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Morphological studies on denervated brown adipose tissue.

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Morphological Studies on Denervated
Brown Adipose Tissue '
BERNARD G. SLAVIN AND SOL BERNICK
Department of Anatomy, University o f Southern California,
School of Medicine, 2025 Zonal Avenue,
Los Angeles, California 90033
ABSTRACT
Intact and denervated brown fat lobes of normal and coldexposed mice were studied by light and electron microscopy. Following two
weeks of denervation in normal and cold-exposed mice, denervated brown fat
cells were hypertrophied because of lipid accumulation. In normal mice there
was a transient increase in glycogen. In cold-exposed mice, the quantity of
glycogen in intact and denervated brown fat was greater than in normal mice.
Mitochondria increased in size, and the number of cristae was greater in intact
fat of cold-exposed mice than in denervated brown fat. Carbon perfusion of
blood vessels demonstrated a decreased capillary bed in denervated fat. Capillary
constriction noted only in denervated brown fat is indicative of reduced blood
flow.
The major role of brown adipose tissue
is one of thermogenesis during arousal
from hibernation and during cold-exposure
(Smith and Horwitz, '69). The thermogenic response is triggered by the innervation of brown adipose cells by sympathetic
nerves (Wirsen and Hamberger, '67; Bargmann et al., '68; Ochi et al., '69; Cottle,
'70; Cottle and Cottle, '70). Several investigators show that surgical removal of
nerves to brown adipose tissue results i n
cellular hypertrophy because of lipid accumulation (Hausberger, '34; Sidman and
Fawcett, '54), transient mobilization during starvation and cold exposure (Hausberger, '34; Sidman and Fawcett, '54; Hull
and Segall, '65), depletion of norepinephrine stores (Weiner et al., '62; Forn et al.,
'70), impaired glucose utilization (Steiner
et al., '70), and reduction of blood flow
(Kuroshima et al., '69).
There are no reports on the fine structure of denervated brown fat, Nor are
there any morphologic data corroborating
the physiologic studies showing reduced
blood flow in denervated brown fat.
One part of this report deals with a light
and electron microscopic study of denervated brown fat cells to correlate known
cytologic and physiologic findings in these
cells (e.g., increased lipid deposition, reANAT. REC., 179: 497-506.
tardation of lipid mobilization, transient
glycogen accumulation) with the cytologic
appearance of the cells. Thus, the morphology of lipid droplets, glycogen and
mitochondria in denervated brown fat cells
is described. In the second part of this report the carbon-perfused vascular trees of
intact and denervated brown f a t is studied
for possible structural alterations in blood
vessels following denervation.
MATERIALS AND METHODS
Forty-eight young male C57BL mice
were used in this study. In each mouse
the right lobe of interscapular brown fat
was surgically denervated (local) ; the left
lobe acted as the intact but sham-operated
control, Twenty-four of these mice were
kept at room temperature ( 2 3 ° C ) and fed
ad lib while a similar number were kept
at 4°C (three mice per cage).
Both lobes of brown fat in each mouse
at 23 "C were examined histochemically
for neutral f a t (oil Red 0 ) and glycogen
(PAS) following one, three, seven, and 14
days of denervation. Intact and denervated brown fat lobes of mice exposed to
4°C for two and five weeks were also exReceived Sept. 7, '72. Accepted Jan.30, '74.
1 Supported by grant (FR-05356)from National Institutes of Health, US. Public Health Service.
497
498
BERNARD G. SLAVIN AND SOL BERNICK
amined, except that denervation preceded
cold exposure by one week. For glycogen
controls, the sections were digested with
diastase.
Five animals each from the room temperature (14 days) and coldexposed (five
weeks) groups were anesthetized with sodium pentobarbital and perfused through
the left ventricle with warm saline followed by a carbon-gelatin suspension for
the demonstration of vascular patterns i n
the intact and denervated brown fat lobes.
Both lobes of f a t were removed in toto,
fixed in a solution of alcohol, formalin,
and acetic acid and embedded in nitrocellulose.
In all cases, brown f a t lobes were removed for study during the mid-morning
hours. Completeness of denervation was
determined by observing the typical hypertrophic cellular response.
