| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Clinical Research Centers |
Division of Endocrinology and Metabolism, Department of Medicine (L.B.W., R.L.F., A.S.W., Z.P., R.V.C.), and Department of Surgery (B.E.K., R.M.J.), Indiana University School of Medicine, Indianapolis, Indiana 46202; and St. Vincent Bariatric Services (R.M.J., M.I., J.H.), Carmel, Indiana 46032
Address all correspondence and requests for reprints to: Robert V. Considine, Ph.D., Indiana University School of Medicine, 541 North Clinical Drive, Clinical Building 455, Indianapolis, Indiana 46202-5111. E-mail: rconsidi{at}iupui.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The data suggest that adipocytes from females are more responsive to the stimulatory effect of dexamethasone in vitro than are adipocytes from males. If adipocytes from females are more responsive to relevant in vivo stimuli for leptin secretion such as insulin or glucose, this could contribute to the gender difference in serum leptin. The data also suggest that leptin release from omental adipocytes may be more responsive to hormonal and nutrient regulation in vivo than are sc adipocytes.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dexamethasone is a potent stimulus for leptin secretion in vitro. Dexamethasone increases leptin production from omental adipose tissue pieces (12, 13), isolated sc adipocytes (14), and preadipocytes differentiated to adipocytes in culture (15). It has been reported that dexamethasone can stimulate leptin release in omental adipose tissue pieces obtained from females, but that the glucocorticoid has no effect on leptin release in omental tissue from males (11). This suggests that a gender-dependent difference in stimulated leptin release may exist.
Troglitazone is a high affinity activating ligand for
peroxisome proliferator-activated receptor-
(PPAR) (16). This
transcription factor is highly expressed in adipocytes and is active in
the differentiation of preadipocytes to adipocytes (17).
Troglitazone inhibits ob gene expression and
leptin release in rodents and cultured cell lines (18, 19, 20, 21). In studies
in humans, troglitazone significantly improved insulin
resistance, but had no effect on serum leptin in two studies (22, 23)
and reduced serum leptin in a third study (24). The direct effect of
troglitazone on human adipose tissue leptin production has
been examined in only one study, in which it was observed that
troglitazone attenuated insulin-stimulated leptin release
(22).
The current studies were conducted to test the hypothesis that leptin synthesis is differentially regulated in sc and omental adipocytes from morbidly obese males and females. Dexamethasone was chosen as a potent stimulus for leptin secretion, and troglitazone was chosen as an inhibitor of leptin production in these studies.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
.
|
Leptin was measured using a commercially available RIA kit (Linco Research, Inc., St. Charles, MO). The limit of detection of this assay is 0.5 ng/mL. The within- and between-assay coefficients of variation are 4.6% and 5.0%, respectively, at 7.2 ng/mL. The culture medium containing 10% FBS contained no detectable leptin, nor did it interfere with the detection of added standard human leptin.
Glucose and lactate in the culture medium were measured using a glucose analyzer (model 2300, YSI, Inc., Yellow Springs, OH). Glucose uptake was determined by measuring medium glucose before and after each medium change and calculating the decrease.
ob messenger ribonucleic acid (mRNA) was determined by RT-PCR as previously described (1). All comparisons between samples were made on the linear portion of the amplification curve (between cycles 20–35), and no product was obtained in the absence of reverse transcriptase. The data are expressed as the ratio of ob complementary DNA (cDNA) to actin cDNA. There was no difference in the amount of actin cDNA among the samples studied.
All data are expressed as the mean ± SEM. The percent inhibition of dexamethasone-stimulated leptin release was calculated by first subtracting basal leptin release from that in the presence of dexamethasone and dexamethasone plus troglitazone. The percent inhibition was then determined by subtracting the ratio of dexamethasone plus troglitazone divided by dexamethasone from 1 and multiplying by 100. All statistical comparisons were made by paired or unpaired t test, using StatView 4.5 (Systat, Evanston, IL) for Macintosh.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adipose tissue was obtained from 30 women and 16 men undergoing
bariatric surgery. As shown in Table 1
, the body mass index (BMI) of
the men was greater than that of the women studied, and the women were
slightly younger than the men. The serum leptin level in the women was
significantly greater than that in the men at the time the adipose
tissue biopsy was taken.
