Plasma insulin rise precedes rise in
ob mRNA expression and plasma
leptin in gold thioglucose-obese mice
Janet M.
Bryson,
Jenny L.
Phuyal,
Deborah R.
Proctor,
Stephen C.
Blair,
Ian D.
Caterson, and
Gregory J.
Cooney
Department of Endocrinology, Royal Prince Alfred Hospital,
Department of Medicine and Human Nutrition Unit, Department of
Biochemistry, University of Sydney, and Garvan Institute of Medical
Research, St. Vincent's Hospital, Sydney, New South Wales 2006, Australia
 |
ABSTRACT |
Circulating leptin
levels are strongly related to the degree of adiposity, with
hyperleptinemia being associated with hyperinsulinemia. In the gold
thioglucose-injected mouse (GTG), hyperinsulinemia is an early
abnormality in the development of insulin resistance and obesity. In
this study, hyperinsulinemia occurred 1 wk post-GTG [GTG, 199 ± 43; age-matched controls (CON), 53 ± 5 µU/ml;
P < 0.001], with leptin levels
not rising until 2 wk post-GTG (CON, 3.2 ± 0.3; GTG, 9.9 ± 1.7 ng/ml; P < 0.001) in parallel with
increases in the size of different fat pads and increased expression of ob mRNA. The ratio of serum leptin to
fat pad weight was significantly higher in GTG mice 12 wk
postinjection. Starvation-induced reductions in serum leptin (50%),
glucose (50%), and insulin (74%) were greater than decreases in fat
pad weight (18%). Adrenalectomy decreased both adiposity and serum
leptin within 1 wk in both CON and GTG and altered the serum leptin
level-to-fat pad weight ratio in CON. Thus hyperinsulinemia preceded
increased ob expression and hyperleptinemia, which occurred in parallel with increasing adiposity, consistent with the role of leptin as an indicator of energy supplies. Changes in hormonal and nutritional status may modify this relationship.
ob gene; adiposity; adrenalectomy; fasting
 |
INTRODUCTION |
IT IS NOW WELL ESTABLISHED in both humans and rodent
models of obesity, except the leptin-deficient
ob/ob mouse, that both the level of
expression of ob mRNA and the
circulating levels of leptin correlate highly with the degree of
adiposity (18). Leptin is produced mainly by adipocytes, with different
adipose sites exhibiting differences in the expression of
ob mRNA (10). Less is known of the
secretion rates of different adipose sites and whether there is
variability in their contribution to circulating leptin levels. The
hormonal and metabolic control of secretion rates is also not fully
understood. Hyperleptinemia is associated with hyperinsulinemia in both
humans and rodents, suggesting a role for insulin in the regulation of
leptin production and secretion. However, whereas both acute and
prolonged exposure to insulin results in increased
ob mRNA expression and leptin
secretion (2, 7, 24), prolonged hyperinsulinemia is required for an
effect on circulating leptin in both humans and rodents (11, 14, 20).
The gold thioglucose-injected (GTG) mouse is a chemically induced model
of obesity in which an infarction of the hypothalamus results in
hyperphagia and weight gain (3, 15). This lesion causes a reduction in
the number of leptin receptors in the hypothalamus (8), and so it is
postulated that the rise in circulating leptin levels is due to leptin
resistance caused by the reduction in receptor number. We have
previously demonstrated that hyperinsulinemia occurs early in the
development of insulin resistance and obesity in this model (3).
Conversely, improvements in glucose tolerance, a decrease in fat
deposition, and loss of body weight are found after adrenalectomy,
implicating glucocorticoids, which upregulate both expression and
secretion of leptin in vitro (25) in the development of obesity in this model.
The aims of this study were to compare the timing of the development of
hyperleptinemia with that of hyperinsulinemia in the GTG-obese mouse,
to observe the relationships between circulating leptin levels and the
size of different fat depots during the development of obesity in both
the fed and fasted states, and to determine the effect of adrenalectomy
and therefore a decrease in glucocorticoids on circulating leptin levels.
