summary, a comprehensive characterization of sarcomeric proteins including multiple isoforms and PTMs in NHP skeletal muscle was achieved by analyzing intact proteins in the top-down MS approach.

Rhesus macaques represent an important species for translational and pre-clinical research studies across a multitude of disease and injury models, including aging. Ketamine anesthesia is used in humans and non-human primates but may be associated with adverse effects, including neuromuscular reactions. The effects of aging on ketamine adverse effects is not well characterized. Urodynamic recordings and electromyography (EMG) studies were performed in aged (>20 years old) and adult (3.9-14.9 years old) female rhesus macaques under an equal and light plane of sedation by constant rate infusion (CRI) of ketamine. A total of 4 of 41 adult subjects (9.7%) showed clinical signs of ketamine-induced abnormal neuromuscular reactivity, whereas a larger portion of 14 of 26 aged subjects showed similar ketamine-induced neuromuscular reactivity (53.8%; P< 0.001). The ketamine CRI rate was 19.8±0.9 mg/kg/h in adults and lower in aged subjects at 16.5±1.4 mg/kg/h (P

Caloric restriction (CR) has been shown to decrease aging and increase the lifespan of several model organisms. However, in two on-going independent, long-term CR studies in non-human primates (NHP) at the National Institute of Aging (NIA) and University of Wisconsin, Madison (UW) the outcome has been more complex. While CR monkeys in the UW study have experienced a significant increase in lifespan relative to their study controls, this was not the case for the NIA study. There, CR did not increase lifespan compared to the already long-lived controls. Yet, in both studies CR was associated with a decrease in the occurrence of age-related diseases. Left ventricle (LV) samples were provided from the rhesus monkeys from both studies to investigate the effect of long-term CR on myocardial pathology and aging in these two cohorts of non-human primates (NHPs). Although CR did not reduce cardiac fibrosis or hypertrophy in either study, there was a 5.9-fold increase in fibrosis (p Abbreviations: CR: Caloric Restriction NHP: Non-human primate mTOR: mechanistic Target of Rapamycin NIA: National Institute on Aging UW: University of Wisconsin, Madison

Estrogen depletion leads to bone loss in almost all mammals with frequent regular ovarian cycles. However, subordinate adult female common marmosets (Callithrix jacchus) undergo socially induced anovulation and hypoestrogenism without clinically apparent adverse skeletal consequences. Thus, we speculated that this non human primate might have evolved a mechanism to avoid estrogen-depletion bone loss. To test this possibility, we performed three experiments in which lumbar-spine (L5-L6) bone mineral content (BMC) and density (BMD) were assessed using dual-energy X-ray absorptiometry: (i) cross-sectionally in 13 long-term ovariectomized animals and 12 age- and weight-matched controls undergoing ovulatory cycles; (ii) longitudinally in 12 animals prior to, 3-4 and 6-7 months following ovariectomy (ovx), and six controls; and (iii) cross-sectionally in nine anovulatory subordinate and nine dominant females. In Experiments 1 and 3, plasma estradiol and estrone concentrations were measured and uterine dimensions were obtained by ultrasound in a subset of animals as a marker of functional estrogen depletion. Estrogen levels, uterine trans-fundus width, and uterine dorso-ventral diameter were lower in ovariectomized and subordinate females than in those undergoing ovulatory cycles. However, no differences were found in L5-L6 BMC or BMD. These results indicate that estrogen depletion, whether surgically or socially induced, is not associated with lower bone mass in female common marmosets. Thus, this species may possess unique adaptations to avoid bone loss associated with estrogen depletion.

In adult female rodents, ovarian estradiol (E2) regulates body weight, adiposity, energy balance, physical activity, glucose-insulin homeodynamics, and lipid metabolism, while protecting against diet-induced obesity. The same E2 actions are presumed to occur in primates, but confirmatory studies have been lacking. We investigated the consequences of ovariectomy (OVX) and E2 replacement in female marmoset monkeys on major metabolic and morphometric endpoints. Sexual behavior and uterine diameters were assessed as positive controls for E2 treatment efficacy. Metabolic parameters were measured 1 mo prior to OVX, and 3 and 6 mo thereafter. During OVX, animals received empty or E2-containing silastic s.c. implants. To test the interaction between E2 and diet, both treatment groups were assigned to either a higher fat diet (HFD) or a low-fat diet (LFD). As anticipated, OVX animals exhibited diminished frequency (p = 0.04) of sexually receptive behavior and increased rejection behavior (p = 0.04) toward their male partners compared with E2-treated OVX females. OVX also decreased (p = 0.01) uterine diameter. There were no treatment effects on total caloric intake. There were no significant effects of OVX, E2 treatment, or diet on body weight, body composition, energy expenditure, physical activity, fasting glucose, or glucose tolerance. Regardless of E2 treatment, serum triglycerides were higher (p = 0.05) in HFD than LFD females. Postmortem qPCR analysis of hypothalamic tissues revealed higher mRNA expression (p

