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and Restores Insulin Sensitivity: Independent Effect from Secondary Weight Reduction in Genetically Obese Zucker Fatty Rats1
The Third Department of Internal Medicine, The Laboratory Animal Facility, Yokohama City University School of Medicine, 3–9 Fuku-ura, Kanazawa-ku, Yokohama 236, Japan
Address all correspondence and requests for reprints to: Shun-ichi Tanaka M.D., Ph. D., The Third Department of Internal Medicine, Yokohama City University School of Medicine, 3–9 Fuku-ura, Kanazawa-ku, Yokohama 236, Japan.
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Abstract |
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In this study, the effects of DHEA treatment on insulin sensitivity
were investigated in genetically obese Zucker rats, an animal model of
insulin resistance, using the euglycemic clamp technique. After 0.4%
DHEA was administered for 10 days to female obese Zucker rats aged 16
weeks, body weight and plasma insulin decreased and glucose disposal
rate (GDR), which was normally reduced in obese rats, rose
significantly compared with age- and sex-matched control obese rats. On
the other hand, although the pair-fed obese rats also showed levels of
weight reduction similar to those of DHEA-treated rats, the increase in
GDR of DHEA-treated rats was significantly greater than in pair-fed
rats, suggesting a direct ameliorating effect of DHEA on insulin
sensitivity of obese rats. Serum concentration of tumor necrosis factor
(TNF)-
, one of cytokines causing insulin resistance, was also
reduced significantly in DHEA-treated, but not in pair-fed obese rats.
In conclusion, our results suggest that DHEA treatment reduces body
weight and serum TNF- independently, and that both may ameliorate
insulin resistance in obese Zucker fatty rats.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, one of cytokines, plays an important role in insulin
resistance in experimental animal models (1, 2, 3) and humans (4). The genetically obese (fa/fa) Zucker rat, a model of human obesity, is characterized by hyperinsulinemia, insulin resistance (5, 6), and the impaired suppression of hepatic glucose production following glucose ingestion in both skeletal muscle (7) and liver (8).
Dehydroepiandrosterone (DHEA) and its sulfate ester are the most abundant circulating adrenal steroids in humans (9), but their precise biological function remains to be elucidated. Administration of DHEA has been reported to have striking beneficial effects on obesity (10), hyperlipidemia, diabetes (11), and atherosclerosis (12) in obese rodents. It has been demonstrated that DHEA reduces weight gain and food intake and ameliorates hyperinsulinemia in obese Zucker rats (13). However, the effects of DHEA on glucose metabolism have remained unclear.
In the present study, to elucidate how and why DHEA restore insulin
sensitivity in obese Zucker rats, the effect of DHEA treatment on
glucose sensitivity was investigated using the
hyperinsulinemic-euglycemic clamp technique, and the effect on serum
TNF- concentration was also investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hyperinsulinemic-euglycemic clamp study
The degree of insulin resistance was evaluated by the
hyperinsulinemic-euglycemic clamp technique. Before the study, the
animals underwent an aseptic surgical procedure for the placement of
polyethylene catheters (0.58 mm ID x 0.965 mm OD;
Becton-Dickinson, San Jose, CA) in the right jugular vein under ip
pentobarbital anesthesia (Nembutal; ARBOTT, North Chicago, IL) (40
mg/kg body wt). The catheter was advanced to the level of the right
atrium. The catheter was filled with heparin-saline (Novo Nordisk,
Mainz, Germany) (50 U heparin/ml 0.9% saline), plugged, tunneled sc
around the side of the neck, and externalized to the back of the head
through a skin incision. The rats were housed individually in cages,
and only those animals that were active, eating normally, and showing
no signs of infection within 36 h after surgery were used. Because
food intake of the rats operated on was fully normal on the second day
after the operation, DHEA treatment was begun 3 days after surgery (day
0), and body weight and food intake were measured every day.
Hyperinsulinemic-euglycemic clamp studies were performed at 1500 h after 16 h of fasting on day 10. Regular insulin (Humulin R, Eli Lilly, Indianapolis, IN) dissolved in 0.9% saline was continuously infused at a rate of 1.67 mU/min·kg of body weight, and 10% glucose was infused to keep blood glucose at 100 mg/dl (14). During the studies, blood was collected from the tail vein at 5-min intervals for the fast determination of blood glucose concentrations.
