Demonstration of a catecholaminergic innervation in human perirenal brown adipose tissue at various ages in the adult.код для вставкиСкачать
THE ANATOMICAL RECORD 215251-255 (1986) Demonstration of a Catecholaminergic Innervation in Human Perirenal Brown Adipose Tissue at Various Ages in the Adult JEFFREY D. LEVER, ROLAND T.JLJNG, JOSEPH 0. NNODIM, PETER J. LESLIE, AND DEBORAH SYMONS Department of Anatomy, University College, Cardiff CFl l X L , Wales (J.D.L., J. 0.N., D.S.); Department of Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SE: Scotland (R.TJ., €?J.L.) ABSTRACT From light and electron microscopic studies on perirenal fat from human donors 27, 39, and 47 years old, unequivocal evidence is found for the presence of islands of multilocular adipocytes. After glyoxylic acid condensation for visualisation of biogenic amines, catecholaminergic nerve plexuses were demonstrated around the arteries of supply to these islands and within the intercellular spaces between their constituent adipocytes. Transmission electron microscopy revealed the cytology of these adipocytes to be similar to that of brown adipocytes in rodents. These findings are viewed in the light of a possible energetic potential for brown adipose tissue in the human adult. Brown adipose tissue (BAT) has been described extensively in adult man in light microscopic studies (Heaton, 1972; Tanuma et al., 1976), and more recently Cunningham et al. (1985) have determined its presence in human perinephric fat depots over a wide adult range by visual inspection, electron microscopy, and nucleotide binding (to the tissue-specific 32-kDa uncoupling protein). The amount of this uncoupling protein in mitochondria from human adult BAT is estimated to be of the same order as that in BAT mitochondria of partially cold-adapted guinea pigs, a finding indicative of some thermogenic potential of adult human brown fat. That the sympathetic nervous system plays a n important role in the adaptational response of BAT in rodents during cold acclimation is suggested by the increase in catecholamine levels (by formaldehyde-induced fluorescence-FIF) demonstrated by Cottle and Cottle (1970) in the nerve plexuses related to rat brown adipocytes and also by the studies by Cottle et al. (1967) which revealed a n increased noradrenaline turnover in rat BAT in the same circumstances. Further, BAT has been identified as the seat of regulatory nonshivering thermogenesis (NST) in mammalia (Smith, 1961; Ball and Jungas, 1961; Afzelius, 1970; Foster and Frydman, 1978).Additionally, Mory et al. (1984) have shown that noradrenaline controls the concentration of the specific 32-kDa uncoupling protein in BAT. A connection between the lipolytic and thermogenic potential of BAT and the regulation of body weight has been postulated by Rothwell and Stock (1979, 1982) on the basis of studies which demonstrated a large increase in the metabolic sensitivity of rat BAT to exogenous noradrenaline in the course of “cafeteria” feeding during which animal body weight did not significantly increase. Although respiratory capacity measurements by Cunningham et al. (1985) suggest that the total perinephric fat in adult man probably only accounts for 0.2% 0 1986 ALAN R. LISS, INC of the whole body respiratory response to infused noradrenaline, the fact that mitochondria from this human adult BAT do possess functional uncoupling protein is sufficient reason to enquire if the tissue is innervated by sympathetic nerves in man at different ages, and this enquiry forms the basis of the present investigation. MATERIALS AND METHODS Perinephric BAT was obtained from three human donors 27, 39, and 47 years old who were operated on for nonmalignant conditions involving the kidney. (Ethical approval was given by the Tayside Health Board Ethical Committee.) Material for light microscopy was fixed in 10% formol saline prior to processing as follows: 1)paraffin embedding and sectioning at 10 pm for either haematoxylin and eosin (H & E) staining or for silver impregnation by the Holmes method, and 2) freeze microtomy a t 15 pm for the application of the oil Red 0 method for lipid demonstration. Material for fluorescence microscopy was frozen by dry ice onto chucks and cryostat sections 12-15 pm thick were cut a t -3O”C, thawed directly onto glass slides (kept a t room temperature) and processed for the demonstration of catecholamines by a modification of the sucrose-potassium phosphate-glyoxylic acid (SPG) technique of de la Torre and Surgeon (1976) employing the following time sequences: five 1-second dips in SPG solution; 5-minute drying time under a warm airstream of a hair dryer; 5 minutes in a n oven a t 80 f 1°C. After mounting in liquid paraffin (B.P.), slides were viewed with the aid of BP 340-380 and suppressor LP 430 filters, in a Leitz Laborlux 12 microscope with a Pleomopak 2.5 fluorescence vertical illuminator. In orReceived September 26, 1985; accepted January 15, 1986. Address reprint requests to Jeffrey D. Lever, Department of Anatomy, University College, Cardiff CF1 1XL, Wales. 252 J.D. LEVER ET AL. perirenal fat. These islets were larger and more numerous in younger donors. In both H & E and oil red 0 preparations, islets were characterised by a n extensive capillary plexus. Contrasting with the adjacent unilocular adipocytes in which nuclei were flattened and peripherally located, the nuclei of multilocular adipocytes were near-rounded in outline (Fig. 1). After silver impregnation, a n abundant vasomotor nerve plexus was discernible on the walls of small arteries of supply and fine nerves were traced in parenchymal distribution between multilocular adipocytes. Within these plexuses were putative catecholaminergic nerve fibres characterised by their blue-green fluorescence in SPG preparations (Figs. 2-4). The distributions of SPGpositive nerve plexuses were both vasomotor to arteries of supply (Fig. 2) and parenchymal (Figs. 2-4) at all ages. Reference to fluorescence and light micrographs of the same fields (see Materials and Methods) enabled a n