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Studies of human adipose tissue in culture I. Incorporation of glucose and release of glycerol

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Studies of Human Adipose Tissue in Culture
I. INCORPORATION OF GLUCOSE AND €ELEASE
OF GLYCEROL
ULF SMITH
Department of Medicine I1 and the Department of Clinical
Chemistry, Sahlgren's Hospital, Gothenburg, Sweden
ABSTRACT
Explants of human adipose tissue were incubated in Parker's
medium 199 and the rates of glucose incorporation into the lipids and release of
glycerol determined. Both of these parameters increased in a linear fashion for
about seven to eight days. After this period of time there was a decrease in the
metabolic rates of explants with a mean cell size larger than about 90-95 pm.
The incorporation of glucose into the lipids was mainly recovered in the glycerol
moiety of the triglycerides. Cellular enlargement was associated with an increase
in glucose incorporation as well as glycerol release, i.e., an increased turnover of
the glyceride-glycerol. Addition of insulin increased the incorporation of label
into the lipids. The stimulatory effect of a single dose of insulin was pronounced
for two days and was only slightly discernible after 14 days. Addition of the enM reduced the incorporazyme inhibitor iodoacetate at a concentration of
tion of glucose to about 5% of the controls. The method of tissue culture used in
the present investigation may be a useful tool for metabolic as well as morphologic studies of human adipose tissue in vitro.
In a previous communication a method pose tissue in vitni. This system would
for long-term investigations of human then provide a unique possibility to study
adipose tissue in vitro was described the metabolic and niorphologic changes of
(Smith, '71). It was reported that the mor- adipose tissue exposed to variations in the
phologic integrity of the adipose cells was biochemical environment for prolonged
maintained during an observation period periods of time. Similar to the findings in
of several weeks. However, adipocytes short-term investigations it was found that
larger than about 90-95 pm decreased in the adipocytes synthesize lipids from glusize during the incubations, which was not cose, are sensitive to the stimulatory effect
the case with the smaller cells. As a pos- of added insulin, and that the turnover
sible reason for this finding metabolic dif- rate of the glyceride-glycerol is increased
ferences between adipose cells of different with increasing cell size. Furthermore,
sizes was suggested (Smith, '71). That lipid synthesis from glucose as well as resuch is the case in short-term investiga- lease of glycerol increased in a linear
tions is now well recognized. For instance, fashion for at least seven to eight days.
the rates of lipid synthesis from glucose
as well as the release of glycerol are corMATERIALS AND METHODS
related to cell size (Bjorntorp and KarlsBiopsies of subcutaneous adipose tissue
son, '70; Goldrick and McLoughlin, '70; were obtained in connection with operaSmith, '70a). However, the responsiveness tions upon patients, mainly suffering from
to insulin is decreased with increasing cell cholecystolithiasis and at exploratory lapsize (Salans et al., '68; Smith, '70a; Salans arotomy. The ages of the patients varied
and Dougherty, '71).
from 26 years to 68 years, and body
The purpose of the present study was to weights from 55 kg to 105 kg. Anesthesia
investigate if the method of tissue culture was induced with a short-acting barbitupreviously described (Smith, '71) may be
useful for metabolic studies of human adiReceived Sept. 7, '71. Accepted Nov. 12, '71.
ANAT. REC., 172: 597-602.
597
598
ULF SMITH
rate and continued with halothane, nitrous
oxide, and oxygen. The biopsies were USUally obtained at the beginning of the operations. After excision, the biopsies were
immediately placed in a sterile vessel containing the same medium that was used
for the incubations. The culture procedure
has been described in detail previously
(Smith, '71 ). Briefly, smaller specimens
weighing about 5-10 mg each were placed
between two cover slips and incubated
in Leighton tubes (Labora, Stockholm,
Sweden) in the presence of 1.5 ml Parker's
medium 199 (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) containing 1.O mM HEPES (N-2-Hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid; Sigma
Chemical Company, St. Louis, Mo.).