Biochemical measurements
Blood glucose levels were measured by the glucose oxidase method
(Glucose analyzer, Elkay Products, Shrewsbury, MA). Blood samples for
insulin measurement were collected from the tail vein, transferred to
capillaries, and centrifuged. Blood samples for DHEA and DHEA-S
measurement were collected from the left jugular vein before and after
the treatment, and centrifuged. Plasma was removed and stored at -20 C
until assay. Plasma insulin levels were assayed with enzyme-linked
immunosorbent assay (ELISA) kits (Morinaga Seikagaku, Yokohama, Japan)
using guinea pig antirat insulin antibodies and rat insulin (Novo
Nordisk) as a standard. Plasma DHEA and DHEA-S concentrations were
assayed by RIA.
Serum TNF- activity
TNF- levels were measured with a bioassay that
quantitates the TNF level by its cytotoxicity to LM cells, a subline of
TNF-sensitive mouse fibroblasts, and by using recombinant mouse TNF-
as a standard, as described previously (15). Briefly, 2 x
105 LM cells suspended in 0.1 ml of RPMI 1640 supplemented
with 5% heat-inactivated FBS were cultured in a 96-well microculture
plate. After confluent cell growth, the same volume of mice serum
containing actinomycin D (Sigma) at a final concentration of 2 µg/ml
was added. Twenty-four hours later, 50 µl of 0.2% crystal violet was
added to each well and left for 3 min to stain the cells. After washing
with water, the plates were dried and the level of cell lysis was
measured at 540 nm with an autoreader (SJeia autoreader, Sanko Junyaku,
Tokyo, Japan). The titer (U/ml) of the sample was defined as the
reciprocal of the dilution resulting in 50% cell survival of LM cells.
The absorbance of control wells to which no rTNF- had been added was
defined as that of 100% cell survival. The reciprocal of the dilution
of the sample corresponding to half of this absorbance (50% cell
survival) was determined with an on-line computer (16).
Statistical analysis
Data were expressed as means ± SEM.
Statistical analyses of differences were performed using one-way ANOVA,
2 test in the comparison study and the paired
t test (two-tailed) to evaluate the effects of DHEA
treatment. Significance was measured using Fisher’s least significant
for the exact P values. Differences were considered
statistically significant at P values less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
Before experimental period, three obese rat groups showed no
significant difference in body weights. After 10 days of experimental
period, there was only a small but significant decrease in body weights
in the DHEA-treated and pair-fed obese animals.
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shows daily food intake
throughout the experiment (day -4 through day 10). Before DHEA was
introduced into the diet, no significant difference was observed
between the DHEA-treated and control obese rats. After experimental
period, however, daily food intake decreased significantly in
DHEA-treated obese rats. DHEA-treated obese rats showed about 60%
reduction in food intake compared with control obese rats. Catheter
placement led to a transient decrease in food intake which returned to
normal after 2–3 days.
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). After 10 days of experimental period,
plasma insulin decreased significantly in DHEA-treated, but not in
pair-fed, obese rats. However, fasting blood glucose showed no
significant difference between DHEA-treated and control obese rats
(data not shown).
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, GDR increased
significantly in DHEA-treated obese rats compared with pair-fed rats,
and suggested a direct ameliorating effect of DHEA on insulin
sensitivity. Despite this increased GDR, however, DHEA-treated obese
rats still showed reduced GDR as compared with lean rats.
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. After the experimental period, serum
DHEA-S, but not DHEA, concentration was significantly increased in
DHEA-treated obese rats as compared with control obese rats. Thus, it
appears that DHEA administrated orally was mainly present as its
sulfated conjugate in obese Zucker rats.
