Experimentally Induced Hyperinsulinemia Fails to Induce Polycystic Ovary Syndrome-like Traits in Female Rhesus Macaques.

As in women with polycystic ovary syndrome (PCOS), hyperinsulinemia is associated with anovulation in PCOS-like female rhesus monkeys. Insulin sensitizers ameliorate hyperinsulinemia and stimulate ovulatory menstrual cycles in PCOS-like monkeys. To determine whether hyperinsulinemia (>694 pmol/L), alone, induces PCOS-like traits, five PCOS-like female rhesus monkeys with minimal PCOS-like traits, and four control females of similar mid-to-late reproductive years and body mass index, received daily subcutaneous injections of recombinant human insulin or diluent for 6−7 months. A cross-over experimental design enabled use of the same monkeys in each treatment phase. Insulin treatment unexpectedly normalized follicular phase duration in PCOS-like, but not control, females. In response to an intramuscular injection of 200 IU hCG, neither prenatally androgenized nor control females demonstrated ovarian hyperandrogenic responses while receiving insulin. An intravenous GnRH (100 ng/kg) injection also did not reveal evidence of hypergonadotropism. Taken together, these results suggest that experimentally induced adult hyperinsulinemia, alone, is insufficient to induce PCOS-like traits in female rhesus monkeys and to amplify intrinsic PCOS-like pathophysiology.

Measurement of 25-hydroxyvitamin D(2&3) and 1,25-dihydroxyvitamin D(2&3) by tandem mass spectrometry: A primate multispecies comparison.

Vitamin D metabolites are widely studied for their roles in bone health, immune functions, and other potential physiologic roles in humans. However, the optimal blood levels of vitamin D metabolites are still unclear. Various methods for measuring vitamin D metabolites have been used and recently liquid chromatography tandem mass spectroscopy (LC-MS/MS) has been adopted as the gold standard for vitamin D metabolite measurement. Here, we report the use of LC-MS/MS to measure 25-hydroxyvitamin D (25(OH)D(2&3)), and 1,25-dihydroxyvitamin D (1,25(OH)2D(2&3)), in three laboratory nonhuman primate species: common marmoset (Callithrix jacchus), rhesus macaque (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis), and compare them to humans using the same technique. The nonhuman primates showed blood levels for 25(OH)D3 and 1,25(OH)2D3 significantly higher than human values with marmosets having the highest levels. Marmoset samples showed significantly more variability among individuals than those from macaques for both metabolites, but all three nonhuman primate species exhibited large variation within species for both 25(OH)D(2&3) and 1,25(OH)2D(2&3). Marmoset females had significantly lower values than the males for 25(OH)D3, while rhesus males showed a significant decrease in 25(OH)D3 with age. The most striking finding is the variation within species for vitamin D levels even in laboratory primates that have a controlled diet, UV exposure, and in some cases, genetic constraints. Similar variation in 25(OH)D responses to a fixed dose of oral vitamin D supplementation has been reported in humans. We suggest that these species can provide primate models for examining the factors influencing variation in the levels of vitamin D necessary for human and nonhuman primate health.

The PPAR-γ agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys.

BACKGROUND: Activation of the peroxisome proliferator-activated receptor gamma (PPAR-γ) has been proposed as a possible neuroprotective strategy to slow down the progression of early Parkinson's disease (PD). Here we report preclinical data on the use of the PPAR-γ agonist pioglitazone (Actos®; Takeda Pharmaceuticals Ltd.) in a paradigm resembling early PD in nonhuman primates. METHODS: Rhesus monkeys that were trained to perform a battery of behavioral tests received a single intracarotid arterial injection of 20 ml of saline containing 3 mg of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Twenty-four hours later the monkeys were assessed using a clinical rating scale, matched accordingly to disability, randomly assigned to one of three groups [placebo (n = 5), 2.5 (n = 6) or 5 (n = 5) mg/kg of pioglitazone] and their treatments started. Three months after daily oral dosing, the animals were necropsied. RESULTS: We observed significant improvements in clinical rating score (P = 0.02) in the animals treated with 5 mg/kg compared to placebo. Behavioral recovery was associated with preservation of nigrostriatal dopaminergic markers, observed as higher tyrosine hydroxylase (TH) putaminal optical density (P = 0.011), higher stereological cell counts of TH-ir (P = 0.02) and vesicular monoamine transporter-2 (VMAT-2)-ir nigral neurons (P = 0.006). Stereological cell counts of Nissl stained nigral neurons confirmed neuroprotection (P = 0.017). Pioglitazone-treated monkeys also showed a dose-dependent modulation of CD68-ir inflammatory cells, that was significantly decreased for 5 mg/kg treated animals compared to placebo (P = 0.018). A separate experiment to assess CSF penetration of pioglitazone revealed that 5 mg/kg p.o. induced consistently higher levels than 2.5 mg/kg and 7.5 mg/kg. p.o. CONCLUSIONS: Our results indicate that oral administration of pioglitazone is neuroprotective when administered early after inducing a parkinsonian syndrome in rhesus monkeys and supports the concept that PPAR-γ is a viable target against neurodegeneration.

