Mass Spectrometry-Based Multiomics Identifies Metabolic Signatures of Sarcopenia in Rhesus Monkey Skeletal Muscle.

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in "omics" technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offers new insights into the molecular mechanisms underlying sarcopenia for the evaluation and monitoring of a therapeutic treatment of sarcopenia.

Cognitive development from infancy to young adulthood in common marmosets (Callithrix jacchus): Effect of age, sex, and hormones on learning and affective state.

Studies looking at individual variability in cognition have increased in recent years. We followed 43 marmosets (21 males, 22 females) from infancy to young adulthood. At 3-months old, marmosets were trained to touch a rewarded stimulus. At 9-, 15-, and 21-months old, they were given visual discrimination and cognitive bias tests, and urine samples were collected to examine hormone levels. Marmosets were significantly more successful learners at 15 months than 9 months. Individuals who were more successful learners at 9 months were also more successful at 15 months, with more male learners than expected at 15 months. At 9 months, learning success was associated with higher cortisol levels. At 15 months, males with higher estradiol levels were more successful learners, whereas at 21 months, females with higher estradiol and cortisol levels tended to be less successful learners and more pessimistic. Nine months, therefore, appears to be an important developmental timepoint for acquiring cognitive control, which has developed by 15 months. Steroids may have differential effects on each sex, with complex interactions between gonadal and adrenal hormones having an influence on cognitive function over the lifespan. This longitudinal study offers new insight into cognition, including its development and biological underpinnings.

Motivational increase of androgens and behavior by infant distress calls in highly responsive common marmoset fathers, Callithrix jacchus.

Common marmoset fathers are highly involved in care of their infants. However, variability exists in their response to infant behavior even in paternally experienced fathers. Using infant distress cries as a motivation test, we investigated: 1. the differences in paternally experienced fathers' motivation to search for the infant vocalization stimuli; 2. the relationship between a father's motivation to search for the source of the infant cries and testosterone levels; and 3. if there is a rapid steroidogenesis pathway leading to increased testosterone and estradiol in the peripheral circulation. Only 44% of the paternally experienced fathers showed a high frequency of searching for the source of the infant distress cries. Through the use of multisteroid analysis, we found high responsive fathers had significantly higher levels of progesterone and testosterone in response to infant distress cries compared to a control stimulus with progesterone and androstenedione correlating with testosterone, while no differences were seen in low responders. The frequency to search for the infant stimuli was positively correlated with higher testosterone compared to control vocal levels. These results suggest that searching for the source of infant cries represents a motivation behavior for fathers that is activated by testosterone and reflects rapid circulating testosterone.

Improving rigor and reproducibility in nonhuman primate research.

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.

Early learning in the common marmoset (Callithrix jacchus): Behavior in the family group is related to preadolescent cognitive performance.

Early environment can have a major impact on development, with family life known to play an important role. Longitudinal studies can therefore help increase our understanding of variance in cognitive abilities in young animals, as well as over time. We followed 22 marmosets (11 male and 11 female) from infancy through to early adolescence. At 3 months old, the marmosets were trained to reliably touch a rewarded stimulus. At 5 months, behavior was observed within the natal group. At 9 months, the marmosets were given a visual discrimination task to assess learning ability. Mann-Whitney U tests found no sex or family size differences in number of errors at 3 or 9 months. While no significant relationships were found between behavior in the family and learning at 3 months, significant negative correlations were found between duration spent in locomotion and learning errors (p = .05), as well as between frequency of calm vocalizations and learning errors (p = .001) at 9 months. A U-shape curve was found between amount of social play and learning at 9 months. Positive family interactions, including moderate amounts of play, as well as calm individual behavior, may therefore be important in learning. This study sheds light on cognitive development in much younger marmosets than previously studied, and helps increase understanding of how individual differences in learning may arise.

Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset).

Vitamin D3 (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D3 due to lack of sunlight exposure. The minimum dietary vitamin D3 requirement and the maximum amount of vitamin D3 that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D3 provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D3 (25(OH)D3 ), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D3 in captive marmosets, but 25(OH)D3 is not the final product of vitamin D3 metabolism. In addition to serum 25(OH)D3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), and the less well understood metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2 D3 ) to characterize the marmoset's ability to metabolize dietary vitamin D3 . We present vitamin D3 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 D3 per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D3 (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)2 D3 (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)2 D3 between the colonies. Serum 1,25(OH)2 D3 increased and 25(OH)D3 decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D3 into the active metabolite 1,25(OH)2 D3 ; excess 25(OH)D3 is metabolized into 24,25(OH)2 D3 . This ability explains the tolerance of high levels of dietary vitamin D3 by marmosets, however, our data suggest that these high dietary levels are not required.

Ovarian estradiol supports sexual behavior but not energy homeostasis in female marmoset monkeys.

OBJECTIVE: 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. METHODS: 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). RESULTS: 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 < 0.001) for PGR in E2-treated monkeys versus OVX controls regardless of diet, but no differences between groups in other selected metabolic genes. In contrast, regardless of E2 treatment, there was a decreased mRNA expression of PGC1α (PPARGC1A), HTR1A, and HTR5A in HFD compared with LFD females. CONCLUSIONS: Our findings, overall, document a greatly diminished role for ovarian E2 in the metabolic physiology of a female primate, and encourage consideration that primates, including humans, evolved metabolic control systems regulated by extra-ovarian E2 or are generally less subject to E2 regulation.

Maintenance of bone mass despite estrogen depletion in female common marmoset monkeys (Callithrix jacchus).

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.

