Departments of 1 Surgery and 2 Medicine, University of California, San Diego, 3 San Diego Veterans Affairs Medical Center; 4 Whittier Diabetes Institute; and 5 Gene Expression Laboratory, Howard Hughes Medical Institute, Salk Institute, La Jolla, California 92093
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ABSTRACT |
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Peroxisome proliferator-activated
receptor- (PPAR
) is the target receptor for
thiazolidinedione (TZD) compounds, which are a class of
insulin-sensitizing drugs used in the treatment of type 2 diabetes.
Paradoxically, however, mice deficient in PPAR
(PPAR
+/
) are more insulin sensitive than
their wild-type (WT) littermates, not less, as would be predicted. To
determine whether PPAR
deficiency could prevent the development of
the insulin resistance associated with increasing age or high-fat (HF)
feeding, insulin sensitivity was assessed in
PPAR
+/
and WT mice at 2, 4, and 8 mo of age
and in animals fed an HF diet. Because TZDs elicit their effect through
PPAR
receptor, we also examined the effect of troglitazone (a TZD)
in these mice. Glucose metabolism was assessed by hyperinsulinemic
euglycemic clamp and oral glucose tolerance test. Insulin sensitivity
declined with age for both groups. However, the decline in the
PPAR
+/
animals was substantially less than
that of the WT animals, such that, by 8 mo of age, the
PPAR
+/
mice were markedly more insulin
sensitive than the WT mice. This greater sensitivity in
PPAR
+/
mice was lost with TZD treatment. HF
feeding led to marked adipocyte hypertrophy and peripheral tissue and
hepatic insulin resistance in WT mice but also in
PPAR
+/
mice. Treatment of these mice with
troglitazone completely prevented the adipocyte hypertrophy and
normalized insulin action. In conclusion, PPAR
deficiency partially
protects against age-related insulin resistance but does not protect
against HF diet-induced insulin resistance.
peroxisome proliferator-activated receptor- deficiency; high-fat
diet; aging; insulin resistance; thiazolidinedione; mice
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INTRODUCTION |
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THIAZOLIDINEDIONE (TZD)
COMPOUNDS are a new class of insulin-sensitizing drugs used in
the treatment of type 2 diabetes. They improve insulin sensitivity,
glucose tolerance, and the lipidemic profile in type 2 diabetic
patients (7, 28), as well as in obese nondiabetic subjects
(19). Similar findings have also been demonstrated in a
number of genetic and nongenetic animal models of diabetes/insulin
resistance (4, 5, 14). TZDs have been shown to elicit
their effect through peroxisome proliferator-activated receptor-
(PPAR
) (15). PPAR
belongs to a subfamily of nuclear receptors involved in the control of various aspects of lipid metabolism (10). These receptors function as heterodimers
with the retinoid X receptor (11, 12, 17) and bind to
cis-acting sequences (peroxisome proliferator response
element) on DNA to initiate transcription (16).
Adipogenesis (30) and other cellular processes of lipid
accumulation (31) are stimulated by PPAR
through the
induction of genes mediating fatty acid metabolism (22, 23,
29). In addition, it plays a critical role in proper placental
vascularization, myocardial health, and embryonic development (1).
We previously studied mice heterozygous for PPAR to further
elucidate the physiological role of PPAR
in glucose homeostasis (homozygous PPAR
-null animals were not viable). Paradoxically, PPAR
+/
mice displayed greater insulin
sensitivity than did their wild-type (WT) littermates
(18). These findings were unexpected and run contrary to
what might have been predicted on the basis of the known biological
effects and mechanism of action of TZDs. This suggests that the
inhibition of PPAR
function could render individuals less
susceptible to the development of insulin resistance due to obesity,
type 2 diabetes, aging, or other factors.
Insulin sensitivity normally declines as rodents (2) and
humans (3) age, and this raises the question whether
PPAR+/
mice exhibit increased insulin
sensitivity at all stages of development or whether these animals are
relatively protected from the natural decline in insulin sensitivity
that occurs with increasing age. A diet high in fat causes insulin
resistance (26, 27), and we also sought to determine
whether PPAR
+/
mice are protected from
high-fat diet-induced insulin resistance. To address these questions,
we measured insulin action in PPAR
+/
and WT
mice from 2 to 8 mo of age and in mature mice fed a high-fat diet.
Last, it was of interest to assess the effects of TZD treatment in
PPAR
+/
mice compared with WT littermates
under these various conditions.
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RESEARCH DESIGN AND METHODS |
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Animals.
