ABSTRACT

Introduction

Brown adipose tissue (BAT) has attracted extensive recent scientific interest due to its characteristic ability to produce heat by non-shivering thermogenesis, a process that consumes calories and could potentially be harnessed to treat human obesity and associated metabolic conditions [1,2]. BAT contains extensive mitochondria expressing uncoupling protein 1 (UCP1) that generates heat and increases overall metabolic activity, with a concomitant dissipation of energy [1].

Temperature regulation through non-shivering thermogenesis in BAT is critical in neonatal mammals. Both rodents and humans have a large interscapular BAT (iBAT) depot during early life [3], and iBAT was first described in human foetuses in 1902 [4]. iBAT persists throughout life in rodents, but in humans regresses with age and is lost in adults. This finding led to the long-term consensus during the early and middle 20^th century that BAT was not important in humans. Although Heaton [5] subsequently reported that BAT was detectable histologically in adult and even elderly humans, BAT attracted only limited interest for many years.

Approximately a decade ago, several imaging studies stimulated interest in this field by demonstrating extensive and highly active BAT depots in thoracic and neck regions of adult humans [6–8]. Critically, the size and activity of BAT depots were increased by cold exposure [8,9] and varied seasonally [10]. In addition, correlations between leanness and BAT volumes were documented in humans, further emphasizing the importance of BAT in metabolic regulation [11].

Recent findings using a human clinical database of over 50,000 patients demonstrated that the presence and abundance of BAT correlate with lower incidences of cardiometabolic diseases, type 2 diabetes and other conditions. The presence of BAT was also associated with improved blood glucose, triglyceride and high-density lipoprotein values [12], indicating that BAT has metabolic benefits over and above weight regulation.

In addition to the long-known iBAT and perirenal BAT depots in mice, recent studies identified new upper thoracic/head/neck BAT depots [13–15]. BAT is present in the subscapular region, near the dorsal neck of the scapula, in the cervical region of the head, and surrounding the thoracic (descending) aorta [15]. Mo et al [13] described supraclavicular BAT depots in mice with histological, molecular and functional attributes of a classic BAT depot, and a recent report from this group described a new BAT depot in the deep neck region [16]. Zhang et al [14] recently used a labelled glucose analogue and PET/CT imaging to visualize BAT depots in mice using the same methodology employed in human subjects. In addition to confirming the presence of BAT depots in thoracic, cervical and axillary regions, they also noted an intense label uptake in the midline region above and below the kidney that is a signature of BAT, which they designated as the ventral spinal BAT depot.

Mice are the most widely used model for studies of both brown and beige adipose tissue. Beige adipose tissue is a third type of adipose tissue that is distinct from BAT and white adipose tissue (WAT) that contains varying amounts of adipocytes with functional and structural characteristics of BAT interspersed among the WAT [1]. Genetic manipulations in mice have provided major insights into BAT physiology [17], and overexpression or deletion of proteins, such as PRDM16 in mice, has increased understanding of BAT development and function [18]. Therefore, the extent and distribution of mouse BAT are important because this is the primary animal model for this field.

During the development of two new transgenic mouse models with altered androgen receptor function [19], we examined the internal reproductive organs of these animals and serendipitously discovered a previously undescribed depot of BAT in the vicinity of these organs. We then reasoned that there might be additional BAT depots in the pelvic/lower abdominal area, and we were able to identify three other new BAT depots in this region that were not associated with reproductive organs. Here, we document the presence and functionality of these previously unknown BAT depots in mice. These depots express classical histological, molecular and functional features of BAT, and necessitate an extensive re-evaluation of our understanding of BAT distribution in the mouse.

