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Demonstration of a catecholaminergic innervation in human perirenal brown adipose tissue at various ages in the adult.

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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.
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Figs. 6, 7. Electron micrographs of human perirenal BAT of 27-year-
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Fig. 6. Mitochondria (arrows) in lipid-freecyto lasmic space of brown
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