Summary The hippocampus is critical for cognition and memory formation and is vulnerable to age‐related atrophy and loss of function. These phenotypes are attenuated by caloric restriction (CR), a dietary intervention that delays aging. Here, we show significant regional effects in hippocampal energy metabolism that are responsive to age and CR, implicating metabolic pathways in neuronal protection. In situ mitochondrial cytochrome c oxidase activity was region specific and lower in aged mice, and the impact of age was region specific. Multiphoton laser scanning microscopy revealed region‐ and age‐specific differences in nicotinamide adenine dinucleotide (NAD)‐derived metabolic cofactors. Age‐related changes in metabolic parameters were temporally separated, with early and late events in the metabolic response to age. There was a significant regional impact of age to lower levels of PGC‐1α, a master mitochondrial regulator. Rather than reversing the impact of age, CR induced a distinct metabolic state with decreased cytochrome c oxidase activity and increased levels of NAD(P)H. Levels of hippocampal PGC‐1α were lower with CR, as were levels of GSK3β, a key regulator of PGC‐1α turnover and activity. Regional distribution and colocalization of PGC‐1α and GSK3β in mouse hippocampus was similar in monkeys. Furthermore, the impact of CR to lower levels of both PGC‐1α and GSK3β was also conserved. The studies presented here establish the hippocampus as a highly varied metabolic environment, reveal cell‐type and regional specificity in the metabolic response to age and delayed aging by CR, and suggest that PGC‐1α and GSK3β play a role in implementing the neuroprotective program induced by CR.

Metabolic syndrome is linked with obesity and is often first identified clinically by elevated BMI and elevated levels of fasting blood glucose that are generally secondary to insulin resistance. Using the highly translatable rhesus monkey (Macaca mulatta) model, we asked if metabolic syndrome risk could be identified earlier. The study involved 16 overweight but healthy, euglycemic monkeys, one-half of which spontaneously developed metabolic syndrome over the course of 2 years while the other half remained healthy. We conducted a series of biometric and plasma measures focusing on adiposity, lipid metabolism, and adipose tissue-derived hormones, which led to a diagnosis of metabolic syndrome in the insulin-resistant animals. Plasma fatty acid composition was determined by gas chromatography for cholesteryl ester, FFA, diacylglycerol (DAG), phospholipid, and triacylglycerol lipid classes; plasma lipoprotein profiles were generated by NMR; and circulating levels of adipose-derived signaling peptides were determined by ELISA. We identified biomarker models including a DAG model, two lipoprotein models, and a multiterm model that includes the adipose-derived peptide adiponectin. Correlations among circulating lipids and lipoproteins revealed shifts in lipid metabolism during disease development. We propose that lipid profiling may be valuable for early metabolic syndrome detection in a clinical setting.

The administration of estradiol-17β (E) to animal models after loss of ovarian steroid production has many beneficial effects on neural functions, particularly in the serotonin system in nonhuman primates (NHPs). E also has anorexic effects, although the mechanism of action is not well defined. In the US, obesity has reached epidemic proportions, and blame is partially directed at the Western style diet, which is high in fat and sugar. This study examined the interaction of E and diet in surgically menopausal nonhuman primates with a 2 × 2 block design. Marmosets (Callithrix jacchus; n=4/group) were placed on control-low fat diet (LFD; 14%kcal from fat) or high fat diet (HFD; 28%kcal from fat) 1 month prior to ovariectomy (Ovx). Empty (placebo) or E-filled Silastic capsules were implanted immediately following Ovx surgery. Treatments extended 6 months. The established groups were: placebo+LFD; E+LFD; placebo+HFD, or E+HFD. At necropsy, the brain was flushed with saline and harvested. The midbrain was dissected and a small block containing the dorsal raphe nucleus was processed for qRT-PCR using Evagreen (Biotinum). Genes previously found to impact serotonin neural functions were examined. Results were compared with 2-way ANOVA followed by Bonferroni post-hoc tests or Cohen’s D analysis. There was a significant effect of treatment on tryptophan hydroxylase 2 (TPH2) across the groups (p=0.019). E stimulated TPH2 expression and HFD prevented E-stimulated TPH2 expression (p