Dexamethasone stimulates leptin production from sc and omental adipocytes
Subcutaneous and omental adipocytes obtained from the same
individual were cultured in suspension, and the leptin secreted into
the medium was quantitated by RIA. As shown in Table 2, sc adipocytes continuously release
leptin into the medium (basal release at 48 h significantly
greater than that at 24 h, P < 0.05). Culture of
sc adipocytes from female subjects with 0.1 µmol/L dexamethasone
resulted in a significant increase in leptin production over that by
untreated cells within the first 24 h of treatment. After 48
h, dexamethasone-stimulated leptin release increased to 207 ±
21% of the control level. In sc adipocytes from male subjects,
dexamethasone had no effect on leptin release within the first 24
h, but significantly increased release to 180 ± 23% of the
control level by 48 h. There was no significant difference in
basal leptin production in sc adipocytes from males and females under
these culture conditions. Dexamethasone-stimulated leptin release as a
percentage of the basal value was also not different between males and
females at 48 h.
|
). There was no difference
in basal leptin release between sc and omental adipocytes of males.
Dexamethasone significantly increased leptin production in omental
adipocytes from females by 24 h (227 ± 53%), and by 48
h, dexamethasone increased leptin production to 398 ± 64% of the
control value. In adipocytes from males, dexamethasone had no
significant effect on leptin release within the first 24 h of
treatment (116 ± 9% of control). After 48 h,
dexamethasone-stimulated leptin release from omental adipocytes of
males was significantly increased to 211 ± 33% of the control
level. The stimulatory effect of dexamethasone on leptin production was
significantly greater in omental adipocytes from females compared to
males (350 ± 46% vs. 194 ± 27% of control;
P < 0.05, unpaired comparison) after 48 h of
treatment. Inhibition of leptin release by troglitazone
Subcutaneous adipocytes were cocultured with 0.1 µmol/L
dexamethasone and 10 µmol/L troglitazone for 48 h.
As shown in Fig. 1, troglitazone significantly inhibited
dexamethasone-stimulated leptin release in adipocytes from both males
and females at 48 h. The extent of
troglitazone-mediated inhibition was not significantly
different in adipocytes from males and females (percent inhibition at
48 h, 49.5 ± 16.8% vs. 62.6 ± 14%).
Troglitazone alone had no significant effect on leptin
production (5.13 ± 0.85 vs. 5.41 ± 1.30 ng/mL
for basal and troglitazone treated, respectively, at
48 h; n = 9). Significant dose-dependent inhibition of
dexamethasone-stimulated leptin production by troglitazone
could be detected as early as 36 h in cultures of sc adipocytes.
At concentrations of 10 and 1 µmol/L, troglitazone was
equally effective at inhibiting dexamethasone-stimulated leptin release
(34 ± 10% and 35 ± 13% inhibition; P <
0.05; n = 6). Troglitazone at 0.1 µmol/L had no
significant effect on dexamethasone-stimulated leptin release
at 36 h.
|
, 10 µmol/L troglitazone
completely inhibited dexamethasone-stimulated leptin production in
omental cells at 48 h. Troglitazone was equally
effective in inhibiting leptin production in adipocytes from males and
females. Troglitazone alone inhibited basal leptin
production in omental adipocytes 15.0 ± 5.2% (5.23 ± 1.19
vs. 4.59 ± 1.15 ng/mL for basal and
troglitazone-treated leptin production by 48 h;
n = 6; P < 0.05). The effect of
troglitazone to inhibit dexamethasone-stimulated leptin
production at 48 h was significantly greater in omental (134
± 26%) than in sc (57 ± 10.7%) adipocytes (P
< 0.005).
|
, dexamethasone
significantly increased the amount of ob mRNA over that in
untreated cells within the first 24 h (P < 0.05).
In the presence of troglitazone, the dexamethasone-induced
increase in ob gene expression was attenuated by 55 ±
12% (n = 7; P < 0.05). These observations
suggest that dexamethasone and troglitazone regulate
leptin production through effects on ob gene transcription
or message stability.
|
Glucose uptake and metabolism are stimuli for ob gene expression and leptin production in rat adipocytes (26). We therefore examined glucose uptake and its metabolism to lactate as a mechanism to explain the difference in leptin synthesis between omental and sc adipocytes in females. There was no difference in the amount of glucose taken up (8.7 ± 1.0 vs. 9.4 ± 1.5 µmol) or lactate released (7.8 ± 0.6 vs. 8.2 ± 1.4 µmol) from paired samples of sc and omental adipocytes, respectively, when examined during the first 24 h (n = 8). It is therefore unlikely that a difference in glucose uptake or metabolism to lactate can account for the difference in basal leptin release between sc and omental adipocytes of females.