Neuropeptide S receptor 1 is a nonhormonal treatment target in endometriosis.

Endometriosis is a common chronic inflammatory condition causing pelvic pain and infertility in women, with limited treatment options and 50% heritability. We leveraged genetic analyses in two species with spontaneous endometriosis, humans and the rhesus macaque, to uncover treatment targets. We sequenced DNA from 32 human families contributing to a genetic linkage signal on chromosome 7p13-15 and observed significant overrepresentation of predicted deleterious low-frequency coding variants in NPSR1, the gene encoding neuropeptide S receptor 1, in cases (predominantly stage III/IV) versus controls (P = 7.8 × 10-4). Significant linkage to the region orthologous to human 7p13-15 was replicated in a pedigree of 849 rhesus macaques (P = 0.0095). Targeted association analyses in 3194 surgically confirmed, unrelated cases and 7060 controls revealed that a common insertion/deletion variant, rs142885915, was significantly associated with stage III/IV endometriosis (P = 5.2 × 10-5; odds ratio, 1.23; 95% CI, 1.09 to 1.39). Immunohistochemistry, qRT-PCR, and flow cytometry experiments demonstrated that NPSR1 was expressed in glandular epithelium from eutopic and ectopic endometrium, and on monocytes in peritoneal fluid. The NPSR1 inhibitor SHA 68R blocked NPSR1-mediated signaling, proinflammatory TNF-α release, and monocyte chemotaxis in vitro (P < 0.01), and led to a significant reduction of inflammatory cell infiltrate and abdominal pain (P < 0.05) in a mouse model of peritoneal inflammation as well as in a mouse model of endometriosis. We conclude that the NPSR1/NPS system is a genetically validated, nonhormonal target for the treatment of endometriosis with likely increased relevance to stage III/IV disease.

Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility.

The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil.

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

An Emergent Integrated Aging Process Conserved Across Primates.

Aging is a complex process emerging from integrated physiological networks. Recent work using principal component analysis (PCA) of multisystem biomarkers proposed a novel fundamental physiological process, "integrated albunemia," which was consistent across human populations and more strongly associated with age and mortality risk than individual biomarkers. Here we tested for integrated albunemia and associations with age and mortality across six diverse nonhuman primate species and humans. PCA of 13 physiological biomarkers recovered in all species a primary axis of variation (PC1) resembling integrated albunemia, which increased with age in all but one species but was less predictive of mortality risk. Within species, PC1 scores were often reliably recovered with a minimal biomarker subset and usually stable between sexes. Even among species, correlations in PC1 structure were often strong, but the effect of phylogeny was inconclusive. Thus, integrated albunemia likely reflects an evolutionarily conserved process across primates and appears to be generally associated with aging but not necessarily with negative impacts on survival. Integrated albunemia is unlikely to be the only conserved emergent physiological process; our findings hence have implications both for the evolution of the aging process and of physiological networks more generally.

Conservation of physiological dysregulation signatures of aging across primates.

Two major goals in the current biology of aging are to identify general mechanisms underlying the aging process and to explain species differences in aging. Recent research in humans suggests that one important driver of aging is dysregulation, the progressive loss of homeostasis in complex biological networks. Yet, there is a lack of comparative data for this hypothesis, and we do not know whether dysregulation is widely associated with aging or how well signals of homeostasis are conserved. To address this knowledge gap, we use unusually detailed longitudinal biomarker data from 10 species of nonhuman primates housed in research centers and data from two human populations to test the hypotheses that (a) greater dysregulation is associated with aging across primates and (b) physiological states characterizing homeostasis are conserved across primates to degrees associated with phylogenetic proximity. To evaluate dysregulation, we employed a multivariate distance measure, calculated from sets of biomarkers, that is associated with aging and mortality in human populations. Dysregulation scores positively correlated with age and risk of mortality in most nonhuman primates studied, and signals of homeostatic state were significantly conserved across species, declining with phylogenetic distance. Our study provides the first broad demonstration of physiological dysregulation associated with aging and mortality risk in multiple nonhuman primates. Our results also imply that emergent signals of homeostasis are evolutionarily conserved, although with notable variation among species, and suggest promising directions for future comparative studies on dysregulation and the aging process.