Summary Caloric restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here, we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic metabolism. We conducted molecular profiling of liver biopsies collected from adult male rhesus monkeys ( Macaca mulatta ) at baseline and after 2 years on control or CR (30% restricted) diet. Quantitation of over 20,000 molecules from the hepatic transcriptome, proteome, and metabolome indicated that metabolism and RNA processing are major features of the response to CR. Predictive models identified lipid, branched-chain amino acid, and short-chain carbon metabolic pathways, with alternate transcript use for over half of the genes in the CR network. We conclude that RNA-based mechanisms are central to the CR response and integral in metabolic reprogramming.

There has been, and continues to be, a dramatic shift in the human population towards older ages necessitating biomedical research aimed at better understanding the basic biology of aging and age-related diseases and facilitating new and improved therapeutic options. As it is not practical to perform the breadth of this research in humans, animal models are necessary to recapitulate the complexity of the aging environment. The mouse model is most frequently chosen for these endeavors, however, they are frequently not the most appropriate model. Non-human primates, on the other hand, are more closely related to humans and recapitulate the human aging process and development of age-related diseases. Extensive aging research has been performed in the well-characterized rhesus macaque aging model. More recently, the common marmoset, a small non-human primate with a shorter lifespan, has been explored as a potential aging model. This model holds particular promise as an aging disease model in part due to the successful creation of transgenic marmosets. Limitations to the use of non-human primates in aging research exist but can be mitigated somewhat by the existence of available resources supported by the National Institutes of Health. This article is part of a Special Issue entitled: Animal models of aging - edited by "Houtkooper Riekelt".

Calorie restriction without malnutrition increases longevity and delays the onset of age-associated disorders in multiple species. Recently, greater emphasis has been placed on healthy life span and preventing frailty than on longevity. Here, we show the beneficial effect of long-term calorie restriction on frailty in later life in a nonhuman primate. Frail phenotypes were evaluated using metabolic and physical activity data and defined using the Fried index. Shrinking was defined as unintentional weight loss of greater than 5% of body weight. Weakness was indicated by decline in high intensity spontaneous physical activity. Poor endurance or exhaustion was indicated by a reduction in energy efficiency of movements. Slowness was indicated by physical activity counts in the morning. Low physical activity level was measured by total energy expenditure using doubly labeled water divided by sleeping metabolic rate. Weakness, poor endurance, slowness, and low physical activity level were significantly higher in control compared with calorie restriction (p < .05) as was total incidence of frailty (p < .001). In conclusion, we established a novel set of measurable criteria of frailty in nonhuman primates, and using these criteria, showed that calorie restriction reduces the incidence of frailty and increases healthy life span in nonhuman primates.

In the cooperative breeding common marmoset monkey, Callithrix jacchus, fathers share the care responsibility and energetic load with their mate from the time their infants are born. However, not all fathers show the same level of participation in direct infant care. Here we present the first results demonstrating that fathering style can improve both survival and growth trajectory of a male's offspring during the first 30 weeks of life and that these infant outcomes are consistent within a father throughout successive births. Twenty-four marmoset fathers were tested for their responsiveness to an infant distress call when their infants were approximately two weeks old. These fathers were categorized as either responsive (RS) or nonresponsive (NRS) based on their response to the calls. Survival past 1 month was then determined and bi-monthly weights of current infants through 30 weeks of age were taken. Infant survival to the first month was significantly higher with RS fathers than with NRS fathers during this critical time period. Infants from RS fathers also had a higher growth trajectory with significant differences in body weight in the 28th and 30th week after birth. Only the RS fathers showed a significant increase in serum testosterone in response to infant cries suggesting a physiological role of testosterone in the motivation to search for the infant. Furthermore, all offspring born to RS fathers from subsequent births also showed a significantly higher survival rate and higher growth trajectory than for offspring of NRS fathers. These results suggest that fathering style is a consistent trait and responsive fathers improve infant survival rate and growth during the first 30 weeks. Such fathering style traits may be passed on to the male offspring due to environmental or genetic factors.