At one, three, seven, and 14 days following surgical denervation, brown f a t
lobes were fixed by cardiac perfusion or
immersion in 3% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.2, and diced into
small blocks (1-2 mm3). Following one to
three hours of fixation, the blocks were
rinsed for one hour in cold 0.1 M phosphate buffer, pH 7.2, and post-fixed in cold
1% OsO, buffered with 0.1 M phosphate,
pH 7.2, for one to two hours. The blocks
of tissue were dehydrated in ethanol and
embedded in Epon and Araldite according
to Mollenhauer ('64). Sections (1-2 p )
were cut on a n MT2 Porter-Blum microtome and stained with Toluidine Blue or
with silver according to the method of
Rosenquist et al. ('71). Blocks were sectioned and stained with uranyl acetate
(Watson, '58) and lead citrate (Reynolds,
'63), and examined with a n AEI EM6B
electron microscope.
OBSERVATIONS
Light microscopy and histochemistry
Adipocytes of intact brown fat lobes
from animals maintained at room temperature were spherical or oval in shape
(fig. 1 ) . Nuclei were usually centrally located, and the cytoplasm was filled with
mitochondria and a number of triglyceride
droplets of varying size, Following PAS
treatment, fine glycogen granules were dis-
persed throughout the cytoplasm (fig. 3 ) .
One day following denervation, the adipocytes enlarged because of the coalescence
of neutral lipid droplets, and heavier deposits of glycogen granules were noted
(fig. 4 ) . By the third day following denervation, cells were filled with abundant lipid
droplets and resembled white fat cells
(fig. 2). Seven and 14 days following denervation, the adipocytes were more hypertrophic and only a n occasional cell contained glycogen granules (fig. 5).
When animals were placed in the cold
for two or five weeks, lipid droplets decreased in size in the adipocytes of innervated lobes. However, cells of the
contralateral denervated lobes were hypertrophic, Glycogen deposition in intact
brown fat of coldexposed mice was greater
than that in intact brown f a t of animals
maintained a t room temperature; and glycogen deposition in denervated brown fat
of cold-exposed mice was the same as that
in intact tissue of cold-exposed mice.
Electron microscopy
In brown f a t cells from intact, nondenervated tissue, the lipid droplets varied
in size and were surrounded by abundant
mitochondria with well organized cristae
(fig. 9). The cells contained scattered free
ribosomes and glycogen particles, while
profiles of tubular elements were rare. The
plasma membrane of the cells, covered by
a typical amorphous cell coat, was smooth
and regular except for occasional micropinocytotic invaginations. Abundant capillaries were in close contact with the cells.
An uniformly narrow interstitial space between fat cells was filled with variable
amounts of connective tissue fibers.
Following one day of denervation, the
ultrastructure of brown fat cells was normal except for additional glycogen granules and larger lipid droplets (fig. 10).
After three days of denervation, glycogen
deposition decreased in some but not all
cells. Glycogen granules were found adjacent to mitochondria and lipid droplets.
Mitochondria of denervated brown f a t cells
were no different from intact cells, except
that they were in closer contact with each
other and contained granules. Brown f a t
cells denervated for seven or 14 days
MORPHOLOGY OF DENERVATED BROWN FAT
499
showed further cellular hypertrophy and a following one day of denervation which
subsided after seven days, Tuerkischer and
reduction in glycogen content.
In brown fat cells of mice exposed to Wertheimer ('42) and Sidman and Fawcold for two or five weeks, the lipid con- cett ('54) found that glycogen levels in
tent was reduced, while greater quantities white and brown fat increased prior to
of glycogen particles were located between lipid deposition in fasted-refed rats. The
mitochondria and lipid droplets. Mito- increased glycogen was greater than that
chondria were generally enlarged and the demonstrated in the present investigation.
cristae were usually more tightly packed The refeeding of fasted animals is a stimuthan that seen in the room temperature lus for lipid and glycogen deposition which
controls (fig. 11). The morphology of mito- would explain the differences in the
chondria i n denervated cells of cold- findings .
The increased glycogen observed in inexposed mice was highly variable. In most
denervated cells, the mitochondria were tact brown fat cells of cold-exposed mice
not as large nor were their cristae as well has not been previously observed, Thomson et al. ('69) reported on the disappearorganized as in intact cells (fig. 12).
