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Stereological assessment of age-related changes in lipid droplet surface area and vascular volume in rat interscapular brown adipose tissue.

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THE ANATOMICAL RECORD 220:357-363 (1988)
Stereological Assessment of Age-Related Changes
in Lipid Droplet Surface Area and Vascular Volume
in Rat lnterscapular Brown Adipose Tissue
JOSEPH 0.NNODIM
Department of Anatomy, College of Medical Sciences, University of Benin, PMB 1154,
Benin City, Nigeria
ABSTRACT The surface area of stored triglyceride and the volume of capillaries
in the interscapular brown fat pad of the rat have been adopted as morphological
indices of the overall thermogenic capability of the tissue. The present study examines the relationship between the chronological profiles of these parameters and
reported senile changes in the biochemical and physiological characteristics of brown
adipose tissue.
Lipid droplet surface density and vascular volume density were estimated by
computer-assisted planimetry with electron micrographs prepared from interscapular brown adipose tissue samples obtained from rats of various ages. The volumes of
the fat pad at these ages were also determined and used to calculate droplet surface
areas and vascular volumes.
Triglyceride surface area showed a 20-fold enhancement in the early postnatal
period (0.44 x lo2 cm2 a t birth; 9.43 x lo2 cm2 at 4 weeks). A further but less
remarkable increase occurred in adulthood (11.67 x lo2 cm2 at 6 months) and, in old
age, only a slight fall was noted (9.66 x lo2 cm2 at 2 years). Intralobular capillary
volume also rose sharply early during the first week after birth, reaching a peak at
4 weeks (2.40 x
cm3). The values recorded in adulthood and old age (1.90 x
lop2cm3 at 6 months; 1.76 x lo-' cm3 at 2 years) were not significantly different
from that obtained at 4 weeks of age.
These results show that the attainment of maximum values of lipid droplet surface
area and vascular volume in rat interscapular brown adipose tissue coincides with
the period of the tissue's peak metabolic capacity. However, although the thermogenic requirements of the older animal are less than those of the young, the relative
constancy of these parameters in adulthood is consistent with the persistent ability
of brown adipose tissue to respond to appropriate stimuli by hyperemia and rapid
intracellular lipid mobilization.
Brown adipose tissue has been established as a major
effector of nonshivering thermogenesis (NST) (Smith,
1961; Ball and Jungas, 1961; Foster and Frydman, 1978)
and the comparable phenomenon of diet-induced thermogenesis (DIT) (Himms-Hagen, 1979; Rothwell and
Stock, 1979). Typically, brown fat is characterized by
mitochondria-rich multilocular adipocytes, a n abundant
blood supply, and a dense catecholaminergic innervation (Fig. 1). In response to a noradrenergic signal, free
fatty acids are mobilized from intracellular triglyceride
stores for mitochondria1 @-oxidation,the thermal yield
of which is greatly enhanced by the presence of a 32-kD
uncoupling protein in the inner membrane of brown
adipocyte mitochondria (Heaton et al., 1978). The large
amounts of oxygen and substrates required by the tissue
are supplied through its extensive vascular network,
which also captures and disperses the heat generated
during thermogenesis.
The multilocular distribution of lipid within brown
adipocytes favors a more rapid mobilization of fatty acids
0 1988 ALAN R. LISS, INC.
from the triglyceride droplets compared to a unilocular
configuration, since in the former, a greater surface area
is exposed to a unit volume of cytoplasm. In hibernators
and many other mammals, the multidroplet storage format is retained throughout life (Afzelius, 1970). However, in the rabbit, Dawkins and Hull (1964) noted a
transformation with age in the pattern of lipid storage
in brown adipocytes such that the large interscapular
organ of the adult animal consists of unilocular cells.
This change occurred in parallel with functional involution of the tissue.