All incubations were performed at 37°C
and at pH 7.4. The gas phase was air.
Medium was changed once a week. In some
experiments 0.1 pC D-[1-14C]glucose (New
England Nuclear Corporation, Frankfurt
am Main, Germany) was added to 1.5 ml
medium. In the set of experiments concerned with the effect of different concentrations of HEPES the tissue specimens
were incubated with labelled glucose for
two hours in Parker's medium 199 modified to a glucose concentration of 1.0 mM
and with the addition of HEPES at a final
concentration of 1.0 mM, 5.0 mM or 10.0
mM. When necessary, the pH was adjusted to 7.4 with 0.5 N NaOH.
After the incubation period the explants
were carefully washed with physiological
saline and the lipids extracted with chloroform-methanol ( 2 : 1 v/v) as described by
Folch et al. ('57). Aliquots of the chloroform phase were evaporated to dryness
and 10 ml scintillation fluid (0.4% 2,5diphenyloxazole and 0.01 % 1,4-bis-2( 5-phenyloxazolyl)-benzene dissolved in
toluene) added. The radioactivity was determined in a Packard Tri-Carb liquid
scintillation spectrometer. Quenching was
corrected for by means of internal standardization.
Aliquots of the chloroform phase were
taken for the determination of the glyceride-glycerol according to the method
described by Carlson ('59). After saponification and acidification the fatty acids
were extracted with n-heptane and the
radioactivity determined by liquid scintillation as described above.
Aliquots of the incubation medium were
taken for the determination of glycerol.
Glycerol was determined enzymatically as
described by Laurel1 and Tibbling ('66).
The release of glycerol was taken as an
index of lipolysis.
Mean cell diameter of the explants was
determined after cell isolation with c01lagenase (Sigma type 1, Sigma Chemical
Company, St. Louis, Mo.) as previously
described (Smith, '71). Since a loss of
cells is unavoidable with this procedure the
cell sizes were determined on explants incubated solely for this purpose. The justification in this procedure may be judged
from the observation that after 14 days
in vitro the mean cell sizes of the digests
of three different explants which had been
prepared from the same biopsy were 58.3
pm, 60.2 pm, and 61.1 pn, respectively. In
another series the mean cell sizes were
82.8 pm, 87.9 pm, and 86.5 pm, respectively.
Incorporation of labelled glucose and
release of glycerol are expressed in terms
of the cellularity of the explants. The number of cells was calculated on the assumption that the cells were spherical. Mean
cellular volume was calculated as suggested by Goldrick ('67) according to the
formula :
v=
(c)
(302+I*)
jz
where v = volume; a* = variance of the
mean cell diameter; x'= mean cell diameter.
Mean cellular weight was calculated on
the assumption that the density of fat cells
is that of triolein (Hirsch and Gallian,
'68). When the glyceride-glycerol content
of the explants is known the number of
fat cells can be calculated.
RESULTS
In order to increase the buffering capacity of Parker's medium 199 HEPES was
added at a concentration of 1.0 mM. This
concentration was chosen since the rate of
lipid synthesis from glucose was unaffected, which does not seem to be the case
at greater concentrations (table 1). Furthermore, repeated determinations have
599
HUMAN ADIPOSE TISSUE IN VITRO
TABLE 1
TABLE 2
Effect of H E P E S on the incorporation of
D-[I -14C] glucose into the
total lipids
Effect of cell size and length of incubation on
the incorporation of D-[I -l*C] glucose into
the total lipids
Glucose incorporation
n moles/l05 cells
Addition
None
1.0 mM HEPES
5.0 mM HEPES
10.0 mM HEPES
Length of
incubation days
7 . 3 2 1.4
7.4 -+ 2.0
5.8 f 1.5
5.1 2 1.1
shown that this concentration is sufficient
to maintain the pH of the medium at
7.4 -C 0.3 during the incubations.