exact topography to be assigned to the fluorescent nerve plexuses (cf. Figs 4, 5). In a comparison of histochemical RESULTS preparations from the three donors, it was clear that the Observations by light and fluorescence microscopy extent and fluorescence intensity of the parenchymal In all cases, islets of multilocular adipocytes were catecholaminergic nerve plexuses diminished with age found surrounded by unilocular adipocytes within the in the present series (Figs. 2-4). der to establish histological identities, coverslips and mountant were removed from sections after fluorescence photography and the same sections were then formoldipped and stained with H & E for light microscopy (see Figs. 4,5). Material for electron microscopy was fixed in ice-cold cacodylate-buffered 3%glutaraldehyde for 4 hours prior to a n overnight buffer wash and subsequent postfixation in 1%buffered osmium tetroxideobefore processing to Araldite. Fine sections (600-900 A thick) were stained with uranyl acetate and lead citrate prior to examination in a Siemens Elmiskop I electron microscope at 60 kV. It should be noted that for clinical reasons the receipt of perirenal fat samples was often delayed for some 30-45 minutes with the result that the optimal fixation of tissues was not always possible. (Color figures 2, 3, and 4 were prepared and appear elsewhere in this issue. Please see pp. 217-229 for these figures and their accompanying legends). Fig. 1. Light micrograph of haematoxylin and eosin (H& E)-stained paraffin section of human (27-yearold) perirenal adipose tissue showing a n islet of multilocular adipocytes (9.Note near-rounded outlines of brown adipocyte nuclei (arrows),regional capillaries (c) and surrounding unilocular fat cells (stars). Scale bar: 10 pm ( x 750). SYMPATHETIC INNERVATION OF HUMAN BROWN FAT 253 Fig. 5. Light micrograph of a n H & E-stained preparation of the section portrayed by fluorescence microscopy in Figure 4 (see Materials and Methods). By comparing locations and profiles in the two figures it is possible to confirm that the distribution (arrows) of fluorescent nerves is indeed between multilocular adipocytes (m). Scale bar: 10 pm ( X 750). Observations by electron microscopy DISCUSSION Because of the ease with which islets of multilocular adipocytes could be located in the youngest (27 years) donor, material for electron microscopy was harvested from this source. The islets of multilocular adipocytes of human perirenal fat showed a n epitheliallike histological arrangement reminiscent of that within rat BAT (Fig. 6). At a parenchymal level, a multilocular lipid distribution was obvious (Fig. 6). Akin again to rat BAT, the bulk of the lipid-free cytoplasm within human multilocular adipocytes was occupied by large mitochondria many of which were characterised by densely packed internal cristae in a shelflike arrangement (Fig. 6). Unmyelinated nerve bundles were found a t pericapillary sites and in interspaces between individual parenchymal cells and between parenchymal cell groups (Fig. 7). Terminal features were observed in many of these nerves (Fig, 7) and included the presence of clusters of 800-1,000- A -diameter dense-sored vesicles as well as a number of smaller (400-600- A -diameter) clear vesicles. Although Stock and Westermann (1963) showed by biochemical extraction that rat BAT contains noradrenaline, similar extraction studies have not so far been applied to man. The justification for such studies is indicated by the present demonstration of catecholaminergic nerves apparently in vasomotor and parenchymal distributions in human BAT: findings similar to those already reported by Wirsen and Hamberger (1967) and Cottle et al. (1974) for sympathetic nerve deployment in the BAT of rodents and other small mammalia. Just as a reduction with age of the fluorescence intensity and, by inference, the catecholamine content of the parenchymal sympathetic plexus was observed by Derry et al. (1972) in the rabbit, the present observations indicate the same changes in man in a comparison between the BAT of the 27-year and 47-year-old donors. The significance of a catecholaminergic innervation within human BAT might, by extrapolation, be viewed in the context of the competence of human brown adipocytes in the performance of the lipolytic and thermogenic roles already demonstrated for BAT in laboratory SYMPATHETIC INNERVATION OF HUMAN BROWN FAT animals by Correll(1963), Flaim et al. (19761, and Foster and Frydman (1978). Since Rothwell and Stock (1979) and Himms-Hagen (1979)have obtained evidence in the rat and other animals which suggests that heat generated in BAT is significant for the dissipation of excess caloric intake during hyperphagia (luxuskonsumption) and that there is, in these animals, a direct relationship between BAT thermogenesis and body weight, speculations on the potential of such tissue in man (Blaza, 1983; Cawthorne, 1983)are pertinent, especially following the discovery of p 3 catecholamine agonist drugs by Arch et al. (1984). Further, any comment on the significance of the terminal features observed within axons (Fig. 7) situated intercellularly in the BAT parenchyma should include the observation that clusters of 800-1,000-A diameter dense-cored vesicles and numbers of smaller clear vesicles are not characteristic of adrenergic nerves and should prompt enquiries as to the exact identity of these neuronal processes. LITERATURE CITED Afzelius, B.A. (1970)Brown adipose tissue: Its gross anatomy, histology and cvtolom. In: Brown Adinose Tissue. 0. Lindbern. ", ed. New York: 2me;;can Elsevier, pp. i-31. Arch, J.R.S., A.T. Ainsworth, M.A. Cawthorne, V. Piercy, M.V. Sennitt, V.E. Thody, C. Wilson, and S. Wison (1984)Atypical a-adrenoceptor on brown- adipocytes as target for anti-obesity drugs. Naiure, 309r163-165. Ball, E.G., and R.L. Jungas (1961) On the action of hormones which accelerate the rate of oxygen consumption and fatty acid release in rat adipose tissue in vitro. Proc. Natl. Acad. Sci. 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