Results

Growth and developmental characteristics of BAT depots

All of the new BAT depots described here are identifiable by postnatal day 4, but the morphology of the various BAT depots changes during subsequent postnatal development. The most striking morphological change with advancing age in the various BAT depots occurs in the pBAT. Although pBAT in adult males occurs only laterally to the urethra and pBAT lobules on the left and right sides of the urethra are anatomically separated (Figure 1b), at postnatal days 4 and 21 pBAT is present not only lateral to the urethra but also extends across the ventral surface of the urethra and is contiguous with the contralateral pBAT (Figure 2). The pBAT on the ventral urethral surface regresses with age, and by adulthood pBAT on the left and right sides of the urethra are separate and anatomically distinct. uBAT is most apparent at the first neonatal age examined, when it is relatively more extensive than at postnatal day 22 or in adulthood. Although pvBAT in adults terminates where the right ovarian vein branches from the caudal vena cava, at postnatal days 4 and 22 it extends all the way to the renal vein, where it is contiguous with the renal BAT.

Figure 2.

BAT depots visualized using a dissecting microscope with transmitted light and darkfield microscopy. The upper panel shows a ventral view of pBAT in a postnatal day 21 male. The pBAT is seen on both sides of the urethra, but at this early developmental stage there is also a bridge of pBAT on the ventral surface of the urethra connecting the contralateral pBAT on the other side of the urethra. DD = ductus deferens, SV = seminal vesicle, UR = urethra, VP = ventral prostate, BL = bladder. Remaining panels show (counterclockwise) ingBAT, iBAT and lower and upper pvBAT from postnatal day 70 mice; note the sacral lymph node (arrow) in the lower pvBAT and the lumbar lymph node (arrow) encased in the upper pvBAT. uBAT from a postnatal day 5 mouse is shown as an inset in the pvBAT-upper image. Note that the magnification bars in images of the ingBAT, iBAT, lower and upper pvBAT and uBAT and uBAT are all equal, allowing direct comparison of relative BAT depot sizes.

Body weight (BW) and weights of the iBAT and pBAT depots from the neonatal period to adulthood are shown in Figure 3. Both depots increase in weight as the mice age (Figure 3b). The iBAT increases are proportionately less than BW increases, and relative iBAT weight (iBAT weight/body weight) declines during development (Figure 3c). The pBAT is smaller than the iBAT at all ages; pBAT weight is approximately 15% of iBAT at postnatal day 4, but by postnatal day 65, pBAT weighs about 30% as much as iBAT. As a result of this weight increase during development, the pBAT/BW ratio remains relatively constant, in contrast to the decline for iBAT. Weights of pvBAT and ingBAT depots were not determined at all ages, but the postnatal day 22 pvBAT depot weighed 3.8 ± 0.9 mg (n = 4) and the postnatal day 65 pvBAT and ingBAT depots weighed 11.4 ± 1.3 mg and 8.2 ± 1.1 mg (n = 4 for both), respectively. Collectively, the overall weights of the pBAT, pvBAT and ingBAT at day 65 are approximately 75% of that of the iBAT at that age, indicating that these BAT depots could be a significant factor in overall metabolism.

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Figure 3.

Body weights and absolute and relative weights (BAT weight/body weight; expressed as %) of iBAT and pBAT depots from postnatal days 4–65. In all cases, iBAT and pBAT values represent samples obtained for weighing from one side of the animal only. Data are shown as mean ± SEM, and n = 4–11 for the various ages and BAT depots.

Expression of UCP1 and induction of UCP1 by an adrenergic agonist in BAT depots

Adult iBAT showed intense immunostaining for UCP1, a hallmark of BAT (Figure 4). In contrast, gonadal WAT did not stain for UCP1 (Figure 4). All other new BAT depots identified in this study (pBAT, pvBAT, ingBAT, uBAT) showed the classic histological structure of BAT as well as intense UCP1 immunostaining (Figure 4). The muscularis externa of the ureter also was UCP1-positive at the neonatal, juvenile and adult ages examined (data not shown). Postnatal day 4 pBAT also stained intensely for UCP1 (data not shown), suggesting that BAT depots described here were functional neonatally, when their thermogenic effects are most critical. As an additional control, another tissue that expresses high amounts of mitochondria (kidney) was also immunostained for UCP1. Kidney did not show UCP1 staining despite high mitochondrial expression in this tissue (not shown), indicating that the strong UCP1 staining seen in BAT depots does not result from non-specific effects of high mitochondrial concentrations.

