The Function and Pathology of Brown Adipose Tissue in Animals and Humans

J Toxicol Pathol 2004; 17: 147–153

IATP Educational Session “Contemporary Issues in Toxicologic Pathology” The Function and Pathology of Brown Adipose Tissue in Animals and Humans

Michael Iatropoulos1, and Gary Williams1

1New York Medical College Valhalla, NY, 10595, USA

Abstract: Brown adipose tissue (BAT), an endocrine tissue, is an important regulator of nonshivering thermogenesis, energy metabolism, mitochondrial biogenesis, extracellular matrix (EMA) homeostasis and signaling, and hibernation. There is a paucity of data about BAT response in laboratory animals because BAT has not been routinely included in regulated safety assessment study protocols. We will present information on the structure, function and pathology of BAT, focusing mainly on younger and older rats from an exploratory study set to study BAT and all other tissues over time, from 6 weeks to 96 weeks of age. The energy supply process is stimulated in mammals mainly by hypothermia and by hypoxia and, in primates, by hypoglycemia. Of these, hypothermia is the main stimulus of brown adipocyte (BA) proliferation. Thermogenesis is achieved in BA in the mitochondria (mt), which are 50 to 100 times more abundant in BA than in any other cell type. The mt response involves a membranous protein in the inner membrane, uncoupling protein (UCP1) or thermogenin. Triiodothyronine (T3) is implicated in the regulation of the UCP1 gene. UCP allows mt to oxidize substrates rapidly without ADP phosphorylation, thus uncoupling oxidation from phosphorylation and promoting instead the dissipation of oxidation energy as heat. Norepinephrine (NEPI) binds to the β3-adrenoreceptor in BA leading to activation of lipoprotein lipase (LPL), which stimulates lipolysis and liberates FFA. Both NEPI and insulin (I) stimulate glucose uptake by BA, and NEPI increases UCP in BA. Nitric oxide (NO) stimulates the biogenesis of mt, redistribution of heat generated by the mt and inhibition of BA proliferation. NO also regulates the binding and releasing of oxygen from hemoglobin (Hgb) and the mt pathway of apoptosis. These effects of NO are mediated by peroxisome proliferator-activated gamma receptor coactivator 1alpha (PPARCA). NO is produced by endothelial NO synthase (eNOS) mainly in BA, but also in the hepatocytes and cells of the zona glomerulosa. PPARCA in turn increases the expression of nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (mtTFA).

Thus, UCP, T3, I, NEPI, NO, PPARCA1, cGMP, eNOS, NRF-1 and mtTFA are all parts of an interactive cascade connecting the BA functions through the EMA of all tissues to controlling the processes of EMA homeostasis and signaling, tissue normoxia and normothermia, apoptosis, proliferation, and differentiation of all cells. (J Toxicol Pathol 2004; 17: 147–153) Key words: brown adipose tissue, structure, function, pathology, laboratory animals, humans

Introduction chemical entities with demonstrated endocrine modulating properties, the need to study BAT over time has significantly Brown adipose tissue (BAT), an endocrine tissue, increased. houses, directs and serves as a gatekeeper of nonshivering In order to address this need, a study was designed and thermogenesis, energy metabolism, mitochondrial carried out in Sprague-Dawley rats24. The experimental biogenesis, extracellular matrix (EMA) homeostasis and design of this study is given in Table 1. The study employed signaling, and hibernation in hibernating animals4,7,20,28,47. two different age groups, a younger group, six weeks old at There is little data about BAT in laboratory animals because study start and an older group, 36 weeks old at study start. BAT has not been routinely included in regulated safety The study duration was 13 months (56 weeks), at the end of assessment study protocols. This undesirable situation has which the younger group was 62 weeks old and the older 92 led to an absurd absence in chronic (2 year) rodent studies of weeks. Hematology, extensive clinical chemistry with morphologic control data from this very important endocrine emphasis on endocrine parameters, complete histopathology tissue, BAT. With the advent of more specialized new focusing on the 3 main BAT locations, PCNA of BAT and adrenals and measurement of the size of intracytoplasmic Mailing address: Michael Iatropoulos, New York Medical College vacuoles in brown adipocytes (BA) was undertaken (Table Valhalla, NY, 10595, USA 1). The UCP/Actin ratio was measured35 in order to assess TEL: 1-914-594-3106 FAX: 1-914-594-4163 the unique BAT protein UCP1. In this paper, we report on E-mail: [email protected] 148 Brown Adipose Tissue Function and Pathology