A similar attenuation in functional competence with
age has been reported for rat interscapular brown adipose tissue (ISBAT) by Barnard et al. (1970). In the
present investigation, the swface area of triglyceride
droplets and the volume of intralobular capillaries in
brown adipose tissue have been used a s measures of the
Received July 15, 1986; accepted June 22,1987.
358
J.O. NNODIM
thermogenic capacity of rat ISBAT. Both parameters 800 System (Cambridge Instruments, Ltd.). These data
have been estimated stereologically in ISBAT samples were used to derive the following:
from rats of various ages to determine their pattern of
Total brown adipocyte cytoplasmic area (Acy)
chronological change.
= Af - (Av Ap + An);
Vascular volume reached a peak a t 4 weeks and lipid
droplet surface area attained 80% of its maximum value
Lipid droplet boundary density [B~(l,cy)]
at that age-a period during which the metabolic capac= Bl/Acy;
ity of rat BAT has been reported to be maximal (Barnard
et al., 1970; Skala et al., 1970). High values for both
Lipid droplet surface density [S,(l,cy)]
parameters were also recorded in adulthood and old age,
= 4/a x B~(1,cy)
(Saltykov, 1958);
reflecting the persistent richness of the tissue's vascular
provisions and the multilocularity of brown adipocytes
Total parenchymal capillary areal density A~(v,pl)
in later life.
= AJAf;
+
MATERIALS AND METHODS
Animals
White Wistar rats were used in the present study.
Room temperature in the Animal House was maintained within a range of 20-25°C and a lightldark cycle
was ensured by use of automatic switching devices. Postweanling and adult rats were housed in pairs in plastic
cages with a wire mesh top. Clean tapwater and a pelleted stock diet were provided ad libitum.
Fetuses of known gestational age were obtained
through timed matings and cesarean delivery by a
breeding procedure described previously (Nnodim and
Lever, 1985).
Pilot Study
In a preliminary study to compare the distributions of
lipid droplet surface density and intralobular vascular
volume density of ISBAT of age-matched rats, fetuses
delivered by cesarean section after 20 days of intrauterine life were assigned to two groups of two animals each.
Similar assignations were made in respect to 18-weekand 6-month-old rat siblings. Samples were taken from
left ISBAT pads and processed for transmission electron
microscopy (TEM). For each animal, one grid of thin
sections was obtained from each of five randomly selected araldite blocks containing tissue. After heavymetal staining, four fields chosen by the grid-square
corner method of Weibel et al. (1966)were photographed
in a Siemens ELMISKOP I electron microscope at a
screen magnification of 1,000. The micrographs were
enlarged to x 3,000 at printing.
Each micrograph was analyzed for general field area
(Af), total brown adipocyte nuclear profile area (An),
total capillary luminal and endothelial cell profile area
(Av), total pericapillary cell profile area (Ap), and totaI
lipid droplet boundary length (Bl) with a QUANTIMET
Parenchymal vascular volume density V,(v,pl)
= A~(v,pl)
(Delesse, 1847).
In each age category, namely the 20-day-old(fetal), 18week-old, and 6-month-old rats, Snedecor's F-test comparisons for Sv(I,cy) and Vv(v,pl) between the two pairs
of age-matched animals showed consistent distributions
of both of these parameters. It was thus considered reasonable to assume that rats of the same age in the colony
would show homogeneity in the distributions of ISBAT
lipid droplet and vascular volume densities.
In further preliminary studies, based on 20 different
samples of various sizes (multiples of 3), it was found
that a random sample of 12 fields would satisfy the
requirements of a confidence interval of 10% with an
error probability of 5%.
Definitive Study
Comparable ISBAT lobule samples were taken from
two rats at each of the following times: a t birth and,
after birth, at 3 and 8 days, 4 weeks, and 2 years.
Specimens were processed for TEM and six representative micrographs were taken for each animal. The micrographs were analyzed as in the pilot study (vide
supra).