Caloric Restriction and Healthy Life Span: Frail Phenotype of Nonhuman Primates in the Wisconsin National Primate Research Center Caloric Restriction Study.

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.

Metabolizable energy intake during long-term calorie restriction in rhesus monkeys.

Calorie restriction (CR) is a dietary intervention shown to increase maximum life-span. The aim of this study was to compare the metabolizable energy of the pelleted semi-purified diet with estimated energy intake from food weight. Energy density of diet, urine and feces were measured by bomb calorimetry in rhesus monkeys (23-29 years old) on CR (CR, n=11) and control (C, n=9). Food moisture was measured to be 2-fold higher (9+/-1%) than indicated on the label (approximately 5%). The measured gross energy of diet was 4.4 kcal/g dry weight of CR and 4.5 kcal/g dry weight of C diets. In a two-day trial, food intake (mean+/-SD) was 112+/-20 g and 136+/-26 g of dry mass/d in the CR and C monkeys, respectively (p=0.003). The fraction of the diet absorbed (CR=0.91; C=0.95) was different (p<0.001) between CR and C monkeys. Using these coefficients, the metabolizable energy intake averaged over 6 months was 450+/-53 and 534+/-97 kcal/d in CR and C monkeys, respectively (Diff=16%; p=0.03). These values were compared with energy expenditure (EE), as measured annually by indirect calorimetry (490+/-61 kcal/d in CR and 532+/-62 kcal/d in C monkeys). Adjusted for changes in body composition (2+/-10 kcal/d in CR and -7+/-12 kcal/d in C), energy balance was not different from zero in CR (-42+/-42 kcal/d) and C (9+/-61 kcal/d) monkeys. Use of diet weight is a reasonable estimate of the level of CR when food waste is assessed.

Pioglitazone improves insulin action and normalizes menstrual cycles in a majority of prenatally androgenized female rhesus monkeys.

PURPOSE OF THE STUDY: To determine whether pioglitazone will improve menstrual cyclicity in a fetal programming model for polycystic ovary syndrome. BASIC PROCEDURES: Eight prenatally androgenized (PA) and 5 control female rhesus monkeys of similar age, body weight and body mass index received an oral placebo daily for 6-7 months followed, after at least 90 days, by daily oral dosing with pioglitazone (3mg/kg) for an additional 6-7 months. Blood was sampled thrice weekly to monitor ovulatory function, and a variety of endocrine challenges were performed to quantify changes in ovarian, gonadotropin and glucoregulatory function. MOST IMPORTANT FINDINGS: Pioglitazone normalized menstrual cycles in 5 out of 8 (62%) PA females (pioglitazone responsive; Pio(RESP)). Pioglitazone increased serum 17alpha-hydroxyprogesterone responses to an hCG injection in Pio(RESP) PA females, while diminishing serum progesterone, and increasing DHEA and estradiol responses to hCG in Pio(RESP) PA and all normal females. PRINCIPAL CONCLUSIONS: Insulin resistance plays a mechanistic role in maintaining anovulation in a majority of PA female monkeys.

Caloric restriction improves health and survival of rhesus monkeys.

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.

Reference body composition in adult rhesus monkeys: glucoregulatory and anthropometric indices.

Rhesus monkeys have been used as models to study obesity and disease. The aim of this study was to define body mass indices for underweight and obesity in rhesus monkeys. Longitudinal data collected over 8-14 years from 40 male and 26 female rhesus monkeys were analyzed. Body weight, insulin sensitivity index, and disposition index were regressed against percent body fat (%BF). A minimal %BF beyond which further loss of body weight resulted in loss of lean mass was determined to be 11.5% in older males, 8% in adult females, and 9% in younger adult males. Insulin sensitivity index and disposition index reached minimum values at 23% fat in older males, 18% in adult females, and 21% in younger adult males, indicating obesity. The estimated reference range for %BF was 9%-23% in male and 8%-18% in female monkeys, corresponding to body mass indices of 32-44 kg/m(2) for male and 27-35 kg/m(2) for female monkeys.