Table 1. Study Design in Younger and Older Control Male and Female Rats over Time

Group identification Initial no. of rats 1 mo. sac. 3 mos. sac. 6 mos. sac. 12 mos. sac. 13 mos. sac. MFMFMFMFMFMF

Age at study start Younger1 age groups 24 24 4 4 4 4 4 4 8 8 4 4 Older2 age groups 24 24 4 4 4 4 4 4 8 8 4 4

M = male; F = female; 1 = 6 weeks old at study start and 60 weeks at study end; 2 = 36 weeks at study start and 96 weeks at study end; Sprague-Dawley CD rats. Animals were observed for clinical signs twice daily and weighed weekly. Postdose rectal body temperatures and feed consumption were also measured weekly. At each scheduled sacrifice, blood samples were collected, analyzed for total protein, globulin, albumin, glucose, triglycerides, urea nitrogen, alanine aminotransferase, alkaline phosphatase, gamma glutamyltranferase, aspartate aminotransferase, cholesterol, total bilirubin, creatinine, calcium, sodium, potassium, phosphorous, chloride, triiodothyronine, thyroxine, thyroid stimulating hormone, parathyroid hormone, aldosterone, corticosterone, epinephrine, and norepinephrine. Adrenals, liver, thyroid/parathyroid and kidneys were weighed. A complete histopathology was performed with emphasis in the 3 BAT locations. Samples from 3 BAT locations from each animal and also from the adrenals were analyzed for evidence of proliferation by PCNA. BAT was also analyzed for levels of a unique BAT protein, UCP. The assessment involved a UCP/Actin ratio to monitor the integrity of the BA cytoskeletal structure. Finally, the size of intracytoplasmic vacuoles in brown adipocytes was also measured.

Table 2. Indices in Younger1 and Older2 Control Male and Female Rats over Time 3 4 5 6 Group identification Temp. UCP/A VS RF Age in weeks at study start M F M F M F M F

One Month on Study Younger age groups 35.1 36.9 6.1 2.7 2.9 2.1 23.7 24.8 Older age groups 35.6 37.2 3.2 6.5 2.5 1.9 23.9 26.6 Three Months on Study Younger age groups 35.0 36.1 4.5 2.7 3.7 3.1 24.3 26.3 Older age groups 35.9 36.6 6.9 3.8 3.3 2.7 20.5 28.9 Six Months on Study Younger age groups 36.3 36.7 3.9 2.6 3.4 3.0 24.8 24.9 Older age groups 36.8 37.6 3.7 2.2 3.5 3.3 25.1 24.2 Twelve Months on Study Younger age groups 36.8 37.6 2.2 1.8 3.4 3.6 24.3 25.0 Older age groups 36.2 37.1 2.5 2.5 3.1 3.3 23.5 25.2 Thirteen Months on Study Younger age groups 36.2 38.0 3.9 2.4 3.8 3.8 21.6 24.3 Older age groups 37.2 38.4 2.3 1.6 3.6 3.0 23.6 25.1

1: 6 weeks old at study start and 60 weeks at study end. 2: 36 weeks at study start and 96 weeks at study end. 3: post dose mean rectal temperature in °C. 4: uncoupling protein/actin ratio monitoring the BAT cytoskeletal integrity. 5: intracytoplasmic vacuolar size/diameter graded 1 to 4, with 4 the largest. 6: replicating fraction in percent indicating PCNA positive nuclei out of total nuclei counted.

the results from this study and discuss in general the role of 40 µ, and that of a white adipocyte (WA) is 120 µ. Unlike BAT in rodents and humans. the univacuolar appearance of WA, BA contain multiple vacuoles with granular cytoplasm with round central nuclei Structure of Brown Adipose Tissue (Fig. 1). The granularity of BA reflects the abundance of mitochondria (mt) which are 50–100 times more numerous BAT arises from a specific anlage at particular body than the mt of other cells. The BAT mt also assume a more sites (upper interscapular, anterior mediastinal and complex ultrastructure42. BA are arranged in distinct lobular retroperitoneal around the adrenals) in the second half of aggregates with prominent extracellular matrix (EMA), gestation and remains in these sites postnatally9,26,50. In vasculature, lymphatic and nervous tissue9. In rats, it is adults of both rodents and primates, BAT has a tendency to estimated that vascularity in BAT is 4 to 6 times greater than become sparse and is more closely linked systemically to the that in WAT1. This is supported with results of the present adrenals9,41. Recently, extensive connections have surfaced study, with the added observation that with increasing age between BAT and a variety of other tissues, identifying BAT vascularity of BAT decreases. The mt of BA have a as a pivotal gatekeeper of energy metabolism membranous protein in their inner membrane called homeostasis4,6,7,13,20,27,29,35,37–39,47–49. uncoupling protein 1 (UCP1), which is specific to The diameter of the average brown adipocyte (BA) is BAT7,18,20,29. Thus, UCP1 helps immunohistochemically to Iatropoulos and Williams 149

