Brief Communication: Histological Assessment of Nonhuman Primate Brown Adipose Tissue Highlights the Importance of Sympathetic Innervation

Objective The objective of this study was to functionally analyze the correlation of key histological features in brown adipose tissue (BAT) with clinical metabolic traits in nonhuman primates. Methods Axillary adipose tissue biopsies were collected from a metabolically diverse nonhuman primate cohort with clinical metabolism-related data. Expression of tyrosine hydroxylase (TH), uncoupling protein 1 (UCP1), cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX IV), beta-3 adrenergic receptor (β3-AR), and adipose cell size were quantified by immunohistochemical analysis. Computed tomography scans were performed to assess body composition. Results Tyrosine hydroxylase was negatively correlated with whole body fat mass as a percentage of body weight (p = 0.004) and was positively correlated with the density of UCP1 (p = 0.02), COX IV (p = 0.006), CD31 (p = 0.007), and cell density (p = 0.02) of the BAT samples. Beta-3 adrenergic receptor abundance had a weak positive correlation with COX IV (p = 0.04) in BAT but did not significantly correlate to UCP1 or TH expression in BAT. Conclusions Our findings highlight that there is a disparity in innervation provided to BAT based on body composition, as seen with the negative association between TH, a marker for innervation, and adiposity. These findings also support the importance of innervation in the functionality of BAT, as TH abundance not only supports leaner body composition but is also positively correlated with known structural elements in BAT (UCP1, COX IV, CD31, and cell density). Based on our observations, β3-AR abundance does not strongly drive these structural elements or TH, all of which are known to be important in the function of brown adipose tissue. In effect, while the role of other receptors, such as β2-AR, should be reviewed in BAT function, these results support the development of safe sympathetic nervous system stimulants to activate brown adipose tissue for obesity treatment.


Introduction
Obesity is associated with health problems such as high blood pressure, type 2 diabetes, heart disease, and other metabolic illnesses. With roughly 42.4% of the United States' adult populace diagnosed with obesity in 2018, new treatment methodologies are essential [1]. An emerging target tissue for treatment of obesity and other metabolic diseases is brown adipose tissue (BAT). BAT is a metabolically active tissue, in which uncoupling protein 1 (UCP1) uncouples mitochondrial oxidation from ATP synthesis and dissipates energy in the form of heat during the natural sympathetic response to cold exposure [2]. Fifty grams of activated brown fat can account for roughly 20% of whole-body energy expenditure [3]. Tere have been attempts to stimulate BAT pharmacologically with the use of sympathomimetics, but adverse cardiologic efects have limited progression of possible agents to clinical use [4].
Tere has been recent imaging success towards understanding activated BAT in vivo, but there is a lack of data concerning the relationship of multiple in situ BAT tissue features, which is vital to identify and understand BAT functionality before it can be utilized in a clinical setting [5][6][7]. Te goal of this study was to assess key BAT histological features relating to mitochondria and its uncoupling, as well as innervation and vascularization from in situ tissues collected from adult nonhuman primates (NHPs) encompassing a range of adiposity and health statuses. NHPs allow for a more relevant interpretation than other animal models due to the close physiological relationship between NHPs and people. With the failure of many pharmaceutical candidates to translate to efective human therapies, particularly for cardiometabolic diseases, understanding BAT biology in NHPs can aid drug development and research directions [8].
We found that axillary BAT in adult NHPs histologically resembles that in humans and functionally compares to previous rodent studies that indicate BAT functionality impacts obesity characteristics [9]. Here, we show that tyrosine hydroxylase, an innervation biomarker in BAT that is a fundamental component of norepinephrine synthesis, has the highest relationship with body composition and that β3-AR abundance does not drive brown fat functionality. Tese fndings provide translational support for pharmacological targeting of the sympathetic nervous system in the treatment of obesity but suggest that other ARs be reviewed for their relationships with brown fat functionality.

Nonhuman Primates.
Te NHP cohort (n � 26) was comprised of 11 female African green monkeys (Chlorocebus aethiops sabaeus) from the Vervet Research Colony at the Wake Forest University School of Medicine (WFUSM) and 15 male rhesus macaques (Macaca mulatta) [10], also housed at the WFUSM. Te cohort was selected to be diverse in age and metabolic health; 7 individuals presented with type 2 diabetes, 4 presented with obesity, and 8 presented with hypertension (Tables S1 and S2). All health phenotypes presented spontaneously, and animal procedures were approved by the Wake Forest University Institutional Animal Care and Use Committee.  Figure S1). Slides were digitally scanned, and image analysis was completed by an operator blinded to health status using Visiopharm software (Hørsholm, Denmark). Analyses included the whole tissue sample and a visually representative region of interest, with results expressed as the percent positive stain by the tissue area. Te UCP1 measures were expressed as z-scores, as data were generated from separate batches of immunostaining. Te β3-AR samples were scored (1: low abundance of β3-AR stain to 4: high abundance of β3-AR stain) based on the overall coverage of an antibody by 5 individuals blinded to health status. An average cell size for each subject was calculated based on a representative region of interest within the sample, and nucleus density was calculated as the total nucleus area divided by the total tissue area. To ensure that BAT depots were comparable, axillary BAT samples were compared to supraclavicular BAT samples in the rhesus macaques, and there were no signifcant regional diferences in brownness, measured by the abundance of the UCP1 antibody present (Table S3).