In denervated brown fat cells of nor- ance of glycogen from brown f a t cells of
mal or cold-exposed mice there were no rats exposed to cold for 24 hours. Not until
changes observed in other cellular organel- two weeks of cold exposure did glycogen
les such as micropinocytotic vesicles, er- levels return to normal, Suter ('69) obgastoplasm, lysosomes, etc., compared to served the disappearance of glycogen from
brown fat cells of rats exposed to cold for
innervated cells.
up to one year. Re-exposure of cold-exposed
Vascular study
rats to room temperature restored the glyThe carbon perfused vascular tree of cogen content of the cells. Our results with
denervated brown fat lobes was less marked mice might represent species differences,
than in intact lobes (fig, 6). Terminal differences in feeding patterns or diet, or
capillary networks were not demonstrable differences in tissue processing.
There was no qualitatively detectable
in many areas. Where present, only a few
terminal capillaries were seen arising from change in glycogen deposition in denerarterioles. In contrast, the vascular distri- vated brown fat of mice at 4°C. Apparently,
bution in the innervated lobes was exten- the metabolic requirements for glycogen by
sive, and the terminal capillary networks brown fat cells of cold-exposed mice is indiffusely encircled the adipocytes. The dependent of an intact nerve supply. Since
capillaries appeared fully patent and inti- it has been established that glycogen formately associated with the adipocytes in mation is a prelude to lipid deposition in
intact tissue (fig, 7). However, the capil- fat cells of animals starved and refed
laries in the denervated lobes were mark- (Tuerkischer and Wertheimer, '42), it
edly constricted (fig. 8). These findings could be that brown fat cells of coldwere observed i n mice maintained at room exposed animals utilize glycogen in a diff eren t manner.
temperature and at 4°C for five weeks.
A dual nerve supply in brown adipose
DISCUSSION
tissue has been described by Derry et al.
The present report shows that local sur- ('69). This consists of intrinsic ganglia
gical denervation altered the morphology with short adrenergic neurons innervating
of brown adipose cells of mice in response the cells, and extrinsic or long adrenergic
to normal and subnormal environmental neurons innervating blood vessels via the
temperatures. Denervation was rapidly fol- sympathetic chain. These investigators
lowed by cellular hypertrophy due to lipid found that immunosympathectomy or loaccumulation. This is i n agreement with cal surgical denervation depleted the catethe morphological and biochemical find- cholamine stores in the extrinsic but not
ings of Hausberger ('34), Sidman and intrinsic neurons. If this is true, then surFawcett ('54), and Kuroshima et al. ('69). gical denervation would not affect the neuIn mice kept at room temperature, there rons innervating individual brown fat cells.
was an increase in intracellular glycogen Of several hundred thin sections of den.
500
BERNARD G. SLAVIN AND SOL BERNICK
ervated tissue, we did note occasional nerve guinea pigs and man (Hauschild et al.,
fibers adjacent to parenchymal cells. This '70), and in pituitary tumors (Schechter,
may have represented faulty surgery or '72). Obviously, a carefully timed morphonerve regeneration. Many more sections logic study (perhaps using stereological
need to be examined, especially since the techniques ) of blood vessels immediately
cells are in a hypertrophic state which following denervation would be helpful to
would reduce the chance of sectioning clarify this point,
through an adjacent nerve fiber. Intrinsic
ACKNOWLEDGMENTS
ganglion cells reported within brown fat
The
authors
wish to express their apprerequire morphologic verification. We noted
ciation to Mrs. Nora Tong and Miss Judy
no cells at the light or electron microscopic Grieshaber for their excellent technical aslevel which resembled typical sympathetic sist ance .
ganglia. Further morphologic studies are
LITERATURE CITED
underway in our laboratory to determine
the presence or absence of intrinsic gan- Bargmann, W., G. v. Hehn and E. Linder 1968
frber die Zellen des braunen Fettgewebes und
glion cells in brown fat.
ihre Innervation. 2. Zellforsch., 85: 601-613.