The fractional volume of parenchymal lobules in the
interscapular brown fat pad, Vv(pl,fp),for each age group
was determined by systematic point counting, employing a coherent test point system constructed according
to Weibel (19791, and was incorporated into the projection screen of a Carl Zeiss ULTRAPHOT I11 microscope.
Serial light microscope sections of whole ISBAT pads
were used for this purpose. The volume of the ISBAT
deposit a t each age, V(fp), was determined by the submersion method of Scherle (1970). Final calculations
TABLE 1. Cascade of calculations'
Phase of interest
Reference space
Cytoplasm (cy)
Brown adipocytes (ba)
Parenchymal lobules (PI)
Interscapular brown fat pad (fp)
Lipid droplets (1)
SV(1,CY)
Sv(1,ba) = Sv(1,cy)
Sv(1,pl) = Sv(1,ba)
Sv(1,fp) = Sv(1,pl)
SV(1)
= Sv(1,fp)
. Vv(cy,ba)
. Vv(ba,pl)
. VV(P1,fP)
. V(fp)
Intralobular capillaries (v)
VV(V,Pl)
VV(V,fP) = VV(V,Pl) . VV(Pl,fP)
= Vv(v,fp) ' V(fP)
VV
~~
~
'Empirically, the fractional volume of brown adipocyte cytoplasm in the parenchymal lobules Vv(cy,pl) was determined
directly rather than in steps [Vv(cy,ba) . Vv(ba,pl)]. Therefore, the working equation for deriving the surface density of lipid
droplets in the fat pad [Sv(l,fp)Jwas SV(1,fp) = Sv(l,cy) . Vv(cy,pl) . VV(p1,fp).
359
AGING CHANGES IN RAT INTERSCAPULAR BROWN FAT
I
THE
BROWN
I
FAT
I
I
I
NERVE AXOTS
6 TERMINALS
I
PA0
PARENCHYMAL
LOBULES
TABLE 2. Rat interscapular brown adipose tissue: Pad
volume [V(fp)],lipid droplet surface [S~(l,fp)],
and
intralobular capillary volume [Vv(v,fp)]densitites at
different ages1
lo3 crn-'1
10-2
V(fPY~ r n ~ )( X Sv(1,fP)
(VV(V,fP)
X 10-9
Age
(X
20 days
(fetus)
2.02 f 0.36
(23)
0.88 f 0.62
1.23 k 0.62
0
3.74 k 0.54
(27)
1.18 fr 0.20
1.81 f 0.35
3 days
6.67 f 1.45
(18)
2.07 k 0.33
2.33 f 0.67
8 days
10.90 f 0.47
(20)
27.83 f 2.04
(13)
31.50 f 3.30
(10)
33.75 f 3.65
(11)
34.75 f 4.69
(6)
2.75 f 0.35
0.94 f 0.27
3.39 fr 0.27
0.86 f 0.28
3.25 f 0.46
0.44 fr 0.27
3.46 f 0.19
0.56 f 0.23
2.65
0.58 f 0.17
(at birth)
VESSELS
I
I
CYTOPLASM
4 weeks
1
7 weeks
6 months
"SPACES"
MEMBRANES
-
LIPID DROPLElS
LIP10 OROPLETS
MITOCHONDRIA
GOLGI
ENOOPLASMIC
RFTICULUM
LYSOSOMES
PEROXISOMES
PAmlCLES
MITOCHONDRIA
-
-
2 years
_+
0.62
'Values are given as means f. standard deviation.
2Numbers in parentheses are numbers of rats used for determining
V(fp).
RIBOSOMES
LYSOSOMES
-
VESICLES
CYTOSOL PLASMALEMMA
PEROXISOMES
VESICLES
-
mates of lipid droplet surface density. It was assumed
that all tissue components would be compressed to the
same extent with no resultant effect on volume density.
Statistical Analysis
Consistency in the distributions of the parameters under
investigation as well as representative micrograph
Fig. 1. Hierarchical model of brown adipose tissue structure.
sample size were determined in preliminary studies (vide
supra).