 |
METHODS |
Animals. Male CBA/T6 mice were
obtained at 6 wk of age from the Blackburn Animal House, University of
Sydney. Obesity was induced by a single intraperitoneal injection (0.5 g/kg) of gold thioglucose (Sigma, St Louis, MO). Mice were kept on a
12:12-h light-dark cycle (light cycle between 0600 and 1800) and were allowed free access to food and water. Groups of GTG-injected mice and
age-matched controls (CON) were weighed and then killed by an overdose
of Nembutal at different times after the induction of obesity ranging
from 1 day to 12 wk. Half of each group (except during the 1st wk) was
starved overnight before being killed, whereas the other half had
normal access to food until being killed. The epididymal
(epi) fat pad, subcutaneous (sc) fat pad from the femoral region, and
retroperitoneal (rp) fat pad were removed, blotted, weighed, and
immediately frozen. Fat pads were always removed by the
same operator. Blood was collected from the chest cavity and spun, and
serum was frozen for subsequent leptin, insulin, and glucose assays. In
a subsequent experiment, groups of mice were killed at 3, 7, and 14 days post-GTG, and epididymal fat pads were removed for determination
of ob mRNA expression. A further group
of GTG mice 4 wk postinjection and their age-matched controls were used
to test the effects of adrenalectomy. Half of each group were
bilaterally adrenalectomized (ADX), and the remaining animals were
subjected to sham-ADX. ADX mice were given access to 0.9% (wt/vol)
saline ad libitum in place of their normal drinking water. Sham-ADX
mice continued to have access to tap water. All animals were killed 1 wk later, and tissues and serum were collected as for the first group.
The number of animals in each group was 8-12.
Oral glucose tolerance test. After an
overnight fast, groups of 5-wk post-GTG-injected obese mice and
age-matched lean mice were given an oral gavage of 200 µl of 50%
glucose (~3 g/kg). Tail vein blood samples (10 µl) were collected
at 15- to 30-min intervals for 120 min, diluted into heparinized
saline, and microfuged, and serum was assayed for glucose and leptin as
detailed in Serum analyses.
Serum analyses. Serum leptin was
measured by RIA (Linco Research, St. Louis, MO). Serum glucose was
measured by a glucose oxidase-peroxidase method with 4-amino-antipyrine
as the dye, and serum insulin was measured by a double-antibody RIA
with rat insulin standards and anti-rat insulin first antibody (Linco Research).
Measurement of ob mRNA expression.
ob mRNA expression was determined in
epididymal fat tissue with semiquantitative RT-PCR techniques. Total
mRNA was extracted from 100 mg of fat tissue with Tri Reagent (Sigma
Biosciences) and was quantitated with the use of Sybr Green II RNA gel
stain (Molecular Probes, Eugene, OR) with 50- to 800-ng RNA molecular
mass markers (Boehringer Mannheim, Germany) to construct the standard
curve. cDNA was synthesized from the RNA with the Superscript
preamplification system for first-strand cDNA synthesis (Life
Technologies) with random hexamers. PCR of cDNA was performed with
Amplitaq Gold (Perkin Elmer, Forster City, CA). Primers for PCR were
designed with the aid of the sequence analysis software Macvector
(Eastman Kodak Company, Rochester, NY). The forward primer for
measuring ob expression was ATG ACC TGG AGA ACC TGC GAG ACC, and the reverse primer was GTC CTG CAG AGA GCC
CTG CAG CCT GCT.
-Actin expression was also measured as a control
with AAT CCT GTG GCA TCC ATG AAA C as the forward primer and CGC AGC
TCA GTA ACA GTC CG as the reverse primer. Cycle parameters were
94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, with 27 cycles used for ob amplification and
25 cycles for
-actin. No genomic contamination was confirmed with
control RNA samples that had not undergone RT. Products were analyzed
after agarose gel electrophoresis and staining with ethidium bromide.
The image was captured with the software package Molecular analyst
(Bio-Rad Laboratories, Hercules, CA) and imported into NIH image
(National Institutes of Health), and the densities of the PCR products
were quantified. Results are expressed as arbitrary intensity units.
Statistics. All results are given as
means ± SE. Comparisons between CON and GTG were performed with
ANOVA followed by Bonferroni-Dunn post hoc tests with repeated measures
where appropriate. Relationships between variables were determined by
simple regression with the Statview IV statistical package.
 |
RESULTS |
Changes in body weight and the size of individual fat
pads during the development of obesity.
Twelve weeks after GTG injection, GTG mice were 20% heavier than
control mice (CON, 36.2 ± 0.9 g; GTG, 43.0 ± 1.1 g;
P < 0.0001). The differences in body
weight between fed GTG mice and their age-matched controls became
significant during the first 3 wk post-GTG as shown in Fig.