The human microbiome is composed of bacteria, archaea, viruses and eukaryotic microbes that reside in and on our bodies, the largest community of which is in the gut. Although the functions of the gut microbiota are not fully understood, they are known to play an essential role in immune, endocrine, and metabolic functions. To begin to understand the relationship between the gut microbiome, aging, and adult-onset, moderate calorie restriction in rhesus macaques (Macaca mulatta), we collected fecal samples at one timepoint from a total of 52 macaques for 16S rRNA gene analyses. Samples were taken from 20 males and 20 females across the natural macaque age range and from 6 males and 6 females enrolled in the long-term study of aging and calorie restriction at the Wisconsin National Primate Research Center. Preliminary data show that, like humans, NHPs exhibit a large interindividual variation in microbiota composition despite well-controlled environmental conditions.

Short-term (1 year) calorie restriction (CR) has been reported to decrease physical activity and metabolic rate in humans and non-human primate models; however, studies examining the very long-term (10 year) effect of CR on these parameters are lacking.The objective of this study was to examine metabolic and behavioral adaptations to long-term CR longitudinally in rhesus macaques.Eighteen (10 male, 8 female) control (C) and 24 (14 male, 10 female) age matched CR rhesus monkeys between 19.6 and 31.9 years old were examined after 13 and 18 years of moderate adult-onset CR. Energy expenditure (EE) was examined by doubly labeled water (DLW; TEE) and respiratory chamber (24 h EE). Physical activity was assessed both by metabolic equivalent (MET) in a respiratory chamber and by an accelerometer. Metabolic cost of movements during 24 h was also calculated. Age and fat-free mass were included as covariates.Adjusted total and 24 h EE were not different between C and CR. Sleeping metabolic rate was significantly lower, and physical activity level was higher in CR than in C independent from the CR-induced changes in body composition. The duration of physical activity above 1.6 METs was significantly higher in CR than in C, and CR had significantly higher accelerometer activity counts than C. Metabolic cost of movements during 24 h was significantly lower in CR than in C. The accelerometer activity counts were significantly decreased after seven years in C animals, but not in CR animals.The results suggest that long-term CR decreases basal metabolic rate, but maintains higher physical activity with lower metabolic cost of movements compared with C.

While moderate calorie restriction (CR) in the absence of malnutrition has been consistently shown to have a systemic, beneficial effect against aging in several animals models, its effect on the brain microstructure in a non-human primate model remains to be studied using post-mortem histopathologic techniques. In the present study, we investigated differences in expression levels of glial fibrillary acid protein (GFAP) and β-amyloid plaque load in the hippocampus and the adjacent cortical areas of 7 Control (ad libitum)-fed and 6 CR male rhesus macaques using immunostaining methods. CR monkeys expressed significantly lower levels (∼30% on average) of GFAP than Controls in the CA region of the hippocampus and entorhinal cortex, suggesting a protective effect of CR in limiting astrogliosis. These results recapitulate the neuroprotective effects of CR seen in shorter-lived animal models. There was a significant positive association between age and average amyloid plaque pathology in these animals, but there was no significant difference in amyloid plaque distribution between the two groups. Two of the seven Control animals (28.6%) and one of the six CR animal (16.7%) did not express any amyloid plaques, five of seven Controls (71.4%) and four of six CR animals (66.7%) expressed minimal to moderate amyloid pathology, and one of six CR animals (16.7%) expressed severe amyloid pathology. That CR affects levels of GFAP expression but not amyloid plaque load provides some insight into the means by which CR is beneficial at the microstructural level, potentially by offsetting the increased load of oxidatively damaged proteins, in this non-human primate model of aging. The present study is a preliminary post-mortem histological analysis of the effects of CR on brain health, and further studies using molecular and biochemical techniques are warranted to elucidate underlying mechanisms.

Higher systemic levels of the proinflammatory cytokine interleukin-6 (IL-6) were found to be associated with lower gray matter volume and tissue density in old rhesus macaques. This association between IL-6, and these brain indices were attenuated by long-term 30 % calorie restriction (CR). To extend these findings, the current analysis determined if a CR diet in 27 aged rhesus monkeys compared to 17 normally fed controls reduced circulating levels of another proinflammatory cytokine, interleukin-8 (IL-8), and raised levels of anti-inflammatory interleukin-10 (IL-10). Further, these cytokines were regressed onto imaged brain volume and microstructure using voxel-wise regression analyses. CR significantly lowered IL-8 and raised IL-10 levels. Across the two dietary conditions, higher IL-8 predicted smaller gray matter volumes in bilateral hippocampus. Higher IL-10 was associated with more white matter volume in visual areas and tracts. Consuming a CR diet reduced the association between systemic IL-8 and hippocampal volumes. Conversely, CR strengthened associations between IL-10 and microstructural tissue density in the prefrontal cortex and other areas, particularly in a region of dorsal prefrontal cortex, which concurred with our prior findings for IL-6. Consumption of a CR diet lowered proinflammatory and increased anti-inflammatory cytokine concentrations, which lessened the statistical association between systemic inflammation and the age-related alterations in important brain regions, including the hippocampus.

