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 .