Food intake in rhesus monkeys following central administration of orexins.

Orexin A has been reported to stimulate food intake in rats while orexin B does not. The purpose of this study was to determine the role of orexin A or orexin B administration on food intake in adult, male rhesus monkeys. Food intake was measured at 2 and 8 h after the morning feeding following central injections of vehicle, orexin A (10, or 20 microg) or orexin B (10, 30, or 100 microg). When compared to vehicle injections, the 10 and 20 microg doses of orexin A decreased food intake at 2 h post-dose by 45% and 64%, respectively. Eight-hour food intake was decreased at only the 20 microg orexin A dose. Orexin B at all doses and time points did not alter food intake when compared to vehicle. These results indicate that orexin A exhibits anorectic activity while orexin B does not affect food intake in the rhesus monkey.

Hyperinsulinemia/diabetes, hearing, and aging in the University of Wisconsin calorie restriction monkeys.

The purpose of this study was to determine the effects of hyperinsulinemia/Type 2 diabetes mellitus (HI-T2DM) on hearing impairment using rhesus monkeys to obtain control over diet and lifestyle factors that confound human studies. The study is a retrospective evaluation of rhesus monkeys from the Wisconsin National Primate Research Center (WNPRC) study on caloric restriction and aging. The research questions were the following: 1. Is HI-T2DM related to hearing impairment? 2. If so, what is the site of lesion in the auditory system? and 3. What physiological factors affect the risk of hearing loss in HI-T2DM? Three groups of eight monkeys each were matched by sex and age; the caloric restricted (CR) monkeys had a reduced risk of diabetes, the normal control (NL) group had a normal risk, and the hyperinsulinemia/diabetes (HI-D) group had already developed HI-T2DM. Auditory testing included distortion product otoacoustic emissions (DPOAEs) with f2 frequencies from 2211 to 8837 Hz and auditory brainstem responses (ABRs) obtained with clicks and tone bursts (8, 16, and 32 kHz). DPOAEs had signal-to-noise ratios 8-17 dB larger in the NL group than in the HI-D and CR groups, signifying that cochlear function was best in the NL group. ABR thresholds were 5-8 dB better in the NL group than in the HI-D group, although no significant differences across the groups were evident for the thresholds, latencies, interwave intervals, or amplitudes. Correlations were significant for quadratic relations between body mass index (BMI) and DPOAE, with largest DPOAEs for animals in the middle of the BMI range. ABR thresholds elicited with 16 and 32 kHz signals were significantly correlated, positively with BMI and HbA1c, and negatively with KG (glucose tolerance), SI (insulin sensitivity index) and DI (disposition index). These findings suggest that the hearing loss associated with HI-T2DM is predominantly cochlear, and auditory structures underlying the higher frequencies are at risk with HI-T2DM. Loss of auditory function begins in the hyperinsulinemia, pre-diabetic state.

Insulin resistance and impaired insulin secretion in prenatally androgenized male rhesus monkeys.

Polycystic ovary syndrome (PCOS) is a familial disease. Affected males harbor some of the metabolic deficits seen in affected females. The prenatally androgenized (PA) female rhesus monkey, an animal model for PCOS, manifests glucoregulatory and reproductive abnormalities similar to those seen in PCOS women. The purpose of this study was to determine whether exposure of fetal male rhesus monkeys to testosterone excess would induce glucoregulatory and reproductive deficits. Seven adult PA males and seven matched controls underwent somatometric measurements, sex steroid analysis, and a frequently sampled i.v. glucose tolerance test. Body measurements were similar in the two groups, although arm circumference was greater in control compared with PA males (P < 0.01). There were no differences in neonatal weight or serum levels of sex steroids between the two male groups. Measures of insulin sensitivity and pancreatic beta-cell compensation (disposition index) were clearly diminished in PA compared with control males [insulin sensitivity: PA, mean 0.8 (95% confidence interval, 0.11, 5.82); controls, 3.06 (1.51, 6.19) x 10(-4)/min/microU/ml; P < 0.05; disposition index: PA, 226.38 (69.54, 383.22); controls, 509.21/min (306.52, 711.89); P < 0.02]. PA males do not exhibit elevated androgens during adulthood, suggesting that insulin resistance and impaired pancreatic beta-cell function may result from fetal reprogramming of key metabolic tissues.