The values of S(1) and V(v) recorded for all age groups
were performed as shown in Table 1 with a program- were examined for real differences by one-way analysis
of variance. Student's unpaired t test was used to evalmable calculator (TI 5511; Texas Instruments).
uate differences between pairs of age groups. All tests
Systematic Errors
were two-tailed.
Since the variance of a small sample (n < 30) tends to
RESULTS AND DISCUSSION
underestimate the true variance of the population, the
Lipid Droplet Surface Area
reason being that the sum of the squares of the deviaTable 3 and Figure 2 illustrate the changes with age
tions of the values in a sample about the sample mean
has a finite minimum value, Bessel's correction [d(n - in the surface area of fat droplets in the interscapular
11, where n = sample size (Moroney, 1951)] was applied brown fat pad of rats. A 20-fold increase was noted in
in each case to the sample variance to obtain the best the early postnatal period, from 0.44 x lo2 cm2 at birth
estimate of the population variance. Thus, the discrep- to 9.43 x lo2 cm2 at 4 weeks of age, and a peak was
ancy between the sample and population means is ad- reached at 6 months (11.67 x lo2 cm2). High levels were
generally maintained in adulthood and although a stajusted for.
The error of underestimation due to finite section tistically significant decrease had occurred by the end of
thickness (Holmes, 1927) was found to be 0.018% for the second year of life (9.66 x lo2 cm2), this final value
SV(1,cy)and 0.009%for VV(v,pl). This was considered to was comparable to that recorded in the first month of
be negligible in both cases and ignored. Similarly, no life, when the tissue exhibits very high metabolic activcorrection was made for tissue shrinkage, which accord- ity (Barnard et al., 1970).
According to Ahlabo and Barnard (1974), it is reasoning to Weibel (1969) is usually less than 5% for resinembedded material. An equally negligible effect of sec- able to assume that in BAT as in white adipose tissue
tion compression during microtomy was inferred from (WAT), hormone-sensitive lipase activity is predomithe nearly geomemtrically perfect circular profiles of nantly located in the cytosol rather than bound to a
free-standing lipid droplets in brown adipocytes. Hence, specific membrane system. Therefore, the surface area
no Loud factor (Loud et al., 1965) was applied to esti- of triglyceride exposed to cytoplasm could serve as a n
T
Atl--\+i-+-*
20d
0
3d
8d
h k s
6mths
7wks
Z4mths
AGE
Fig. 2. Changes with age in ISBAT lipid droplet surface area. Note sharp increase during
first month of life. Thereafter, triglyceride surface area remains high, decreasing only slightly
in old age.
i
20d 0
3d
Bd
\
4bks
I
7bks
6rths
24mtile
AGE
Fig. 3. Changes with age in ISBAT intralobular vascular volume. A marked increase is
evident early in the first week after birth. Subsequently, capillary fractional volume diminishes
sharply.
AGING CHANGES IN RAT INTERSCAPULAR BROWN FAT
361
TABLE 3. Rat interscapular brown adipose tissue: Lipid droplet surface area [%I)]
and intralobular capillary volume [V(v)] at different ages','
Age
20 days
(fetus)
Parameter
S(1)
( x 10' cm2)
0.25
V(V)
(x
0 (at birth)
3 days
8 days
4 weeks
7 weeks
6 months
24 months
(A)
(B)
(C)
(D)
(E)
(F)
(GI
(HI
0.14 k 0.03 0.44 f 0.08 1.38 f 0.22 2.94 f 0.34 9.43 k 0.76 10.23 f 1.45 11.67 f 0.64 9.66 f 1.67
cm3)
* 0.13
0.68 f 0.13 1.55 f 0.45 1.03 f 0.29 2.40 k 0.78
1.38 f 0.84
1.90 f 0.78 1.76
* 0.58
+
'Values are d v e n a s means standard deviation.
2Differences10ne-wayanalysis of variance revealed significant differences (P < 0.005) among the age groups with regard to both S(1) and V(v).