Glucose uptake and lactate release were also examined as a mechanism
for dexamethasone- and troglitazone-regulated leptin
synthesis. In sc adipocytes, dexamethasone significantly decreased
glucose uptake (6.9 ± 1.1 vs. 5.8 ± 1.0 µmol;
P < 0.01) and lactate release (6.9 ± 0.8
vs. 5.8 ± 0.6 µmol; P < 0.05;
n = 6) in the first 24 h. As shown in Table 3, glucose uptake and lactate
production in the presence of dexamethasone continued to be inhibited
at 48 h in culture. The combination of dexamethasone and
troglitazone did not result in significant inhibition of
glucose uptake. Troglitazone alone had no significant
effect on glucose uptake or lactate production in sc adipocytes.
|
, glucose uptake and lactate production in the presence
of dexamethasone continued to be inhibited at 48 h in culture.
Coincubation of dexamethasone with troglitazone also
significantly inhibited glucose uptake and lactate release.
Troglitazone alone significantly reduced glucose uptake
and lactate release in omental adipocytes at 48 h (Table 3). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Basal leptin release from sc adipocytes of females was significantly greater than that from omental adipocytes of females. In contrast, there was no difference in the basal release of leptin from omental and sc adipocytes of males. Several studies using adipose tissue obtained primarily or entirely from females also observed that ob gene expression/leptin production is greater in sc than omental adipocytes from the same individual (8, 9, 10). The size of the adipocytes obtained from females in these studies may be important in explaining these observations; ob gene expression is greater in larger adipocytes than in smaller adipocytes isolated from the same piece of adipose tissue (27), and leptin secretion is strongly correlated with fat cell volume (28). It has been observed in morbidly obese subjects that sc adipocytes from females are significantly larger than omental adipocytes from the same subject, but there was no difference in the size of omental and sc adipocytes from males (29, 30). The lack of difference in adipocyte size most likely explains our observation that basal leptin release from sc and that from omental adipocytes from males were the same. In support of this, Montague et al. observed that the ratio of sc to omental adipose tissue ob mRNA was 1.9 ± 0.2 in males vs. 5.5 ± 1.1 in females (8).
Casabiell et al. (11) recently observed a gender-dependent
difference in spontaneous leptin production in omental adipose tissue
pieces cultured for 48 h in vitro. In contrast, we did
not find a gender-related difference in basal leptin release from
isolated sc or omental adipocytes during 48-h cultures. It is possible
that the three-dimensional architecture of the adipose tissue pieces
was an important determinant of basal leptin production in the study by
Casabiell et al. Alternatively, a gender-dependent
difference in basal leptin production may be more readily detectable in
adipose tissue from normal weight subjects (BMI: females, 27.3 ±
0.8; males, 26.9 ± 0.6 kg/m2)
vs. the extremely obese subjects in our study (BMI: females,
48.8 ± 1.4; males, 59.4 ± 3.4 kg/m2).
Indeed, although serum leptin is in general 2- to 3-fold greater in
females than in males of similar fat mass when examined in a general
population, as shown in Table 1 this difference between the genders is
attenuated in subjects with extreme obesity. The mechanism resulting in
the attenuation of the gender difference in extremely obese subjects is
not known. Casabiell et al. (11) also reported that
dexamethasone did not stimulate leptin release from omental adipose
tissue pieces from males. In contrast, we observed significant
dexamethasone-stimulated leptin production from both sc and omental
adipocytes from males. An explanation for the discrepancy between the
two studies is not readily apparent.
In both sc and omental adipocytes from females, dexamethasone significantly increased leptin release within 24 h. In contrast, a significant effect of dexamethasone on adipocytes from males was not observed until 48 h of treatment. Omental adipocytes from females responded to dexamethasone with a greater fold increase in leptin release over basal than sc adipocytes from females. In contrast, there was no difference in the extent of response between omental and sc adipocytes of males. Although complete dose-response studies to examine the effect of dexamethasone were not performed, Halleux et al. determined that 0.1 µmol/L dexamethasone (the concentration used in these studies) is a maximally stimulating dose in omental adipose tissue pieces (12). These observations, therefore, suggest that adipocytes from females are more responsive to the dexamethasone challenge. If adipocytes from females are more responsive to stimuli for leptin secretion such as insulin or glucose in vivo, this could contribute to the observed gender difference in serum leptin.