Figure 4.

Immunohistochemical staining of UCP1 in various adipose depots. Expression of UCP1 (brown staining) was strong in postnatal 70 iBAT (positive control), as well as in pBAT, pvBAT, ingBAT and uBAT at the same age, but could not be detected in gWAT. Panels A-D are all at the same magnification, and magnification bars for all images are shown in the bottom right hand corner.

Western blotting indicated significant baseline UCP1 expression in iBAT, pBAT, pvBAT and ingBAT (Figure 5, a and b). Treatment with the adrenergic agonist CL 316,243 increased UCP1 in all BAT depots. In contrast, UCP1 could not be detected in gonadal (g)WAT following either vehicle or CL 316,243 treatment. The uBAT consists of thin discontinuous strands of BAT that are intimately associated with the ureter and could not easily be separated from it, so western blotting for uBAT was not performed.

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Figure 5.

Protein and gene expression are similar in iBAT and the newly identified BATs. Western blotting (a) and quantitation (b) for UCP1 expression in iBAT, pBAT, pvBAT, ingBAT and gWAT in postnatal day 35 female mice given vehicle or the β3-adrenoceptor agonist CL 316,243 (1 mg/kg/day for 3 days). Adipose tissues were collected 24 h after the last injection. The housekeeping protein β actin was used as a loading control. UCP1 expression in all groups was expressed relative to the UCP1 value in vehicle-treated iBAT, arbitrarily set as 1. The n = 3 for all groups used in the Western blotting in (a) and (b) and n = 4 for all groups used in the qPCR studies (d and e). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Transmission electron microscopy (TEM) of iBAT and pBAT

Brown adipose tissue is characterized by extensive mitochondria and multilocular lipid droplets. Transmission electron microscopy indicated that these typical BAT features occurred in both iBAT (Figure 7, left panel) and pBAT (Figure 7, middle and right panels). In contrast to WAT, in which the nucleus is highly eccentric due to the single large lipid droplet pushing it to the cell periphery, BAT nuclei are more central, and this was apparent in both iBAT and pBAT.

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Figure 7.

Transmission electron micrographs (TEM) of iBAT and pBAT. Left panel: iBAT had extensive cytoplasmic mitochondria (M) and multilocular lipid droplets (L). Middle panel: lower power view of pBAT showing extensive mitochondria, multilocular lipid droplets and a nuclear morphology that were all similar to iBAT. Right panel: higher power view of pBAT showing a greater magnification of the region highlighted by the square in the middle panel. Magnification bars shown in lower left corner of all images.

Discussion

Our findings indicate that there are four heretofore undescribed BAT depots in the pelvis, lower abdomen and upper hindlimb of the mouse, anatomical regions where BAT has not been reported previously. pBAT is associated with the male and female reproductive tract in both sexes and with the urethra in the male. uBAT envelops the ureter and the pvBAT lies adjacent to major blood vessels in the lower abdomen and pelvis, whereas the ingBAT is a small depot found between adjacent muscles at the juncture of the upper hindlimb and abdominal body wall. These results indicate that BAT is more widely distributed in this species than previously realized.

All new BAT depots described are present neonatally and then increase in overall size and weight as the animal develops. The weights and sizes of the pBAT and each of the other newly described BAT depots are significantly smaller than iBAT, where the largest amount of BAT is found in the body. However, in aggregate, these four new BAT tissues are comparable in size to the iBAT. Considering their total size in concert with our results showing that these new BAT depots are active metabolically suggests that these BAT depots likely have significant roles in overall BAT function and physiology in the mouse and should be taken into account in studies of BAT activity in this animal.