1A.
GTG-injected mice initially lose weight in response to the GTG
injection but regain this weight within 1 wk and are significantly
heavier than controls by 2 wk.

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Fig. 1.
Changes in body weight (A) and in
weights of epididymal (B),
subcutaneous (C), and
retroperitoneal (D) individual fat
pads after injection of gold-thioglucose (GTG; ) and in age-matched
controls (CON, ). Each point is means ± SE for 8-12 mice.
** P < 0.01, *** P < 0.001 for differences
between GTG and CON groups.
|
|
The changes in body weight in the GTG mice were paralleled by similar
changes in all three fat pads (Fig. 1,
B-D),
with significant increases in the size of all fat pads at 2 wk. Strong
correlations were seen in the GTG mice between body weight and the
weights of all three fat pads during these first 3 wk (epi,
r = 0.949; sc,
r = 0.905; rp,
r = 0.880;
P < 0.0001). Significant
correlations were also seen in CON (epi,
r = 0.771; sc,
r = 0.541; rp,
r = 0.608, P < 0.0001).
Changes in serum glucose, insulin, and leptin during
the development of obesity. Serum glucose was reduced
in the first few days post-GTG, reflecting the fall in food intake
after injection. Serum glucose was then normalized, and the
GTG-injected mice did not become significantly hyperglycemic until 4 wk
postinjection (CON, 17.1 ± 2.6; GTG, 25.0 ± 2.0 mM;
P = 0.028). Fed serum levels of
insulin and leptin during the first 3 wk after GTG injection when
differences between the groups became significant are shown in Fig.
2. Serum insulin in GTG was unchanged
during the first few days despite the loss of body weight that occurs
after injection, but it was significantly elevated in GTG at 7 days
(P = 0.0008), preceding the changes in
body and fat pad weight. Serum leptin also showed no fall in the early
stages with signs of an increase at 7 days and with levels threefold
higher than controls by 14 days (P = 0.0001). These changes paralleled the changes that occurred in the size
of all three fat pads and occurred after the rise in serum insulin.
Strong correlations were seen between serum leptin and the weight of
all three fat pads during these first 21 days (epi,
r = 0.810; sc,
r = 0.768; rp,
r = 0.774;
P < 0.0001). The ratio of serum
leptin to the weight of each fat pad was constant from 7 days to 12 wk
in both lean and obese mice as shown for the epididymal fat pad in Fig.
3, indicating a constant leptin secretion
rate that tended to be higher in the GTG-obese mice and was
significantly higher 12 wk post-GTG (P < 0.0007).

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Fig. 2.
Changes in serum insulin (A) and
leptin (B) after injection of GTG
( ) and in age-matched controls ( ). Each point is means ± SE
for 8-12 mice. ** P < 0.01, *** P < 0.001 for
differences between GTG and CON groups.
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Fig. 3.
Ratio of circulating leptin level to epididymal fat pad weight in GTG
(shaded bars) and CON (open bars) at different time points after GTG
injection. Each point is means ± SE for 8-12 mice.
*** P < 0.05 for differences
between GTG and CON groups.
|
|
Changes in ob expression in epididymal adipose tissue
during the development of obesity. Body weight,
epididymal weight, and serum insulin and leptin levels in the mice used
for determination of ob expression
were similar to those found in the longer time course experiment, with
the rise in serum insulin in GTG-injected mice preceding the rise in
serum leptin (Table
1). There was no
difference in the expression of
-actin between CON and GTG (CON,
52.0 ± 2.6; GTG, 56.2 ± 2.8 U) and no effect of time. Changes in ob expression in epididymal fat
pads in GTG-injected mice 3, 7, and 14 days postinjection and in
age-matched controls are shown in Fig. 4.
ob mRNA expression was significantly
reduced at 3 days (P = 0.0168) and
significantly increased by 14 days post-GTG
(P = 0.0268). There was no change in
ob expression in the control mice.
Strong correlations were found between circulating leptin levels and
ob expression at 7 days
(r = 0.764, P = 0.0002) and 14 days
(r = 0.837, P = 0.0096), with a weaker association
at 3 days (r = 0.562, P = 0.069). An association between
ob expression and epididymal weight
was only seen in GTG mice at 7 days (r = 0.897, P = 0.0002).
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Table 1.