One possible explanation for the increased response of adipocytes from females to dexamethasone could be that these adipocytes have more adipose tissue glucocorticoid receptor than adipocytes from males, although there is no evidence to date to support such a hypothesis. However, Rebuffe-Scrive et al. (31) found that omental adipocytes of females contain more glucocorticoid receptor than sc adipocytes of females, thus explaining our observation of greater dexamethasone-stimulated leptin production in omental vs. sc adipocytes from females. A second hypothesis to explain the greater responsiveness of the adipocytes from females is that the cells are preconditioned by exposure to reproductive hormones in vivo. Estrogens have been proposed to stimulate, and androgens to inhibit, leptin synthesis (3, 4, 5, 32, 33, 34). In vitro, estrogen stimulates leptin release from cultured adipose tissue of both rodents and humans (11, 35, 36). It is, therefore, possible that exposure to estrogen before adipose tissue biopsy could up-regulate the responsiveness of the cells from the females to dexamethasone. Alternatively, testosterone in males could reduce the subsequent in vitro responsiveness of the adipocytes, although a direct effect of testosterone on ob gene expression was not detected in rodent adipocytes (35, 36). The mechanism through which the reproductive hormones regulate leptin production remains to be determined.
Troglitazone inhibited dexamethasone-stimulated leptin
production to a greater extent in omental adipocytes (134 ± 26%)
than in sc adipocytes (57 ± 10.7%). Furthermore,
troglitazone significantly inhibited basal leptin release
in omental, but not sc, adipocytes. There was no gender-related
difference in the effect of troglitazone in either sc or
omental adipocytes. These observations suggest that omental adipocytes
may be more responsive to troglitazone than are adipocytes
from the sc depot. However, no depot-related difference in PPAR mRNA
expression was found in a small study of six individuals with BMI
greater than 30 kg/m2 (9). Additional experiments
will be necessary to conclusively determine whether omental adipocytes
contain more PPAR than sc adipocytes and whether this results in a
greater sensitivity to troglitazone of other metabolic
functions of omental adipocytes.
In the current study dexamethasone significantly increased
ob gene expression, and troglitazone attenuated
the dexamethasone-induced increase in ob gene expression to
regulate leptin production. The routine use of dexamethasone to
differentiate preadipocytes to adipocytes in vitro (17)
raises the possibility that the effects of the glucocorticoid to
increase leptin production in primary cultures of adipose tissue
explants or isolated cells could be related to the prevention of
dedifferentiation during the culture period. This is unlikely based on
the observation that dexamethasone increases ob gene
expression over that at the time of isolation in omental adipose tissue
pieces from humans (12) and isolated rat adipocytes (37).
Thiazolidinediones have been demonstrated to reduce the promoter
activity of the human ob gene transfected into rat
adipocytes through activation of PPAR (20). Negative regulation of
the ob promoter by the thiazolidinedione AD-5075 maps to the
proximal promoter at -65 to +9, a region that binds C/enhancer binding
protein 
, but not PPAR or retinoid X receptor (21).
Hollenberg et al. (21) suggest that
thiazolidinediones may act via functional antagonism of C/enhancer
binding protein binding, although the exact mechanism for
such an interaction remains to be elucidated. An action of
troglitazone to inhibit the ob promoter at a
proximal site could explain the ability of this compound to inhibit
both dexamethasone- and insulin-induced leptin production (22).
Glucose uptake and metabolism are important determinants of leptin production in rodent adipocytes in vitro (26); however, we did not find a difference in glucose uptake or lactate release in paired samples of sc and omental adipocytes from females. Therefore, depot dependent differences in glucose metabolism to lactate do not appear to account for the difference in leptin synthesis between sc and omental adipocytes. We also found that dexamethasone decreased glucose uptake and lactate production, but increased leptin release in both sc and omental adipocytes. These findings argue against a role for increased glucose uptake as the mechanism by which dexamethasone stimulates leptin production. Troglitazone alone had no effect on glucose uptake or lactate release in sc adipocytes, but significantly attenuated glucose uptake in omental adipocytes. Alterations in glucose uptake and its metabolism to lactate do not appear to be involved in the regulation of leptin production by troglitazone.