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levels concentration was examined in
three obese rat groups. As shown in Table 2, serum TNF-, which could be detected
in control obese and pair-fed obese rats, was not detected in
DHEA-treated obese rats or in lean rats. In each of the control obese
and pair-fed obese rats, 7 of 10 rats detected serum TNF- (70%),
whereas none in DHEA-treated obese rats (P 
0.001). It could not
be concluded from the experimental results alone that TNF- levels of
DHEA-treated obese rats were same in lean rats. We tested the effect of
DHEA and DHEA-S on the survivability of the LM cells; the results
showed that neither DHEA nor DHEA-S with physiological concentration
had a direct influence on the LM bioassay (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and subsequently increased GDR in obese Zucker rats. Body
weight also decreased significantly in the pair-fed obese rats, but the
increase in GDR was significantly lower than in DHEA-treated rats.
These results suggest that the increase in insulin sensitivity in
association with DHEA treatment may be, at least in part, a direct
effect of DHEA as well as a secondary effect of the weight
reduction.
To our knowledge, this is the first report of DHEA administration
reducing serum TNF- and ameliorating insulin resistance in obese
rats via the euglycemic hyperinsulinemic clamp. The euglycemic glucose
clamp technique offers significant advantages over the commonly used
technique for assessing insulin sensitivity, the glucose tolerance test
(14). In the oral version of the test, a rise in plasma glucose
stimulates the [E1]-cell release of insulin, the resultant rise in
plasma insulin stimulates the cellular uptake of glucose, and the
plasma glucose falls. Because this technique places the plasma glucose
under the investigator’s control and thus breaks the simple
glucose-insulin feedback loop, the euglycemic glucose clamp technique
provides a more reliable estimate of tissue sensitivity to insulin.
DHEA is an adrenal and gonadal hormone intermediate whose sulfated form is the most abundant steroid in human plasma. This precursor to such classical steroids as estradiol and testosterone has been shown to have an antiobesity effect in several species (11, 17). Previous studies reported that DHEA treatment reduced food intake, body weight, and plasma insulin in hyperinsulinemic obese rats (7, 18). Plasma insulin levels were reduced by DHEA treatment with little or no effects on blood glucose (19). However, it was also reported that the antiobesity effect of DHEA was not mediated by the reduction in food intake but was primarily due to loss of fat stores (18, 20). In several studies, lower body fat was found in DHEA-treated rats even when body weight was not altered (21). In addition, lowered body fat has been found in DHEA-treated rats even when body weight was not altered (21). In addition, lowered body fat has been found in DHEA-treated was more than just a consequence of the lower body weight rats (7). It is not clear how these effects of DHEA on adipose tissue growth and metabolism are mediated, whether it is an indirect or direct action. The fundamental mechanism of this antiobesity effect of DHEA has not yet been elucidated. In this study, serum DHEA-S, but not DHEA, concentration was significantly increased in DHEA-treated obese Zucker rats. The result may suggest an ameliorating effect of DHEA-S on insulin sensitivity of obese rats.
On the other hand, recent studies have shown that TNF- plays a
key role in the pathophysiology of obesity. Administration of
TNF--inhibited gastric emptying (22), and body weight reduction in
obese subjects after dietary restriction was associated with a decrease
in TNF- mRNA(23). Spiegelman et al. demonstrated that
TNF- from adipose tissue may play a crucial role in the systemic
insulin resistance of obese Zucker rats (1, 24). In these studies,
obese Zucker rats did not detectable serum levels of TNF-. As shown
in Table 2
, serum TNF- concentration was detectable, although low
levels, in the control obese and pair-fed obese rats, but not in lean
and DHEA-treated obese rats in our study. Well known overexpression of
TNF- in adipose tissue of obese animals may be relevant to increased
circulating TNF- in obese animals, which may suggest an importance
of an endocrine mechanism as well as autocrine-paracrine mechanism
discussed in these studies. These results suggest that DHEA treatment
may reduce body weight and serum TNF- independently, and that both
ameliorate insulin resistance in obese Zucker fatty rats.
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Acknowledgments |
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Footnotes |
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Received December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: direct role
in obesity-linked insulin resistance. Science 259:87–90 in obese rats.
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and obesity-induced
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hyperplasia and hyperinsulinemia in genetically obese Zucker rats. Proc
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), DHEA-treated obese (•),
pair-fed obese (
), and lean (
) rats. Numbers of animals studied
were 10 in each group. Results are presented as means ±
SE.
), and lean rats (
). Numbers of animals
studied were 10 in each group. Results are presented as means ±
SE. *, P < 0.05 vs.
baseline.