Metabolic assessment of a non-human primate model of metabolic syndrome and obesity requires the necessary biomarkers specific to the species. While the rhesus monkey has a number of specific assays for assessing metabolic syndrome, the marmoset does not. Furthermore, the common marmoset (Callithrix jacchus) has a small blood volume that necessitates using a single blood volume for multiple analyses. The common marmoset holds a great potential as an alternative primate model for the study of human disease but assay methods need to be developed and validated for the biomarkers of metabolic syndrome. Here we report on the adaptation, development, and validation of commercially available immunoassays for common marmoset samples in small volumes. We have performed biological validations for insulin, adiponectin, leptin, and ghrelin to demonstrate the use of these biomarkers in examining metabolic syndrome and other related diseases in the common marmoset. Am. J. Primatol. 75:500-508, 2013. © 2013 Wiley Periodicals, Inc.

The process of angiogenesis, new capillary formation from existing blood vessels, is critical for maintenance of the microvasculature and cardiovascular homeostasis. Previous studies in laboratory rodents demonstrate that aging is associated with a progressive deterioration of microvascular homeostasis due to age-related impairment of angiogenic processes (1–5). We and others have proposed that these changes have a key role in age-related microvascular rarefaction (6), decreasing tissue blood supply, impairing adaptation to hypoxia (7–9), and suppressing wound healing. Age-related decreases in microvascular density in the heart are thought to contribute to a severe impairment in cardiac pump function during aging leading to an increased occurrence of ventricular failure (10). Age-related impairment in endothelial angiogenic capacity and decreases in cerebromicrovascular density in the hippocampus and the cingulate, retrosplenial, and motor cortex have also been causally linked to a decline of local cerebral blood flow (11) and an age-related impairment of higher brain functions, including cognitive decline (12). These results are consistent with findings in elderly patients demonstrating that age-related microvascular alterations and impairment of regional cerebral blood flow contribute to the development of mild cognitive impairment in humans (13,14). Moderate caloric restriction (CR) without malnutrition has been shown to slow the rate of aging and increase life span and/or healthspan in most species studied, including invertebrate model organisms (15), laboratory rodents, and nonhuman primates (16–27). In laboratory rodents, moderate CR was shown to exert significant microvascular protective effects (28,29) increasing microvascular density in the heart (30) and the brain (11,12). Furthermore, explanted aorta segments isolated from rats maintained on an every other day feeding regimen, compared with control vessels, showed increased endothelial sprouting in collagen matrix (31). CR was also shown to promote revascularization in response to hindlimb ischemia in mice (32) and increase angiogenic activity in the rabbit testis (33). The aforementioned findings suggest that CR exerts beneficial effects on microvascular health (29), but the underlying mechanisms by which CR regulates angiogenic processes and confers endothelial protection remain elusive. CR is associated with changes in the level of neuroendocrine mediators present in the circulation, which readily reach microvascular endothelial cells and elicit a variety of cytoprotective responses (29,34). CR-induced changes in circulating factors were also shown to be responsible for activation of evolutionarily conserved survival pathways in rats and mice (35,36). Important to the present discussion are our previous observations that in vitro treatment of cultured endothelial cells with sera from CR-fed rats mimics many of the vascular effects observed in vivo during CR in rodents (28). These studies support a key role of neuroendocrine factors in CR-mediated endothelial protection. However, no studies have investigated whether CR-induced changes in circulating factors mediate proangiogenic effects of CR. Furthermore, despite the rapid progress of aging research in the last few years (37–73), there are no studies to our knowledge investigating the role of CR-induced changes in circulating factors in endothelial protection in primates. The present study was designed to elucidate the role of circulating factors induced by CR in the nonhuman primate Macaca mulatta in modulation of endothelial angiogenic capacity. Using cultured human coronary arterial endothelial cells (CAECs) as a model system, we tested the hypothesis that factors present in sera derived from CR monkeys stimulate angiogenic processes (including cell proliferation, adhesion, migration, and formation of capillary-like structures) through (a) an increase in NO bioavailability, (b) activation of Nrf2-dependent pathways, and/or (c) upregulation of vascular endothelial growth factor (VEGF) signaling.