Differences in body and epididymal fat pad weights and serum insulin
and leptin levels between gold thioglucose-injected mice 3, 7, and 14 days postinjection and age-matched controls used for
estimation of ob expression
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Fig. 4.
ob mRNA expression in GTG (shaded
bars) 3, 7, and 14 days postinjection and in age-matched CON (open
bars). Expression is expressed as arbitrary density units determined
from image analysis of bands after agarose-gel electrophoresis of PCR
products. Results are means ± SE.
* P < 0.05 for differences
between GTG and CON groups.
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|
Effects of overnight starvation on body and fat pad
weights and on serum glucose, insulin, and leptin
levels. Significant and consistent starvation-induced
reductions in body weight, individual fat pad weights, and serum
glucose, insulin, and leptin levels were seen at all time points.
Results for 12-wk mice that were typical of all time points are shown
in Table 2. The average percent changes in
leptin (50%), glucose (50%), and insulin (74%) were similar in both
CON and GTG and were much greater than the changes in body weight
(12%) and epididymal weight (18%).
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Table 2.
Differences in body and epididymal fat pad weights and serum glucose,
insulin, and leptin levels between fed and overnight-starved CON
and 12 wk GTG-obese mice
|
|
Effects of an oral glucose tolerance test on serum
leptin in controls and GTG-treated mice. Serum glucose
and leptin levels during the oral glucose tolerance test are shown in
Fig. 5. Serum glucose rose in
both groups, peaking about 30 min postgavage, with a higher peak
response being seen in the GTG mice. Although not measured in this
study because of the difficulty of obtaining sufficient blood from
tail-vein bleeds, we have previously shown parallel changes in serum
insulin during an oral glucose tolerance test, with an insulin peak
three times the basal level in CON and fourfold higher in GTG (5).
Fasting leptin levels before the glucose gavage were 2.06 ± 0.20 ng/ml in CON and 10.60 ± 1.62 ng/ml in the GTG-injected mice. There
was no change in leptin levels in either group throughout the 2-h
period.

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Fig. 5.
Serum glucose (2 top
curves; and ) and leptin (2 bottom
curves; and ) levels in CON
(solid
curves) and 5 wk GTG
(dashed
curves) after an oral gavage of 200 µl of 50% glucose. Each point is means ± SE for 8-12
mice.
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Effects of ADX on body and fat pad weights and on
serum glucose, insulin, and leptin levels. Body weight,
epididymal weight, and serum glucose, insulin, and leptin levels in the
ADX and sham ADX mice 1 wk after ADX are summarized in Table
3. ADX resulted in significant
reductions in body and epididymal weight in both CON and GTG mice. No
change occurred in serum glucose in CON, but a reduction did occur in
the hyperglycemia of the GTG mice. Insulin levels were halved in both
CON and GTG mice, whereas leptin levels were reduced by 57% in CON and
24% in GTG mice. Once again, the changes in leptin more closely
resembled the changes in insulin than the changes in fat pad weight. In
this experiment, the ratio of leptin to epididymal weight was
significantly higher in GTG mice (CON, 8.45 ± 0.65; GTG, 13.34 ± 0.61; P < 0.0001). Whereas ADX
decreased this ratio in CON (CON + ADX, 5.35 ± 0.43, P < 0.0001), it had no effect in GTG
(GTG + ADX, 12.30 ± 0.41, P = 0.209).
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Table 3.
Effect of ADX on body and epididymal fat pad weight and serum glucose,
insulin, and leptin levels in CON and 5-wk GTG mice 1 wk
postsurgery
|
|
 |
DISCUSSION |
This study shows that elevated ob
expression and hyperleptinemia develop in the GTG obese mouse in
parallel with increased fat deposition and after the development of
hyperinsulinemia. Parallel development of hyperinsulinemia and
hyperleptinemia (1, 21, 26) or elevated
ob expression (7, 12, 19, 21) has been
demonstrated in other studies where measurements were made before and
after the development of hyperinsulinemia. By more frequent testing
during the early stages of the development of insulin resistance, we
have shown in this model that the rise in insulin precedes the rise in
ob expression and leptin.
Hyperinsulinemia in the GTG mouse is accompanied by a period of
increased insulin sensitivity, resulting in enhanced lipogenesis (3).