An important question to consider for all the new BAT depots described is why they were not reported previously. The BAT depots associated with the distal regions of the male and female reproductive tract described here were initially identified using a dissecting microscope with transmitted light and darkfield microscopy, which allowed clear differentiation of BAT from surrounding WAT and was crucial for initial identification of BAT in regions where it was not reported earlier. When viewed under a dissecting microscope with normal brightfield illumination and incident light, the extensive adipose tissue in the urethral/vaginal area in the female and the urethral/prostatic area in the male appears fairly homogeneous and there is little indication that BAT tissue is present. However, under darkfield illumination with transmitted light, the BAT can be easily differentiated from the surrounding WAT tissue, allowing the extent and distribution of the BAT in this region to be clearly visualized.

pBAT is anatomically diffuse and discontinuous and consists of numerous lobular structures and is more structurally similar to subclavicular BAT than the iBAT depot, which is clearly defined anatomically without obvious lobules. Although pBAT volumes appear comparable in males and females, the sexually dimorphic nature of the reproductive organs in this region results in the anatomical distribution of this BAT depot differing in males and females. Whether the thermogenic activity of BAT associated with the male and female reproductive tract has any specific function in these organs is currently unknown.

pvBAT and pBAT are the largest of the new BAT depots described here. At weaning, the pvBAT depot is contiguous cranially with the perirenal BAT and extends bilaterally along the descending aorta and caudal vena cava from just beneath the kidney to the pubic symphysis, following the iliac veins after they branch from the caudal vena cava. Although a mediastinal BAT lying around the thoracic aorta in the thoracic region was described previously [21], the pvBAT described here is anatomically distinct from that thoracic BAT depot. Zhang et al [14] used positron emission tomography-computed tomography (PET/CT) with radioactively labelled compounds to identify areas of intense glucose and fatty acid metabolism corresponding to BAT. These studies suggested intense fatty acid uptake in the midline below the kidneys, especially following β3 adrenergic stimulation, and indicated that there could be BAT in this region. However, this imaging study did not allow the anatomical location of the signal to be precisely determined. Our results indicate that the previously investigated region likely reflects the pvBAT we report here, which is located below the kidneys in the midline.

The pvBAT consists of two thin bilateral strips of tissue. The small diameter of this structure may have impaired detection in previous human and animal studies utilizing tracer methodologies. However, the extensive length of pvBAT, which extends from the renal artery to the pubic symphysis, indicates that it could be physiologically significant. A similar argument can be made for pBAT, which is diffuse and anatomically discontinuous and thus also may be problematical to resolve by current imaging methodologies.

The ingBAT is the only one of the newly described BAT depots that occurs outside the body cavity, although other more peripheral BAT depots such as in the head and neck have been reported [10]. In adults, the size of the ingBAT is less than 25% that of the iBAT, and the ingBAT depot also weighs less then the pBAT depot described here. The ingBAT may be a significant contributor to overall BAT activity, as suggested by previous studies in which increased focal metabolic activity was detected at the anatomical location of the ingBAT. The BAT imaging studies of Zhang et al [14] identified one punctate area of the leg that showed intense metabolism. This activity was ascribed to the presence of beige adipocytes in the inguinal WAT, but the inguinal WAT is much more anatomically diffuse than the sharply delineated signal reported in this study. In the light of the present results, this activity likely emanated from the ingBAT.

The uBAT envelops the ureter and is most pronounced during neonatal and juvenile life. Although it is reduced during adulthood, uBAT around the ureter is still demonstrable. In addition, our results also indicate that the smooth muscle layers of the ureter express UCP1, consistent with a previous report [22]. Because of its close association with the ureter, it is problematic to quantitate the uBAT tissue compared to the other new BAT depots described here. The uBAT that envelops the ureter, especially during neonatal and juvenile life, suggests that this BAT may play a role in ureter development or function, but the specific roles of uBAT in normal ureter physiology, remain to be determined. Work from the early 20th century demonstrated that ligation of the ureter led to bone formation in this tissue [23]. This process, known as heterotopic ossification, has more recently been shown to involve brown adipocytes [24]. Thus, the uBAT described here may be mechanistically involved in the ureter heterotopic ossification following ligation that has previously been documented.