establish cells of origin37. UCP2 and UCP3, with high Table 3. Comparative Measurements of Metabolism homology to UCP1, can also be found in other tissues13,48. 2 The cytoskeletal integrity of BA can be monitored through Species Mass (kg) kcal/day kcal/kg kcal/m 35 the UCP/Actin ratio . Mouse 0.02 3.7 185 465 The average BA is multivacuolar in all known BAT Rat 0.25 25.0 100 625 locations. Yet, the BAs of the interscapular location have Rabbit 2.00 120.0 60 750 bigger vacuoles, on average, than those of either the Dog 15.00 535.0 36 1160 mediastinal or retroperitoneal BA locations. Younger male Man 65.00 1,600.0 25 1020 and female rats (6 weeks) have bigger BA vacuoles than Modified from Iatropoulos MJ, Drug Toxicokinetics, 245-266, 1993. older rats (36 weeks), and this remains true over time (Table 2). The rate of proliferation of BA, measured by the proliferating cell nuclear antigen (PCNA), remains steady ratio value than females (Table 2)24. over time, in a similar fashion to the adrenocortical cells. In general, in mammals, the smaller the animal the Both the size of the vacuoles in BA and the rate of higher the rate of metabolism and the greater the need for proliferation over time denote that the rate of BAT available energy. For example, the metabolic weight of involution is very slow, unlike involution in the thymus, humans is 25 Kcal/kg, of dogs 36, of rats 100, and of mice, under normo- or slightly hyperthermic conditions of 1 to 2°C 185 Kcal/kg. Thus, the energy needs of the rat are 4 times (Table 2). greater than energy needs of humans (Table 3)22,27,35. BAT directs mitochondrial biogenesis with the help of Function of Brown Adipose Tissue the peroxisome proliferator- activated gamma receptor coactivator 1α (PPARCA), which is abundant in BA, In adult rodents and primates, BAT is linked hepatocytes, and skeletal and cardiac myocytes and most systemically to the adrenals, pancreas, thyroids, and other parenchymal cells6,36. PPARCA together with nitric regionally to nervous and lymphatic tissue and to the oxide (NO) increases the expression of nuclear respiratory extracellular matrix throughout the body6,19–21,27,40,45,47,49. factor 1 (NRF1) together with an increase in cellular calcium Specifically, norepinephrine (NEPI) increases UCP1 and, and the signaling molecule cAMP. This results in more together with insulin, stimulates glucose uptake into BAT45. PPARCA which favors mitochondrial biogenesis, which Glucagon increases thermogenesis in glucagon-sensitive contain their own nitric oxide synthase (mtNOS) and further BAT, which is present in rats and mice, but not in hamsters49. promote the process of biogenesis via cGMP. Moreover, This hormone is both gluconeogenic and lipolytic, mainly in PPARCA, NO via NRF-1 and cAMP stimulate mt the liver, very similar to T3, which is also involved in the biogenesis in BA through upregulation of PPARCA and regulation of the UCP gene family21,47. These hormones mitochondrial transcription factor 1 (mtTF 1), and through provide free fatty acids (FFA) and acyl CoA as substrates upregulation of NOS in all the other parenchymal cells. which constitute the signal for activation of High levels of NO inhibit mt respiration and thermogenesis35,47. phosphorylation. Thus, NO serves as a gatekeeper of mt The initial event in the stimulation of thermogenesis is biogenesis and apoptosis6,33,36. 45 the binding of NEPI to the β3-adrenoreceptor . The receptor It is apparent that BAT acts as a systemic gatekeeper of binding in BA leads to activation of lipoprotein lipase (LPL), the extracellular matrix (EMA) homeostasis and signaling at which stimulates lipolysis liberating FFA and acyl CoA27. the operational tissue unit (OTU) level. OTU is the smallest Thus systemic antigluconeogenesis, fat anabolism and unit of operation within a given sector of an organ/tissue reduction of the intermediary metabolism in the liver go subunit, e.g. acini, lobules, etc. It comprises a certain together23. Further, during sustained stress, there is general number of parenchymal cells, nonparenchymal cells, protein catabolism and concomitant upregulation of heat supporting cells and EMA. The nonparenchymal cells are shock protein (HSP) 70 in the liver (critical temperature BA, fibroblasts and resident immune cells. Supporting cells 42°C). The energy supply process is stimulated in mammals make up the vasculature. EMA consists of matrix mainly by hypothermia, but also by hypoxia, and in primates metalloproteins, proteoglycans, adhesive proteins and other also by hypoglycemia (hunger). Rodents in general, and rats complex proteins and a milieu of cytokines and growth in particular, do not eat to warm up19,47. UCP1 factors3,25,40,43. Thus, processes like normoxia, (thermogenin) of BA allows mitochondria to oxidize normothermia, cell polarization, number of cell per OTU and substrates rapidly without ADP phosphorylation, thus tissue, cell-to-cell communication, differentiation, uncoupling oxidation from phosphorylation and instead apoptosis, proliferation and repair are maintained effectively promoting the dissipation of oxidation energy as heat35,47. at the OTU level. Older rats have a higher temperature (+1.1°C) than younger Finally, BAT helps achieve the protective status of rats, and females have a higher temperature than males. The hibernation in certain animal species19,20,28,35,47. The UCP/A ratio value, monitoring BAT cytoskeletal integrity, protection consists of tolerance of gradual prolonged decreases with age, with male rats having a higher UCP/A hypothermia, hypoxia, hypoglycemia and general sustained 150 Brown Adipose Tissue Function and Pathology