Thus it is impossible to say from this study whether the
hyperleptinemia is directly due to the hyperinsulinemia or indirectly
due to the increasing fat content of the adipocytes.
Many studies have shown that exogenous insulin given either acutely or
chronically is able to increase ob
expression in rodents in vivo (7, 24) and both
ob expression and leptin secretion in
vitro (2, 22). However, it appears that whereas insulin has a rapid
effect on expression levels, it requires prolonged exposure to produce
any change in circulating leptin levels (11, 20), as shown by the lack
of change of leptin levels during the oral glucose tolerance test in
this study. Studies in humans suggest that prolonged hyperinsulinemia
is necessary for the elevation of plasma leptin (14), whereas in the
short term, hyperinsulinemia may prevent the postabsorptive decline in
leptin levels (23). A recent study looking at
ob expression and protein production rates in suckling Zucker rats was able to show an increase in leptin
secretion earlier than any change in expression rates (21), suggesting
that increased secretion is not due to elevated production leading to
an efflux of leptin, but rather a separate mechanism is controlling
release into the circulation.
The strong relationship seen between circulating leptin and the size of
the different fat pads gives further support to the theory that the
primary role of leptin is to indicate fatness. The same association
also occurred in the lean mice and has been shown in lean humans and
other models although not in lean
Psammomys obesus (6). The constant ratio of
leptin to fat pad weight over the 12 wk of this study does not suggest
an effect of age independent of increasing adiposity as has been
reported in other studies (1, 16). The ratio tended to be higher in the
GTG mice, and this difference is probably a reflection of the
resistance induced by decreased receptor numbers in the hypothalamus.
Despite the degree of adiposity being the strongest determinant of
leptin levels in both rodents and humans, other factors have been shown to influence the rate of production or secretion per unit of fat, including strain differences (26), dietary composition (1, 9, 26), and
gender (9). It is of interest then that adrenalectomy had an effect on
the ratio of leptin to fat pad weight in the lean mice but not the
obese mice. Rat studies have shown an increased sensitivity to leptin
after ADX (27), and the lower production rates in the lean mice may be
a reaction to this increased sensitivity. In the GTG mice, ADX is not
able to increase sensitivity as the leptin receptors are destroyed,
and, therefore, higher production rates are maintained. Glucocorticoids
stimulate both leptin expression and secretion (25) in vitro, and,
therefore, the fall in leptin in both ADX groups may reflect the
removal of a direct effect of glucocorticoids on the adipocytes. Once
again the relationship between adiposity and circulating leptin is maintained.
Although insulin may not acutely increase circulating leptin levels,
starvation-induced decreases in insulin are accompanied by dramatic
falls in leptin as well as in ob
expression in all models tested except the Zucker rat (1, 7,
10-12, 17, 19, 24). The starvation-induced changes in leptin in
this study more closely paralleled the fall in serum glucose and
insulin levels than the changes in body weight or the weight of
individual fat pads. Whether the trigger is the fall in insulin or a
decrease in total calories or a particular nutrient requires further
study. Human studies suggest a change in carbohydrate intake is
involved (13), and attenuation of the response in high-fat fed mice (1) is consistent with this hypothesis. In vitro studies suggest a role for
free fatty acids in inhibiting leptin production (22), and this is also
consistent with the increased lipolysis seen in starvation. The primary
role of leptin is to indicate the sufficiency of the energy stores of
the body; the administration of exogenous leptin results in reduced
food intake (18), and the inhibition of endogenous leptin action
increases food intake (4). Thus a sudden fall in leptin could be part
of a protective mechanism designed to trigger increased eating to
prevent any change in the degree of adiposity.
In summary, in the GTG-injected obese mouse, hyperinsulinemia precedes
increased ob expression and
hyperleptinemia, which is strongly associated with adiposity. The ratio
of circulating leptin to fat pad weight is unaffected by age, reduced
by fasting and by the absence of glucocorticoids, and slightly raised
in the absence of hypothalamic leptin receptors. Therefore, whereas leptin remains a strong indicator of body fatness, changes in hormonal
and nutritional status may modify this relationship.
 |
ACKNOWLEDGEMENTS |
This study was supported by a project grant from the National
Health and Medical Research Council of Australia.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. Bryson, Human Nutrition Unit, Dept of
Biochemistry, Univ. of Sydney, Sydney, NSW 2006, Australia.
Received 7 May 1998; accepted in final form 30 September
1998.
 |
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