S(1): (B) vs (E), P < 0.001; (E) vs (G), P < 0.001;(G) vs (H), P < 0.001.(E) vs (H) not significant a t P = 0.1.
V(v): (C) vs (D), P < 0.01; (E) vs (F),P < 0.01. (E) vs (G), (E) vs (H) not significant at P = 0.1.
estimate of the potential of the tissue to mobilize substrate (fatty acids) from storage under stimulation. In
the distribution of a given volume of lipid, the surface
area achievable is proportional to the locularity of that
distribution. Thus, the multidroplet arrangement of triglyceride in the typical brown adipocyte may be considered to represent a more active disposition, metabolically, than is the case with the typical white adipocyte,
in which a unilocular distribution is closely approached.
In the BAT of adult hamsters exposed to a temperature of 4°C for 1week, Ahlabo and Barnard (1974) noted
a redistribution of lipid within brown adipocytes into
smaller and more numerous droplets. This reorganization was estimated to result in a two- to threefold increase in lipid droplet surface density. The transition
from the womb to a n extrauterine environment at birth
can be regarded as constituting a thermal challenge to
the neonate of a nature comparable to cold exposure. In
the present study, the sharpest increase in lipid droplet
surface density was recorded during the first week of
life (75.4% between day 0 and day 3, Table 2). Total
droplet surface area also increased considerably (threeto fourfold, Table 3 and Fig. 2) during the same period,
which coincides with the phase of rapid augmentation
in the specific activities of respiratory enzymes in rat
BAT (Barnard et al., 1970; Skala et al., 1970). According
to Jansky (19611, the specific activities of these enzymes
are valid indices of the theoretical maximum metabolic
capacity of the tissue.
Brown adipose tissue lipid droplet surface area was
relatively constant after the first month of life, diminishing only slightly in old age. This finding, coupled
with the electron microscope appearance of the tissue
(Figs. 4-6), refute the claim by Simon (1965) that rat
brown adipocytes generally transform into white adipocytes with age. Nevertheless, lipid droplet coalescence
has been observed in rat brown adipocytes from as early
as postnatal day 3, resulting in a progressive increase
in average droplet size (Nnodim and Lever, 1985). It is
conceivable that very large droplets could arise in this
way but in those cells where one or more droplets reach
a considerable size, there are numerous smaller-sized
additional globules. It may well be that these smaller
droplets compensate for the relatively low interfacial
area contributed by the larger droplets to the total surface density of lipid within the tissue. It is thus probable
that brown adipocytes are able to redistribute their lipid
droplets into various size categories in order to maintain
the surface density of the triglyceride store during maturation and aging, a phenomenon comparable to the
multilocular reorganization observed by Ahlabo and
Barnard (1974) in the brown adipocytes of cold-exposed
hamsters.
Vascular Volume
The volume of intralobular capillaries in rat ISBAT,
V(v), increased markedly in the first 3 days of postnatal
life (0.68 x lop2 cm3 a t birth, 1.55 x lop2 cm3 on day
3; Fig. 3). A peak value of 2.4 x
cm3 was recorded
at 4 weeks of age, a time at which biochemical indices
show that the metabolic potential of BAT is at a
maximum.
According to Barnard (1969))capillaries, together with
pericapillary cells and extracellular spaces, account for
about 30% by volume of rat BAT at birth. In the present
study, a fractional volume of 18.1% was obtained for
capillaries alone at the same age (Table 2). Capillary
fractional volume reached a peak of 23.3% on day 3
before declining sharply. This decline may be accounted
for by a n opposite trend in the growth of parenchymal
elements. According to Suter (1969) and Schneider-Picard et al. (1980), the diameter of brown adipocytes increases with age from birth onwards. The decline in
vascular volume density was steepest in the latter half
of the first postnatal week and in the second month
(Table 2), resulting in a significant but transient decrease in tissue vascular volume during both periods
(Table 3, Fig. 3). However, the levels of V v) maintained
in adulthood and old age (1.9 x
cm at 6 months;
1.76 x lop2 cm3 at 2 years) were not significantly different from that at 4 weeks of age.