Fig. 1. Mediastinal BAT containing BA with multiple vacuoles Fig. 4. High magnification of a hibernoma located in the anterior and granular cytoplasm with round central nuclei. PCNA mediastinum. The cells are pleiomorphic, with stain × 400. anisocytosis and anisokaryosis, with centrally located rounded nuclei and prominent nucleoli constituting areas of anaplasia. H&E × 400.

Fig. 2. Hibernoma located in the anterior mediastinum. The Fig. 5. High magnification of a different mediastinal hibernoma, neoplasm is composed of multivacuolated BA exhibiting a with anaplastic, pleiomorphic cells, with anisocytosis and lobular pattern with prominent vascular supply. anisokaryosis with karyomegaly and prominent nucleoli. H&E × 40. H&E × 400.

Fig. 3. Higher magnification of the previous hibernoma. The Fig. 6. Liposarcoma of the anterior mediastinum. Clusters of cells are uniform, round or oval, intermixed multi- and pleiomorphic and anaplastic BA-like cells with foam cells, univacuolar, with a granular cytoplasm and a dark giant cells, and fibroblast-like cells. The anaplastic BA- nucleus. H&E × 400. like cells have karyomegaly with a nucleolus demonstrating vesicular arborization. H&E × 400. Iatropoulos and Williams 151 suppression of metabolism with concomitant decrease in the (Figs. 2 and 3). Ultrastructurally, there is abundance of immune response capabilities and increase in antioxidant mitochondria, which are round to tubular with parallel defenses. Furthermore, hibernation enables the liver to transverse cristae and a very prominent Golgi apparatus, utilize fat as an energy substrate in the presence of protein making them very similar to adrenocortical cells. Each BA catabolism (≥ 10%). Hibernation in some ways resembles has a well-defined basal lamina and scarcity of RER9,14,44,50. the status of neoplasia and neoplastic angiogenesis32,40. Based on recent unpublished experience in a 2-year Sprague- Dawley rat study with hibernomas, the rate of proliferation Pathology of Brown Adipose Tissue of BA measured by PCNA is significantly increased within the neoplasm compared to the BA immediately outside. From the few available data from laboratory animals to Moreover, within the hibernoma, the number of apoptoses date, hypertrophy of BA constitutes the initial mode of were decreased. response to increased functional demands. This is an To date, malignant hibernomas have never been adaptive process51. The rate limiting step is that enlarged conclusively described in humans or rodents11,16,26,50. On the BA have fewer receptors31. Recently, agonists of other hand, the IARC classification of rodent tumors peroxisome proliferator-activated receptor gamma (PPARγ) describes as malignant hibernoma a neoplasm consisting of have been found to induce BA hypertrophy49. PPAR-γ is a lobules of clusters of pleiomorphic cells with centrally member of a family of nuclear receptor genes that encode located dense and rounded nuclei and varying amounts of receptors for steroid and thyroid hormones, vitamin D and intracytoplasmic vacuoles showing infiltrative growth with retinoic acid. They are expressed in BA, WA, colonic evidence of dissemination and metastases34. The authors, enterocytes, immunocytes and vascular endothelial cells10. instead, because of lack of conclusive evidence of The first group of agonists includes the thiazolidinediones, dissemination and metastasis, even in the presence of e.g. Troglitazone/Rezulin, Rosiglitazone, and infiltrative growth within the immediate vicinity, would Pioglitazone5,17. The second group includes L-tyrosine rather call this neoplasm