The present data show that denervation Cottle,
M. K. W., and W. H.Cottle 1970 Adrenof brown fat leads not only to cellular hyergic fibers i n brown f a t of cold-acclimated
rats. J. Histochem. Cytochem., 18: 116-119.
pertrophy, but also to a reduction in the
W. H. 1970 The innervation of brown
vascular bed. Kuroshima et al. ('69) dem- Cottle,
adipose tissue. In: Brown Adipose Tissue.
onstrated a decreased rate of blood flow
0. Lindberg, ed. American Elsevier Publishing
through denervated brown fat in coldCo., New York, pp 155-178.
exposed rats compared to intact controls. Derry, D. M., E. Schonbaum and G. Steiner 1969
Two sympathetic nerves supplies to brown adiIn addition, Herd and Goodman ('68)
pose tissue of the rat. Can. J. Physiol. Pharmafound that blood flow rates fell considerC O ~47:
. , 57-63.
ably in denervated white fat depots in fast- Forn, J., G. L. Gessa, G. Krishna and B. B. Brodie
1970 Increased lipolytic response to norepiing rats. In a recent report by Steiner et
nephrine i n isolated brown fat cells after symal. ('70) and Hollenberg et al. ('70) denpathetic denervation. Life Sci., 9: 429435.
ervated brown fat was slightly impaired in Hausberger, F. X. 1934 Uber die Innervation
its ability to take up labeled glucose and
der Fettorgane. 2. Mikranat. Forsch., 36: 231265.
convert it to lipid in vivo in response to
cold. But, in vitro, denervated brown fat Hauschild, U., M. F. Baghirzade and U. Kirsch
1970 Capillarkompression als Ischamiefolge.
showed an equal or an even higher reElektronenoptische Untersuchungen a n Papelcovery of label in lipids compared to inlarmuskeln des Menschen und des Meerschweinchens. Virchows. Arch. Path. Anat., 351: 205tact tissue. The authors postulated that the
224.
neural influence is exerted on blood flow
Herd, J. A., H. M. Goodman and S. A. Grose
rather than directly on the metabolism of
1968 Blood flow rates through adipose tissues
the brown fat cell.
of unanesthetized rats. Am. J. Physiol., 214:
263-268.
Whether the reduction of vascularity is
C. H., A. Angel and G. Steiner 1970
due directly to loss of innervation or Hollenberg,
The metabolism of white and brown adipose
whether it is a result of mechanical imtissue. Can. Med. Assoc. J., 103: 843-849.
pingement upon capillaries by the hyper- Hull, D., and M. M. Segall 1965 Sympathetic
nervous control of brown adipose tissue and
trophic fat cells, remains to be determined.
heat production in the new-born rabbit. J.
Although endothelial cells of capillaries
Physiol., 181: 458467.
are known to contract, there appears to Kuroshima, A., K. Doi and S. Itoh 1969 Nerbe no concomitant reduction in the caliber
vous and humoral influences on the blood flow
response of brown adipose tissue to cold in the
of the vessels (Majno et al., '69; Poirier,
rat. Jap. J. Physiol., 19: 392402.
'69). It is our belief, based on the mor- Majno,
G., S. M. Shea and M. Loventhal 1969
phology of capillaries at the fine structural
Endothelial contraction induced by histaminelevel, that constriction, or rather, comprestype mediators: A n electron microscopic study.
J. Cell Biol., 42: 647-672.
sion of the capillaries is a result of exH. H. 1964 Plastic embedding
panding brown fat cells. Compression of Mollenhauer,
mixtures for use in electron microscopy. Stain
capillaries has been reported adjacent to
Techn., 39: 111-114.
contracted anoxic myocardial Cells O f Ochi, J., M. Konishi and H. Yoshikawa 1969
MORPHOLOGY OF DENERVATED BROWN FAT
Morphologischer Nachweis der Sympathischen
Innervation des braunen Fettgewebes bei der
Ratte. 2. Anat. Entwick1.-Gesch., 129: 259-267.
Poirier, J. 1969 Les cellules endotheliales des
capillaires et des veinules se contractent-elles?
Press. Med., 77: 2122.
Reynolds, E. S. 1963 The use of lead citrate at
high pH as a n electron-opaque stain i n electron microscopy. J. Cell Biol., 17: 208-212.
Rosenquist, T. H., B. G. Slavin and S. Bernick
1971 The Pearse silver-gelatin method for
light microscopy of 0.5-2 p plastic sections.
Stain Techn., 46; 253-257.
Schechter, J. 1972 Ultrastructural changes in
the capillary bed of human pituitary tumors.
Am. J. Pathol., 67: 109-126.
Sidman, R. L., and D. W. Fawcett 1954 The effect of peripheral nerve section o n some metabolic responses of brown adipose tissue i n mice.
Anat. Rec., 118: 487-507.