Non-human primates as a model for aging.

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".

Syndecan-1 is required to maintain intradermal fat and prevent cold stress.

Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38α in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation in vitro. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARγ agonist rosiglitazone rescues Sdc1-/- intradermal adipose tissue, placing PPARγ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.

Plasma diacylglycerol composition is a biomarker of metabolic syndrome onset in rhesus monkeys.

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.

A calorie-restricted diet decreases brain iron accumulation and preserves motor performance in old rhesus monkeys.

Caloric restriction (CR) reduces the pathological effects of aging and extends the lifespan in many species, including nonhuman primates, although the effect on the brain is less well characterized. We used two common indicators of aging, motor performance speed and brain iron deposition measured in vivo using magnetic resonance imaging, to determine the potential effect of CR on elderly rhesus macaques eating restricted (n=24, 13 males, 11 females) and standard (n=17, 8 males, 9 females) diets. Both the CR and control monkeys showed age-related increases in iron concentrations in globus pallidus (GP) and substantia nigra (SN), although the CR group had significantly less iron deposition in the GP, SN, red nucleus, and temporal cortex. A Diet X Age interaction revealed that CR modified age-related brain changes, evidenced as attenuation in the rate of iron accumulation in basal ganglia and parietal, temporal, and perirhinal cortex. Additionally, control monkeys had significantly slower fine motor performance on the Movement Assessment Panel, which was negatively correlated with iron accumulation in left SN and parietal lobe, although CR animals did not show this relationship. Our observations suggest that the CR-induced benefit of reduced iron deposition and preserved motor function may indicate neural protection similar to effects described previously in aging rodent and primate species.

Development of metabolic function biomarkers in the common marmoset, Callithrix jacchus.

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.

Detrusor underactivity is associated with metabolic syndrome in aged primates.

Lower urinary tract (LUT) dysfunction is prevalent in the elderly population, and clinical manifestations include urinary retention, incontinence, and recurrent urinary tract infections. Age-associated LUT dysfunction is responsible for significant morbidity, compromised quality of life, and rising healthcare costs in older adults, but its pathophysiology is not well understood. We aimed to investigate the effects of aging on LUT function by urodynamic studies and metabolic markers in non-human primates. Adult (n = 27) and aged (n = 20) female rhesus macaques were evaluated by urodynamic and metabolic studies. Cystometry showed detrusor underactivity (DU) with increased bladder capacity and compliance in aged subjects. Metabolic syndrome indicators were present in the aged subjects, including increased weight, triglycerides, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and high sensitivity C-reactive protein (hsCRP), whereas aspartate aminotransferase (AST) was unaffected and the AST/ALT ratio reduced. Principal component analysis and paired correlations showed a strong association between DU and metabolic syndrome markers in aged primates with DU but not in aged primates without DU. The findings were unaffected by prior pregnancies, parity, and menopause. Our findings provide insights into possible mechanisms for age-associated DU and may guide new strategies to prevent and treat LUT dysfunction in older adults.

Mass spectrometry-based multi-omics identifies metabolic signatures of sarcopenia in rhesus monkey skeletal muscle.

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in omics technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified numerous proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified multiple metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offer new insights into the molecular mechanism underlying sarcopenia for the evaluation and monitoring of therapeutic treatment of sarcopenia.

Evaluating depression- and anxiety-like behaviors in non-human primates.

Depression and anxiety are some of the most prevalent and debilitating mental health conditions in humans. They can present on their own or as co-morbidities with other disorders. Like humans, non-human primates (NHPs) can develop depression- and anxiety-like signs. Here, we first define human depression and anxiety, examine equivalent species-specific behaviors in NHPs, and consider models and current methods to identify and evaluate these behaviors. We also discuss knowledge gaps, as well as the importance of evaluating the co-occurrence of depression- and anxiety-like behaviors in animal models of human disease. Lastly, we consider ethical challenges in depression and anxiety research on NHPs in order to ultimately advance the understanding and the personalized treatment of these disorders.

Aromatase Inhibition Eliminates Sexual Receptivity Without Enhancing Weight Gain in Ovariectomized Marmoset Monkeys.

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.

Development and validation of an LC-MS/MS based quantitative assay for marmoset insulin in serum.

Insulin is a peptide hormone that is secreted by the ß 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 (13C, 15N) 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.

Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice.

The loss of skeletal muscle function with age, known as sarcopenia, significantly reduces independence and quality of life and can have significant metabolic consequences. Although exercise is effective in treating sarcopenia it is not always a viable option clinically, and currently, there are no pharmacological therapeutic interventions for sarcopenia. Here, we show that chronic treatment with pan-adiponectin receptor agonist AdipoRon improved muscle function in male mice by a mechanism linked to skeletal muscle metabolism and tissue remodeling. In aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle functional measures in vivo and ex vivo. Improvements were linked to changes in fiber type, including an enrichment of oxidative fibers, and an increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon treatment improved contractile force and activated the energy-sensing kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome proliferator-activated receptor gamma coactivator one alpha). In cultured cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated with increased mitochondrial membrane potential, reorganization of mitochondrial architecture, increased respiration, and increased ATP production. Furthermore, the ability of AdipoRon to stimulate AMPK and PGC1a was conserved in nonhuman primate cultured cells. These data show that AdipoRon is an effective agent for the prevention of sarcopenia in mice and indicate that its effects translate to primates, suggesting it may also be a suitable therapeutic for sarcopenia in clinical application.

Ceramides are early responders in metabolic syndrome development in rhesus monkeys.

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.