Mice carrying the PPAR-null allele are described elsewhere
(1). Genotypes were determined by PCR of tail DNA
(1). Animals used in our physiological studies were
age-matched WT (PPAR
+/+) and
PPAR
+/
male offspring of eight consecutive
back-crosses onto a C57BL/6J strain background (30:1
C57BL/6J-to-129/SvJae allelic ratio). Mice were housed under controlled
light (12:12 h) and temperature conditions, and had free access to food
and water. All procedures were in accordance with the Guide for
the Care and Use of Laboratory Animals of the National Institutes
of Health and approved by the Animal Subjects Committee of the
University of California, San Diego.
Age-related study.
PPAR+/
and WT mice were studied at 2, 4, and 8 mo of age. In addition, 8-mo-old mice were treated with and
without troglitazone (a TZD) for 4 wk and underwent glucose clamp
testing and an oral glucose tolerance test (OGTT) according to methods
described previously (9). The drug was given as a 0.2%
food admixture and was freshly mixed with regular powdered rodent chow
(Rodent Diet no. 8604, Harlan Teklad, Madison, WI) in small amounts
every week and stored at 4°C. The 2-mo-old mice underwent a modified
glucose clamp, because their small size precluded the use of glucose tracer.
High-fat feeding study.
Eight-month-old PPAR+/
and WT mice were fed
regular chow or a high-fat diet (TD 85418; Harlan Teklad) with and
without troglitazone for 4 wk. Fifty-six percent of the calories of the
high-fat diet came from partially hydrogenated vegetable oil, and a
complete description of the diet is described elsewhere
(9). Troglitazone was given as a 0.2% admixture and was
freshly mixed with the fat diet or powdered rodent chow in small
amounts every week and stored at 4°C. Three weeks into the diet,
animals underwent an OGTT and, 1 wk later, a glucose clamp experiment
as described elsewhere (18). The epididymal fat pads were
harvested after the glucose clamp.
Assays. Plasma glucose concentration was measured with a YSI 2300 STAT Glucose/Lactate Analyzer (YSI, Yellow Springs, OH). Insulin and leptin were measured using radioimmunoassay kits (Linco, St. Charles, MO). Plasma glucose specific activity was measured after deproteinization with barium hydroxide and zinc sulfate (21). Epididymal fat cell size was determined using the osmium tetraoxide method after digestion with collagenase (6).
Calculations. Hepatic glucose production (HGP) and glucose disposal rate (GDR) were calculated for the basal period and the steady-state portion of the glucose clamp by use of the Steele equation for steady-state conditions (25). Values presented are means ± SE. Statistical analysis was performed by using a two-way analysis of variance (ANOVA) for unbalanced data. Significance was assumed at P < 0.05.
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RESULTS |
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Animals.
Our studies were performed with WT and PPAR-heterozygous mice, and
animals were fully developed, fertile, and healthy. Due to effects of
sporadic genetic variations between different mouse strains on the
susceptibility to metabolic disorders, experiments were conducted on
animals back-crossed for eight consecutive generations against a
C57BL/6J strain background. The control group was comprised of WT
siblings of the heterozygous mice.
Age-related effects of PPAR deficiency: comparison of 2-, 4-, and 8-mo-old mice.
We have previously shown (18) that, in animals 8 mo of
age, heterozygous PPAR
+/
mice show enhanced
peripheral and hepatic insulin sensitivity compared with WT mice. It is
known that insulin sensitivity normally declines as rodents and humans
age, and, because 8-mo-old mice are postmature, the question
arises whether PPAR
+/
mice exhibit
increased insulin sensitivity at different stages of development or
whether these animals are relatively protected from the natural decline
in insulin sensitivity that occurs with age.
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Effect of TZD treatment on 8-mo-old WT and
PPAR+/
mice.
The PPAR
receptor is the target of insulin-sensitizing TZD agents,
and it is notable that animals with a 50% genetic deficiency of this
receptor display enhanced insulin action on glucose metabolism. It was,
therefore, of interest to assess the effects of TZD treatment in
PPAR
+/
mice compared with WT littermates.
Accordingly, chow-fed 8-mo-old WT and
PPAR
+/
animals were given troglitazone for
4 wk or not, and various measurements were made in these groups of animals.
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Effect of PPAR deficiency on high-fat diet-induced insulin
resistance.
In 8-mo-old PPAR
+/
and WT mice, a
diet high in fat increased body weight, fat pad weight, fat cell size,
and FFA and leptin levels compared with chow-fed mice (Fig.