hibernoma with areas of anaplasia based compounds, e.g. GI262570X10. Moreover, gradual (Figs. 4 and 5). increases in plasma catecholamines, which occur in cachetic A malignant neoplasm of lipomatous origin is the and sustained chronic hypoxemic states, also induce BA liposarcoma. This neoplasia consists of lobular clusters of hypertrophy12. pleiomorphic BA-like cells with foam cells, giant cells, With further increased functional demands due to myxoid cells, fibroblast-like cells and stellate cells with hypothermia, sustained upregulation of glucose utilization, either one cell type predominating or consisting of a mix of down regulation of the de novo fatty acid synthesis, all these cell types. The anaplastic BA-like cells have a large suppression of lipoprotein lipase, NO downregulation and central nucleus and a nucleolus demonstrating vesicular reduction in intermediary metabolism, the conditions for arborization (Fig. 6). In the stroma are usually myxoid stimulation of BA proliferation are set. This BA hyperplasia changes and in the periphery, areas of infiltration, was studied extensively in B6C3F1 mice and Wistar rats in dissemination and expansion into the vicinity. In humans, two 2-year studies with Troglitazone17. Based on results liposarcoma, although rare, is seen occasionally. The from preliminary shorter-term studies which preceded the 2- neoplasm occurs predominantly in males and is usually year studies, BA hyperplasia was present in both genders of located in thighs or retroperitoneum26,50. In rats, it occurs rats and mice. This BA hyperplasia did not result in BA equally in both genders and has been observed in many neoplasia/hibernoma after 24 moths of gavage with locations with an incidence of less than one percent16,34. Troglitazone (up to 800 mg/kg/d in male and female mice Recently, male and female transgenic (Gγ/T) mice with and male rats, and up to 400 mg/kg/d in female rats). the hybrid gene constructs including human fetal Gγ globin Unfortunately, in these 2 studies, the rectal temperature and promoter linked to the simian virus 40 T antigen, developed the plasma catecholamine levels were not obtained17. It is hibernomas at an incidence of 40–75% within the first 4 conceivable that increases in BA proliferation, without months of age. The location of the hibernomas was concomitant BA decreases in apoptosis are not strong interscapular, periadrenal and pericardial. In addition, 20% enough stimulus of BA neoplasia. Perhaps with the advent of males developed prostatic neoplasms within 4 to 5 months of new PPARCA agonist compounds, which result in of age. All the hibernomas had a high mitotic index and high increases in intracellular calcium and downregulation of expression of UCP38. NO36, the opportunity to study BA hyperplasia in rodents To date, no chemical has been conclusively shown to will be made possible. induce BAT preneoplastic lesions or early (small) neoplasms BAT neoplasms, known as hibernomas, are very rare or neoplasms in conventional strains of rats or mice. The benign neoplasms in both rodents and humans2,8,9,12,14– thiazolidinediones, which induce dose-related BA 16,26,30,34,44,46,50. They are composed of multivacuolated BA hyperplasia in both rats and mice, do not stimulate exhibiting a lobular pattern with prominent vascular supply. progression into neoplasia even after 2 years of continuous The cells are uniform, round or oval, intermixed multi- and exposure17. uni-vacuolar, with a granular cytoplasm and a dark nucleus 152 Brown Adipose Tissue Function and Pathology

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