It would therefore seem that the morphological parameters measured in the present study, namely, lipid droplet surface area, S(l), and intralobular capillary volume,
V(v), do not reflect senile functional involution in rat
ISBAT. The maintenance of S(1) and V(v) at a high level
into old age is conducive to rapid substrate mobilization
from storage and to hyperemia. Numerous studies have
shown that although the thermogenic capacity of rat
BAT tends to wane with age, the tissue remains readily
responsive to acute stimulation and to trophic influences
(see Barnard et al., 1980, for review). In the fully grown
Q
362
J.O. NNODIM
Figs. 4-6. Electron micrographs showing general features of rat
interscapular brown adipose tissue at age 3 days (Fig. 41, 18 weeks
(Fig. 5), and 2 years (Fig. 6). Note diminishing number of capillary
profiles per field, increasing size of brown adipocytes and individual
lipid droplets through the series. The characteristic multilocular ar-
rangement of intracellular lipid is retained at all ages. e, erythrocytes;
Id, lipid droplets. In Figure 6 note lipofuscin bodies (arrowhead) and
brown adipocyte nucleus (asterisk) indented by neighboring lipid droplets. All figures X 1,700. Scale bar: 5.0 pm.
AGING CHANGES IN RAT INTERSCAPULAR BROWN FAT
animal, it has been suggested that BAT may continue
to play a physiologically significant role in the dissipation of wempty” dietary calories, such as are derived
from calorie-rich food with otherwise no nutritional
value (Himms-Hagen, 1979; Rothwell and Stock, 1979;
Cannon and Nedergaard, 1985).
363
Vol. I. Practical Methods for Biological Morphometry. Academic
Press, London, New York, Toronto, Sydney, San Francisco.
Foster, D.O., and M.L. Frydman (1978)Nonshivering thermogenesis in
the rat. 11. Measurements of blood flow with microspheres point to
brown adipose tissue as the dominant site of the calorigenesis
induced by noradrenaline. Can. J. Phvsiol. Pharmacol.. 56:llO122.
Heaton, G.M., R.J. Wagenvoord, A. Kemp, and D.G. Nicholls (1978)
Brown adipose tissue mitochondria: Photoafinity labelling of the
regulatory site of energy dissipation. Eur. J. Biochem., 82515-521.
ACKNOWLEDGMENTS
Himms-Hagen, J. (1979) Obesity may be due to a malfunctioning of
This study was partially accomplished during the tenbrown fat. Can. Med. Assoc. J., 121r1361-1364.
ure of a scholarship of the Federal Government of Ni- Holmes, A. (19271 Petrographic Methods and Calculations. Murby,
London.
geria (ED/SC/PG/80/0354) a t University College, Cardiff, Jansky,
L. (1961)Total cytochrome oxidase activity and its relation to
U.K.
basal and maximal metabolism. Nature, 189:921-922.
The assistance of the late Dr. P.C.R. Hughes in the Loud, A.V., W.C. Barany, and B.A. Pack (1965) Quantitative evaluation of cytoplasmic structures in electron micrographs. Cited by
development of the stereological technique used in this
E.R. Weibel(1979)Stereological Methods, Vol. I. Practical Methods
investigation is duly acknowledged. I a m also grateful
for Biological Morphometry. Academic, London, New York, Toto Professor J.D. Lever for the use of facilities in his
ronto, Sydney, San Francisco.
department and to Mr. P. Hire and Ms. D. Symons for Moroney, M.J. (1951) Facts From Figures. Penguin, Harmondworth,
Middlesex.
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Nnodim, J.O., and J.D. Lever (1985)The pre- and post-natal development and ageing of interscapular brown adipose tissue in the rat.
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