Lipid synthesis from glucose. Incorporation of labelled glucose into the lipids
was initially determined after 2, 4, 6, and
14 days in vitro. As show in figure 1 the
incorporation of label into the total lipids
increased in a linear fashion for the first
six days but the linearity of the system
was not maintained throughout the observation period. It is also shown in figure 1
that there were large variations in the rate
of lipid synthesis from glucose between
different explants. The effect of cell size
on this parameter was then studied.
600.0-
-
500.0
400.0-
5
eg
.
?
- 300.0200.0100.0I
1
I
2
4
i
14
I
6
75.5
91.6
110.8
Incorporation of glucose
n moles/lW cells/24 hours
~
Specimens of human adipose tissue were incubated
in quadruplicates for two hours at pH 7.4 i n Parker’s
medium 199 containing 1.0mM glucose and 0.15pC
D-[l - l4Cl glucose per 2 ml medium. The concentration of HEPES is shown above. The results are the
means 2 S.E.M.
2
Mean cell
sue, pm
Length of Incubation
Days
Fig. 1 Incorporation of labelled glucose into
the total lipids. Explants of human adipose tissue
were incubated in Parker’s medium 199 with the
addition of 0.1 pC D-[l-14C] glucose per 1.5 ml
medium for 2, 4, 6, and 14 days. The results
shown are the means -t S.E.M. of seven duplicate determinations. Mean cell sizes of the explants ranged from 75.5 pm to 110.8 pm.
2
4
6
14
18.9
23.6
15.1
141.6
46.1
44.1
50.4
30.6
~~
~
87.6
79.3
77.0
53.6
Explants of human adipose tissue were incubated in
Parkers’s medium 199 with the addition of 0.1 pC D[ l - W] glucose per 1.5 ml medium. Mean cell size
was determined on isolated cells.
Throughout the observation period explants with a large mean cell size have
greater rates of glucose incorporation than
those with a smaller mean cell size. Representative experiments are shown in
table 2. Expressed on a per day basis the
explants with a small mean cell size
(75.5 pm) only showed minor variations
in the rate of glucose incorporation
throughout the observation period, i.e., the
linearity of the system was maintained.
However, after 14 days the explants with
the larger mean cell sizes showed a decrease in the rate of glucose incorporation
(table 2). In separate experiments where
the determinations were performed daily
this decrease in the metabolic rates of the
larger adipose cells was found after about
eight days in vitro.
The incorporation of ‘Y: was mainly recovered in the glycerol moiety of the triglycerides. Irrespective of the length of incubation about 5% of the radioactivity
was recovered in the glyceride - fatty
acids. Addition of insulin (0.15 I.U.) to
the incubation medium increased the incorporation of glucose into the lipids. The
average increase was about 40%. This
effect was due to an increase in the synthesis of glyceride-glycerol as well as
glyceride-fatty acids. No significant correlation between cell size and the stimulatory effect of insulin was found. The
effect of a single dose of insulin decreased
gradually after two days and was only
slightly discernible after 14 days.
In some experiments an enzyme inhibitor of glucose metabolism, iodoacetate,
was added to the incubation medium at
different concentrations. At a concentra-
600
ULF SMITH
tion of lO-'M the incorporation of glucose
into the lipids was reduced to about 5%
of the controls.
Explants with a mean cell size larger
than about 90-95 pm decreased in cell size
during the incubation period. After 14 days
in vitro the mean cell sizes of the explants
with initial means of 75.5 pm, 91.6 pm, and
110.8 pm (shown in table 2) were 74.3
pm, 85.7 pm, and 100.9 pm, respectively.
Release of glycerol. Analogous to the
results obtained with glucose incorporation
the release of glycerol was found to increase in a linear fashion for at least
seven to eight days. After this period of
time the lipolytic rates of the larger adipose cells decreased. Similar to the effect
of cell size on glucose incorporation enlargement of the adipocytes was associated
with an increase in the lipolysis. Representative experiments are shown in table
3. In table 3 it is also shown that on a
per day basis explants with large mean
cell sizes showed a decrease in the rate
of glycerol release.