Because uBAT is continuous with the renal BAT cranially and the distal juncture of the ureter is near where pBAT is located at the base of the bladder, all three of these BAT depots may have a common embryological origin, although this remains to be directly addressed.

A hallmark of BAT is UCP1 expression, which is essential for thermogenesis in this tissue [25,26]. Importantly, UCP1 expression in BAT is inducible by cold stress or β3-adrenoceptor agonists such as the endogenous hormone norepinephrine or synthetic ligands such as CL 316,243 that stimulate glucose uptake, heat production and oxygen consumption [22,23]. All of the four newly identified BAT depots robustly express UCP1 by immunohistochemistry, a control gonadal WAT sample does not. Kidney, which has extensive mitochondria, also does not express specific UCP1 staining l Western blotting also showed that UCP1 is detectable in all control BAT depots (without CL 316,243 stimulation). Furthermore, UCP1 is strongly inducible by CL 316,243 in iBAT and all of the new BAT depots reported, with the magnitude of the increases in UCP1 in response to CL 316,243 in the four new BAT depots being similar in magnitude to those in iBAT. UCP1 was not detectable in WAT either before or after stimulation with CL 316,243.

Mestres-Arenes [27] recently described perivascular adipose near the abdominal midline that has a WAT histological appearance with some multilocular adipocytes visible. This tissue expressed UCP1 under thermoneutral conditions, but at a level only about 10% of what is seen in iBAT. Both the intense baseline immunohistochemical staining of our pvBAT, as well as our western blotting results indicating that the pvBAT expresses UCP1 in a basal and CL 316,243-stimulated state, suggest that this tissue is classical BAT, and not the WAT with some BAT characteristics previously described in the area near the kidney.

In addition to UCP1 measured by western blotting, our newly identified BAT depots also show a molecular signature consistent with classical BAT. Expression of Ucp1 and Cidea mRNAs, which are diagnostic for BAT, is high in control iBAT as well as the new BAT depots described here, but absent in WAT. ASC-1, a cell surface marker of WAT that is minimally expressed in BAT [28], is robustly expressed in gWAT, but not in iBAT or the other BAT depots. These results confirm that the BAT depots described here have classic molecular characteristics of BAT, although minor WAT contamination of depots such as the pBAT, which occur in vivo interspersed within WAT, is likely inevitable and may account for the low Asc-1 mRNA signal in pBAT.

The norepinephrine analog CL 316,243 is a potent and highly selective β3-adrenoceptor agonist that stimulates UCP1 expression in BAT, but not WAT [1]. The marked stimulation of UCP1 by CL 316,243 in all BAT depots, in conjunction with other results presented here, demonstrate that the new BAT depots described express an extensive number of characteristics that are unique to BAT but not seen in WAT.

Both the iBAT and pBAT contain extensive mitochondria and multilocular lipid droplets as demonstrated by TEM. These are classical characteristics of BAT and confirm that pBAT expresses the unique features of BAT [20]. Another classic characteristic of BAT is extensive sympathetic innvervation. The thermogenesis that is the hallmark of BAT is induced by the release of the neurotransmitter noradrenaline from extensive sympathetic fibres present in this tissue and subsequent stimulation of UCP1. Conversely, WAT does not express the extensive sympathetic innervation characteristic of BAT. The rate-limiting step in catecholamine synthesis is regulated by TH, and is typically used as an immunohistochemical marker for sympathetic postganglionic motor neurons and motor axons [20]. The extensive sympathetic innervation seen in pBAT is comparable to that seen in control iBAT sections and far exceeds that in WAT, further establishing pBAT as classical BAT.