Dietary restriction and beta-cell sensitivity to glucose in adult male rhesus monkeys.

We examined the effects of dietary restriction (DR) and age on ss-cell function and peripheral insulin sensitivity in rhesus monkeys. A semipurified diet was provided either ad libitum for approximately 8 hours/day to controls (C) or as approximately 70% of baseline intake to restricted (R) animals for 10 years. The minimal model of C-peptide secretion and kinetics and the labeled 2-compartment minimal model of glucose kinetics were identified using plasma glucose, C-peptide, and insulin concentrations during an intravenous glucose tolerance test. R monkeys had less body fat, lower basal ss-cell sensitivity to glucose (Ø(b)), greater insulin sensitivity, and lower first-phase plasma insulin response. DR did not significantly affect first-phase and second-phase ss-cell sensitivity to glucose. Indices of body fatness were highly predictive of the effect of DR on Ø(b), fasting insulin concentration and insulin responses to glucose. Enhanced peripheral insulin sensitivity among R monkeys was strongly correlated with lower Ø(b).

Effects of caloric restriction and aging on the auditory function of rhesus monkeys (Macaca mulatta): The University of Wisconsin Study.

The present study is part of a larger project that investigates the effect of caloric restriction on longevity in the rhesus monkey. The purpose of the present study was to document presbycusis and the effect of caloric restriction on presbycusis in monkeys. The control group had 35 monkeys allowed to eat freely and the caloric-restricted group (CR) had 33 monkeys with a 30% reduction in caloric intake. Monaural and binaural auditory brainstem response (ABR) and middle latency response (MLR) were obtained from 27 female and 41 male monkeys that were 11-23 years of age and had been in the study for 102, 42, or 36 months when tested. Significant findings were the following: (1) wave I amplitudes were larger for females and for younger monkeys, and amplitudes decreased in aging males but not in aging females; (2) wave IV amplitudes were larger for females than males, and amplitudes for CR females were larger than for female controls, whereas the amplitudes from control and CR males were not different; (3) wave Pa latencies were shorter for females, and shorter latencies were maintained for aging females but not for aging males; (4) interwave interval IV-Pa was shorter for females, and intervals lengthened for aging males but not aging females; (5) binaural wave IV amplitude decreased faster with age for control monkeys than for CR monkeys, and the L+R Pa amplitude decreased with age. Additional trends were identified for longitudinal monitoring as monkeys enter old age.

Sex hormones, insulin sensitivity, and diabetes mellitus.

Sex differences and the role of gonadal hormones in modulating insulin sensitivity and glucose tolerance are of increasing interest and importance because of the increasing prevalence of type 2 diabetes mellitus and the metabolic abnormalities associated with aging. Body composition is closely associated with insulin sensitivity, and increased body fat, particularly in the visceral compartment, is a risk factor for developing type 2 diabetes mellitus. Sex differences in body composition and/or insulin sensitivity are evident in humans throughout the lifespan. Ovarian hormones influence insulin sensitivity across the menstrual cycle, during pregnancy, and in the menopausal transition. Similarly, estrogens and progestins used for contraception and hormone replacement therapy affect glucoregulation. Nonhuman primates and humans have similar life histories and reproductive characteristics. As a result, nonhuman primates provide a valuable model for investigating factors related to insulin sensitivity. Studies of nonhuman primates have contributed significantly to our understanding of sex differences and the influence of sex steroids in this context. This brief review surveys present knowledge of the sex differences in body composition, insulin sensitivity, and risk for development of type 2 diabetes mellitus derived from studies in humans and nonhuman primates. The influences of endogenous and exogenous gonadal steroids are emphasized.

Assessment of glucoregulation in rhesus monkeys sedated with ketamine.

Glucose, insulin, and cortisol were measured in the blood of rhesus monkeys under several conditions. Glucose and insulin concentrations were higher and cortisol lower shortly after the morning meal than after an overnight fast. Ketamine had no detectable effect on basal (fasting) glucose or insulin values in normal monkeys, even when sedation was maintained for more than 2 h. Ketamine did not appear to affect the response to a glucose load or to insulininduced hypoglycemia. It is concluded that ketamine is a useful agent for pharmacological restraint of rhesus monkeys during studies of glucoregulation.