DISCUSSION
In order to increase the buffering capacity of Parker's medium 199 HEPES was
added at a concentration of 1.0 mM. The
usefulness of HEPES as a tissue culture
buffer for several lines of cells has been
shown recently (Shipman, '69; Danynkiewicz and Jacobson, '71). Generally, greater
concentrations than 1.0 mM are desirable
(Shipman, '69). However, it has been
found that the morphology of the adipose
cells is unaltered at this concentration
(Smith, '71) and, as shown in the present
study, the rate of lipid synthesis is unaffected, which does not seem to be the
case at greater concentrations.
TABLE 3
Effect of cell size and hngth of incubation on the
release of glycerol to the medium
Mean cell
size, &m
Length of
incubation days
7
14
86.8
96.0
99.3
Release of glyceml
n moles/l05 cells/24 hours
50.7
54.7
117.9
83.1
136.2
92.4
Explants of human adipose tissue were incubated in
1.5 ml Parker's medium 199. Mean cell size was determined on isolated cells.
Short-term studies have revealed that
adipocyte size is an important parameter
for the rate of lipid synthesis from glucose,
release of glycerol, and responsiveness to
insulin (Salans et al., '68; Bjomtorp and
Karlsson, '70; Goldrick and Mchughlin,
'70; Smith, '70; Salans and Dougherty,
'71). In the present study utilizing the
tissue culture technique it was found that
increasing cell size was accompanied by
an increase in lipid synthesis as well as in
glycerol release i.e., an increased turnover
of the glyceride-glycerol. Since the specimens were isolated from nervous and
hormonal controls for prolonged periods
of time it seems reasonable to assume that
the greater metabolic activities of the
larger adipose cells are associated with cellular enlargement per se and not the fact
that large adipose cells are generally obtained from obese individuals.
Incorporation of glucose into the lipids
and release of glycerol to the medium increased in a linear fashion for at least
seven to eight days. After this period of
time there was a decrease in the metabolic
rates of the larger adipose cells (larger
The reason for this
than about 90 q).
discrepancy can only be speculated upon
at present. One possibility, however, may
be a greater intercellular accumulation of
acid metabolites leading to a greater local
decrease in the pH. This concept is supported by the observation that the larger
adipose cells have greater rates of glucose
metabolism as well as lipolysis. The conversion of glucose to lipids is decreased if
the pH is lowered (Longmore et al., '68).
Rudman and Shank ('66) have shown that
stimulation of the lipolysis in vitro as well
as in uivo is associated with a decrease in
the pH, presumably caused by the dissociation of the released free fatty acids (FFA)
into the carboxyl and hydrogen ions. Furthermore, Dieterle et al. ('70) have reported that the lipolysis is inhibited by increasing concentrations of lactate. Since
the incubations were performed in the
absence of albumin (see below) another
possibility for the differences under discussion may be due to an excessive intracellular accumulation of FFA in the larger
adipose cells, which may lead to a decrease
in the metabolic activities (Angel et al.,
'71a,b). However, this possibility seems
HUMAN ADIPOSE TISSUE IN VITRO
less likely since the depressing effect of
FFA-accumulation occurs rapidly (Angel
et al., '71a,b) and the metabolic rates of
the larger adipose cells were linear for
about seven to eight days.
In agreement with a previous study
(Smith, '71) it was found that adipose
cells larger than about 90-95 pm decreased in size during the observation
period of 14 days. The finding of the present investigation that cellular enlargement
is associated with an increase in the metabolic activities of the cells is suggested as
a possible reason. In addition, the variations in the metabolic rates of the larger
adipose cells during the incubation period
may also contribute. Support for the concept that adipose cell size is affected by
the metabolic rates of the cells is offered
by the investigations of Slavin and Elias
('69, '70). It was found in their in vitro
studies of adipose tissue from mice that
in the presence of lipolytic hormones fat
cell size was significantly decreased after
three to four days. Although the release of
FFA was not determined in the present
study i t seems quite probable that the
larger adipose cells have greater rates of
FFA release, as has been found in shortterm incubations using Parker's medium
199 (Smith, '70b).