CGRP is a neuropeptide that has been previously used as a marker of sensory peptidergic neuronal fibres of interscapular and perirenal BAT and is believed to be involved in BAT thermogenesis, energy metabolism, food intake and lipid homoeostasis [19,29,30]. These actions may be mediated by effects on the morphological characteristics of adipocytes and depots along with mediating inflammatory and metabolic responses [31]. Our results show that pBAT also expresses CGRP. The CGRP sensory axons are much less numerous than the sympathetic axons and often occur in close proximity to the more numerous TH axons. However, CGRP does not co-label the same axons as TH and these axons innervate different areas of the BAT, again consistent with previous findings in iBAT [20].

It has been known for several years that mice can have UCP1-positive (beige) cells in their WAT depots that express UCP1 and resemble BAT histologically [32]. Although numbers of these beige adipocytes can be increased by cold exposure and other means, beige fat has only patchy occurrence of UCP1-expressing cells that show histological characteristics of BAT. These are distributed thoughout the larger tissue that shows WAT characteristics. In contrast, our newly described BAT depots show intense UCP1 immunohistochemical staining and classic BAT histological characteristics throughout and thus are clearly distinct from beige adipose tissue.

The common and unique features of WAT, BAT and beige adipose tissue development have generated extensive interest [21,33–35]. The developing iBAT expresses Myf5, a transcription factor involved in muscle differentiation, suggesting iBAT and muscle may be derived from a common embryonic lineage [36]. However, subsequent work indicated that some other BAT depots did not express Myf5 developmentally, suggesting a different embryonic lineage [37]. Cells with characteristics of brown adipocytes in beige adipose tissue appear to arise from a different developmental lineage and evidence has been presented supporting both de novo differentiation of brown adipocytes [38] as well as transdifferentiation of existing white adipocytes to cells with characteristics of brown adipocytes [39]. Thus, brown adipocytes appear to develop from several different lineages.

Interscapular BAT strongly expresses Zic1 mRNA, and this was initially suggested as a marker to identify BAT and differentiate it from beige adipose tissue. However, subsequent work indicated that Zic1 appeared to only be expressed in anterior BAT depots, but was reduced or absent in more posterior BAT depots (reviewed by [34]). In addition, Zic1 could be transiently expressed in adipocytes undergoing beiging in WAT under some conditions [40].

Our results indicate that Zic1 mRNA is abundantly expressed in iBAT, in agreement with previous findings. Reduced amounts of Zic1 mRNA were also expressed in pvBAT, but Zic1 mRNA expression was absent in the pBAT and ingBAT. These results are consistent with previous findings that Zic1 mRNA expression in BAT is preferentially expressed in more anterior BAT depots, which may be indicative of different developmental origins for these depots.

Our data indicating that pBAT also occurs in the rat suggest that this BAT depot is not confined to mice but occurs in other species as well. A critical question that remains to be addressed is whether the new BAT depots described here are unique to rodents or occur in humans and other mammals. Imaging studies to examine BAT distribution in humans have shown signalling in the lower abdominal region as well as the pelvic region around the bladder [10,14], although these studies focused on BAT depots in the upper thoracic region. The lack of reports indicating BAT presence in the lower abdominal/pelvic region may have suggested to the authors that the signals detected in this region were non-specific and did not represent BAT, or the signals in this region may simply not have been the focus of the study and thus were not noted. Additional studies are required to determine whether human BAT depots are present in similar anatomic locations to the new rodent depots described here. Nevertheless, the extensive nature of the lower abdominal/pelvic depots in the mouse, the previous suggestions of BAT in these regions from earlier studies and the strong correlation between the anatomical locations of thoracic BAT depots in the mouse and human [14] suggest the new-found BAT may be clinically relevant.

Methods

References