Body Composition.
Computed tomography scans were performed within 3 months of tissue collections (Siemens SOMATOM Defnition Flash, Munich, Germany) to establish total body composition characteristics including total body fat and lean mass percentages in relation to body weight at the time of scans (Table S1, Figure S2). Te scans were reconstructed as DICOM images using AquariusNet Tin Client (TeraRecon, Durham, NC) and then analyzed as previously described [10] using Materialise's interactive medical image control system ((Mimics) Materialise, Leuven, Belgium) (Table S1, Figure S2).

Data Analysis.
Statistical analysis was performed using Statistica v13 (StatSoft, Tulsa, OK). Body weight, fasting blood glucose, and diastolic blood pressure were analyzed as z-scores due to species or sex-specifc diferences. All continuous variables were assessed for normality and were log transformed if needed to achieve a normal distribution. Signifcant correlations were assessed by Pearson's correlation coefcients, controlled for age, and signifcance was assumed when p ≤ 0.05; trends were indicated when p ≤ 0.10 for interval scale data. Signifcant correlations were assessed by Spearman Rank correlation coefcients, and signifcance was assumed when p ≤ 0.05; trends were indicated when p ≤ 0.10 for ordinal data.

Results
We characterized the metabolic diversity of our NHP cohort by using metrics comparable to human metabolic outcomes (Table S1). We measured age, body weight, systolic and diastolic blood pressures, fasting blood glucose, hemoglobin A1c, triglyceride concentration, high-density lipoprotein cholesterol (HDLC) concentration, adiposity represented by fat mass as a percentage of body weight, and cell density (Table S1). We found that our cohort was diverse and presented healthy subjects, as well as those with hypertension, hyperglycemia, and obesity across both species (Table S2).
We completed the immunohistochemical analysis of adipose tissue stained with UCP1, COX IV, CD31, TH, and β3-AR antibodies to measure brown fat abundance (UCP1), mitochondrial abundance (UCP1 and COX IV), perfusion 2 Journal of Obesity (CD31), sympathetic innervation provided to tissue (TH), and adrenergic receptor abundance (β3-AR) (Figure 1(a)). We found that BAT TH was the only histological feature that had a signifcant correlation with a clinical metabolic measure, as it was negatively correlated with adiposity (p value � 0.004) (Figure 1(b)). We found that BAT TH density was in turn positively correlated with other key features of BAT, including UCP1 (p value � 0.02), COX IV (p value � 0.006), CD31 (p value � 0.007), and cell density (p value � 0.02) (Figure 2). Te β3-AR abundance in BAT had a strong positive relationship with CD31 coverage in WAT (p value � 0.007) and a weak yet signifcant positive relationship with COX IV in BAT (p value � 0.04) (Figure 3).

Discussion
Tere is a gap in knowledge regarding the association of key BAT histological features, UCP1, COX IV, TH, CD31, and β3-AR, and expressed clinical phenotypes. To date, there has not been a study that analyzes innervation, endothelial, and mitochondrial markers within BAT explants. Innervation is highly signifcant in BAT functionality as it is responsible for catalyzing the reaction of events necessary for UCP1 activation, and thus the energy expensive process of UCP1dependent thermogenesis itself. Tis occurs through the stimulation and release of norepinephrine by the sympathetic nervous system in response to cold exposure, which binds to beta-adrenergic receptors (β-ARs), leading to the production of cyclic adenosine monophosphate and activation of UCP1 [11].
Tere have been attempts to increase BAT activation with sympathomimetics, as it is a tissue capable of extensive energy expenditure [3], in order to treat obesity and diabetes by mimicking the efect of norepinephrine on β3-AR. However, side efects including increased heart rate and elevated blood pressure have hampered drug development to date [4]. Tere has also been recent debate as to the prevalence of β2-AR over β3-AR in human brown fat, which has been hypothesized why a promising β3-AR agonist, mirabegron, has displayed conficting results in preclinical testing [12][13][14].
Tere are several reasons why BAT innervation may vary between individuals. It is known that age can be related to impairment of the sympathetic nervous system, as well as to changes in endocrine signals and infammation [15,16]. Exercise also plays an important role in sympathetic tone, and previous research suggests that exercise increased sympathetic tone in BAT [17].
Te fndings of this study were reproducible across two NHP species that were diferent in size, sex, and age; however, each NHP species displays the same range of metabolic health from lean and healthy to obese and type 2 diabetic (Tables S1 and S2). Tis demonstrates the reproducibility and generalizability of the relationship of BAT TH, and other histological features, with obesity and related metabolic disorders. Our fndings concerning tyrosine hydroxylase provide evidence that supports the use of sympathomimetics to activate BAT in the treatment of diabetes, obesity, and other cardiometabolic disorders, as they illustrate the importance of innervation in the functionality of brown adipose tissue. Furthermore, our results suggest that the abundance of β3-AR does not drive brown adipose tissue functionality, indicating that other β-ARs should be reviewed in relation to BAT functionality and considered for sympathomimetic drug development. Tese characterizations of nonhuman primate adipose also highlight the need for further study into the reasons for variation in BAT innervation.

Data Availability
Tere are immunohistochemical and imaging data, as well as historical clinical data on the subjects, available upon request.