In the present study the incorporation
of glucose was mainly recovered in the
glyceride-glycerol which is in agreement
with the results obtained in short-term
incubations (Galton, '68). Addition of insulin increased the incorporation of label
into the lipids. The stimulatory effect of a
single dose of insulin (0.15 I.U.) decreased gradually after two days and was
only slightly discernible after 14 days.
There was no significant correlation between cell size and the stimulatory effect
of insulin which is in disagreement with
the results obtained in short-term studies
(Salans et al., '68; Smith, '70a; Salans
and Dougherty, '71 ). Preliminary results
indicate that one reason for this discrepancy may be due to the fact that the larger
adipose cells decrease in size during the
incubation period.
In the presence of the enzyme inhibitor
iodoacetate, the incorporation of glucose
was almost completely inhibited. The concentration used to obtain this effect
60 1
(10-4M) is well in agreement with that
found in short-term studies (Mirsky and
Perisutti, '62 ) .
In agreement with the suggestions of
several investigators (Hellman et al., '63;
Martinsson, '68; Salans et al., '68) the
incorporation of glucose and release of
glycerol are in the present study expressed
in terms of the cellularity of the specimens
rather than the ciistomary manner, i.e.,
per unit of tissue lipid. The reasons for
this are the great variations in cell size between different individuals and, as also
found in the present study, cell size is an
important parameter for metabolic rates.
All incubations were performed in the absence of albumin. This diminishes but does
not inhibit the release of FFA (Vaughan
et al., '64). In fact, a significant positive
correlation between cell size and release
of FFA of tissue specimens incubated in
Parker's medium '199 in the absence of
albumin has been demonstrated (Smith,
'70b). One possibility for the release of
FFA to the medium in the absence of albumin may be that certain amino acids
present in Parker's medium 199 are capable of forming bonds with the fatty acids
to increase the water solubility. This would
be in agreement with the FFA-binding
properties of albumin as discussed by
Goodman ('58). Experimental support for
this concept is obtained by the studies of
Bjorntorp ('66).
With the method of tissue culture used
in the present study the morphologic integrity of the adipose cells is maintained
for prolonged periods of time (Smith,
'71). In the present report it was demonstrated that the adipose cells are sensitive
to insulin, and that the incorporation of
glucose into the lipids as well as the release
of glycerol increase in a linear fashion for
at least seven to eight days. Lipid synthesis
from glucose was depressed by the adddition of the enzyme inhibitor iodoacetate.
Furthermore, the greater metabolic rates
of large adipose cells as demonstrated in
short-term studies were also found in the
present investigation. For these reasons it
is suggested that tlhe method of tissue culture used in the present investigation may
be a useful tool for metabolic and morphologic studies OF human adipose tissue
in nitro. At present, the long-term effects
602
ULF SMITH
Hirsch, J., and E. Gallian 1968 Methods for
the determination of adipose cell size in man
and animals. J. Lipid Res., 9: 110-119.
Laurell, S., and G. Tibbling 1966 A n enzyACKNOWLEDGMENTS
matic fluorimetric micromethod for the deThe expert technical assistance of Miss
termination of glycerol. Clin. Chim. Acta, 13:
C . Liljequist and Mrs. I. B. Hilmersson is
317-322.
Longmore, W. J., B. R. Landau, E. S. Baker,
gratefully acknowledged.
A. B. Hastings, D. M. Lum and H. R. Williams
This investigation was supported by
1968 Effect of pH and Con concentration on
grants from the Swedish Medical Research
glucose metabolism by rat adipose tissue
Council (Project B 72-13X-3506).
in vitro. Am. J. Physiol., 215: 582-586.
Martinsson, A. 1968 Methods of isolation and
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