Smith, R. E., and B. A. Horwitz 1969 Brown
fat and thermogenesis. Physiol. Rev., 49: 330425.
501
Steiner, G., M. Loveland and E. Schonbaum 1970
Effect of denervation on brown adipose tissue
metabolism. Am. J. Physiol., 218: 566-570.
Suter, E. R. 1969 The fine structure of brown
adipose tissue. 1. Cold-induced changes i n the
rat. J. Ultrastructural Research, 26; 216-241.
Thomson, J. F., D. A. Habeck, S. L. Nance and
K. L. Beetham 1969 Ultrastructural and biochemical changes i n brown fat in cold-exposed
rats. J. Cell Biol., 41: 312-334.
Tuerkischer, E., and E. Wertheimer 1942 Glycogen and adipose tissue. J. Physiol (London),
100: 385409.
Watson, M. L. 1958 Staining of tissue sections
for electron microscopy with heavy metals. J.
Biophys. Biochem. Cytol., 4: 475-478.
Weiner, N., M. Perkins and R. L. Sidman 1962
Effect of reserpine on noradrenalin content of
innervated and denervated brown adipose tissue of the rat. Nature (London), 193: 137-138.
Wirsen, C., and B. Hamberger 1967 Catecholamines i n brown fat. Nature (London), 214:
625-626.
PLATE 1
EXPLANATION OF FIGURES
Figures 1 and 2 represent brown fat fixed in glutaraldehyde, postfixed in OsO, and embedded in Epon and Araldite. Sections were cut a t 1 p, de-plasticized, and impregnated with
silver nitrate. x 1,000.
1 Typical innervated brown adipose tissue from a mouse kept at room temperature. Note
numerous lipid vacuoles ( L ) of varying size surrounded by coarse granular material
(presumably mitochondria). Arrow points to a red blood cell within a capillary.
2
Brown adipose cells following three days of denervation. Note extensive cellular hypertrophy due to lipid accumulation. L, lipid vacuole.
Figures 3-5 represent brown fat of mice kept at room temperature. Periodic acid-Schiff
(PAS) stain. x 500.
Intact (non-denervated) brown fat showing fine glycogen granules dispersed throughout the section.
Following 24 to 48 hours of denervation there appears to be heavier deposits of glycogen
in most of the cells. This transient increase in glycogen deposition is most marked following 24 hours of denervation.
A reduction i n glycogen is noted following seven days of denervation. Here, only a n occasional cell is found to contain glycogen.
Section through both lobes of interscapular brown fat following carbon-gelatin perfusion.
Lobe in the upper half is the intact, innervated one; i n the lower half is the contralateral denervated lobe. Note the sparseness of vascular supply in the denervated lobe
compared to the innervated one. x 25.
One micron-thick plastic embedded section of innervated brown fat. Mouse a t 4°C.
Note numerous patent capillaries ( c ) adjacent to multilocular fat cells. L, lipid droplet.
Toluidine Blue. X 1,000.
Similar section as above of denervated brown fat from mouse kept at room temperature.
Note the drastic cellular hypertrophy (very similar to that at 4°C). Capillaries ( c ) i n
this case appear flattened and smaller i n caliber. Note two cells which indent a capillary (arrows). L, lipid droplet. Toluidine Blue. x 1,000.
502
MORPHOLOGY OF DENERVATED BROWN FAT
Bernard G. Slavin and Sol Bernick
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
9 Portions of two cells from innervated brown fat. Animal kept at room temperature.
Note typical mitochondria ( M ) and lipid droplets (L). x 17,500.
10 Brown f a t cell following 24 hours of denervation. Glycogen (GLY) is found in clumps
adjacent to mitochondria ( M ) and lipid droplets ( L ) . Mitochondria are found in greater
juxtaposition. x 17,500.
11
Portions of two brown f a t cells from innervated lobe of mouse exposed to 4°C for two
weeks. Note the large mitochondria1 size and numerous cristae. GLY, glycogen. L, lipid
droplet. x 22,5QO.
12 Brown f a t cell of contralateral denervated lobe from same animal as i n figure 11. In
general, mitochondria are not as large and do not undergo organizational changes noted
above. L, lipid droplet. GLY, glycogen. x 22,500.
504
MORPHOLOGY OF DENERVATED BROWN FAT
Bernard G. Slavin and Sol Bernick
PLATE 2
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