5). Compared with the chow diet,
high-fat feeding also led to an increase in basal glucose and insulin
concentrations (t = 0) to similar levels in both groups (Fig. 6). During the OGTT (Fig. 6), both
groups showed a greater and equal increase in plasma glucose and
insulin concentrations compared with the chow-fed animals (compare Fig.
3 with Fig. 6). This suggests that
PPAR
+/
and WT animals fed a high-fat diet
are equally insulin resistant. As seen in Fig.
7, basal glucose turnover in the
PPAR
+/
and WT groups were slightly but
significantly elevated compared with the chow-fed groups. The
insulin-induced increase in GDR and decrease in HGP of the
PPAR
+/
and WT groups were similar but
significantly less than those of the chow-fed groups (Fig. 7). These
results indicate that the liver and the peripheral tissues of
PPAR
+/
and WT mice are equally insulin
resistant on a high-fat diet.
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Effect of TZD treatment on high-fat diet-induced insulin
resistance.
In 8-mo-old mice fed a high-fat diet, troglitazone treatment equally
decreased body weight, fat pad weight, fat cell size, and FFA and
leptin levels in both PPAR+/
and WT groups
(Fig. 5). Treatment also equally decreased basal and postglucose
challenge glucose and insulin levels to the values seen in chow-fed WT
mice (Fig. 6, bottom). Furthermore, troglitazone treatment
had a comparable effect of enhancing insulin sensitivity in both groups
(WT and PPAR
+/
) of high fat-fed mice. Thus
the insulin-induced increase in GDR and the suppression of HGP were
enhanced equally in both groups, and the values were comparable to
those seen in the chow-fed WT mice (Fig. 7).
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DISCUSSION |
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Insulin-sensitizing thiazolidinediones are high-affinity ligands
for the PPAR nuclear receptor (15), which regulates the transcription of genes involved in lipid and glucose metabolism (22, 23, 29). We have previously shown (18)
that a 50% reduction in PPAR
receptor content did not result in
insulin resistance, as one might predict, but rather led to an increase in insulin sensitivity. Therefore, we postulated that PPAR
deficiency might prevent or attenuate the insulin resistance associated
with type 2 diabetes, obesity, aging, and other factors. In this study, we examined the effect of PPAR
deficiency on two physiological causes of insulin resistance: increasing age and high-fat diet.
Age-related insulin resistance.
It is well established that insulin sensitivity normally declines with
age in both humans and animals (2, 3), and this may be due
to obesity, physical inactivity, or other age-related factors. The mice
in our study were no exception. At 2 mo of age, insulin sensitivity, as
measured by Glcinf values, of the
PPAR+/
and WT mice were similar and
progressively declined by 4 and 8 mo of age. The rate of decline of the
PPAR
+/
mice, however, was less than that
for the WT mice, such that by 8 mo of age, insulin sensitivity of the
PPAR
+/
mice was greater than in WT mice.
PPAR deficiency and high-fat diet-induced insulin resistance.
Four weeks of high-fat diet feeding led to the expected effects of
glucose intolerance, increased adiposity, and both peripheral tissue
and hepatic insulin resistance in WT mice. Although the results in Fig.
1 show that PPAR
deficiency confers protection against age-related
insulin resistance, it did not protect against high-fat diet-induced
insulin resistance. Thus the PPAR
+/
mice
became just as glucose intolerant and insulin resistant as their WT
littermates fed a high-fat diet. In fact, because these animals were
more insulin sensitive before high-fat feeding was initiated, the
diet-induced decrease in peripheral and hepatic insulin sensitivity was
actually greater in the PPAR
+/
animals.
Furthermore, fat pad weight and fat cell size, as well as circulating
FFA and leptin levels, were all comparable between TZD-treated WT and
PPAR
+/
mice on the high-fat diet.
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ACKNOWLEDGEMENTS |
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We thank Michael C. Nelson for excellent mouse colony management and genotyping.
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FOOTNOTES |
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Y. Barak was supported by a European Molecular Biology Organization fellowship and by funds from the Charles and Anna Stern Foundation. R. M. Evans is an Investigator of the Howard Hughes Medical Institute at the Salk Institute and March of Dimes Chair in molecular and developmental biology. This work was supported in part by grants from the National Institutes of Health (DK-33651 and HD-27183) and the Veterans Administration Research Service, Department of Veterans Affairs.
Address for reprint requests and other correspondence: P. D. G. Miles, Dept. of Surgery (8400), UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 92103 (E-mail: pmiles{at}ucsd.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00312.2002
Received 12 July 2002; accepted in final form 14 November 2002.
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