In the experiments described in this study we used a pharmacological
concentration of dexamethasone as a stimulus, and
troglitazone as an inhibitor, of leptin production. One
must exercise caution in directly extrapolating our in vitro
findings to the regulation of leptin in vivo. Although
pharmacological doses of dexamethasone increase serum leptin in humans
(38, 39, 40, 41, 42), infusion of hydrocortisone at normal daily levels in patients
with primary adrenal failure had no effect on serum leptin (43). In
this same study reversal of the diurnal pattern of serum cortisol had
no effect on serum leptin. Based on these and other observations it
does not appear that physiological levels of cortisol directly regulate
leptin production. Troglitazone was used in the current
studies to inhibit leptin production at a concentration (10 µmol/L)
slightly higher than the peak serum concentration (6.3 µmol/L)
attained in humans (44). Although leptin production is also inhibited
by 15-deoxyprostaglandin J2 (45), a putative natural ligand of PPAR,
there is no evidence to date that regulation of leptin production by
PPAR is physiologically relevant in vivo. However,
despite the immediate lack of physiological relevance, dexamethasone
and troglitazone are valuable tools to understand the
mechanisms regulating leptin production.
In summary we have shown that dexamethasone increases leptin production in omental and sc adipocytes from both males and females in vitro. Dexamethasone-induced leptin release occurs more rapidly and is greater in adipocytes from females than in those from males in vitro. This observation suggests that leptin synthesis in females in vivo may be more responsive to stimulatory signals and that this increased responsiveness may contribute to the elevation in serum leptin in women compared to that in men with similar fat mass. Troglitazone attenuates the dexamethasone-induced increase in leptin production in adipocytes from the sc and omental depots of both males and females. The effects of dexamethasone and troglitazone on leptin synthesis occur through alterations in ob gene expression, but not through effects on glucose uptake. The dexamethasone-induced increase in leptin production and the inhibitory effect of troglitazone are both greater in omental than in sc adipocytes in vitro, suggesting that leptin production in this adipose tissue depot may be more responsive to nutrient and hormonal signals in vivo.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received December 20, 1999.
Revised March 27, 2000.
Accepted April 18, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J
Clin Invest. 98:1004–1009.[Medline]
and
peroxisome proliferator-activated receptor- on the leptin promoter. J Biol Chem. 272:5283–5290.
(12, 14) prostaglandin J2: a putative endogenous promoter
of adipogenesis suppresses the ob gene. Metabolism. 48:786–791.[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  B J Kendall, G A Macdonald, N K Hayward, J B Prins, I Brown, N Walker, N Pandeya, A C Green, P M Webb, D C Whiteman, et al. Leptin and the risk of Barrett's oesophagus Gut, April 1, 2008; 57(4): 448 - 454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Pasarica, N. Mashtalir, E. J. McAllister, G. E. Kilroy, J. Koska, P. Permana, B. de Courten, M. Yu, E. Ravussin, J. M. Gimble, et al. Adipogenic Human Adenovirus Ad-36 Induces Commitment, Differentiation, and Lipid Accumulation in Human Adipose-Derived Stem Cells Stem Cells, April 1, 2008; 26(4): 969 - 978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. MacLaren, W. Cui, S. Simard, and K. Cianflone Influence of obesity and insulin sensitivity on insulin signaling genes in human omental and subcutaneous adipose tissue J. Lipid Res., February 1, 2008; 49(2): 308 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Teta, A. Tedjani, M. Burnier, A. Bevington, J. Brown, and K. Harris Glucose-containing peritoneal dialysis fluids regulate leptin secretion from 3T3-L1 adipocytes Nephrol. Dial. Transplant., July 1, 2005; 20(7): 1329 - 1335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lundgren, J. Buren, T. Ruge, T. Myrnas, and J. W. Eriksson Glucocorticoids Down-Regulate Glucose Uptake Capacity and Insulin-Signaling Proteins in Omental But Not Subcutaneous Human Adipocytes J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2989 - 2997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C McCowen, P. R. Ling, C. Friel, J. Sternberg, R A. Forse, P. A Burke, and B. R Bistrian Patterns of plasma leptin and insulin concentrations in hospitalized patients after the initiation of total parenteral nutrition Am. J. Clinical Nutrition, May 1, 2002; 75(5): 931 - 935. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, K.-Y. Guo, P. A. Diaz, M. Heo, and R. L. Leibel Determinants of leptin gene expression in fat depots of lean mice Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R226 - R234. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
