Effect of heterozygous PPARgamma deficiency and TZD treatment on insulin resistance associated with age and high-fat feeding

Philip D. G. Miles1, Yaacov Barak5, Ronald M. Evans5, and Jerrold M. Olefsky2,3,4

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptor-gamma (PPARgamma ) 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 PPARgamma (PPARgamma +/-) are more insulin sensitive than their wild-type (WT) littermates, not less, as would be predicted. To determine whether PPARgamma deficiency could prevent the development of the insulin resistance associated with increasing age or high-fat (HF) feeding, insulin sensitivity was assessed in PPARgamma +/- and WT mice at 2, 4, and 8 mo of age and in animals fed an HF diet. Because TZDs elicit their effect through PPARgamma 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 PPARgamma +/- animals was substantially less than that of the WT animals, such that, by 8 mo of age, the PPARgamma +/- mice were markedly more insulin sensitive than the WT mice. This greater sensitivity in PPARgamma +/- 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 PPARgamma +/- mice. Treatment of these mice with troglitazone completely prevented the adipocyte hypertrophy and normalized insulin action. In conclusion, PPARgamma deficiency partially protects against age-related insulin resistance but does not protect against HF diet-induced insulin resistance.

peroxisome proliferator-activated receptor-gamma deficiency; high-fat diet; aging; insulin resistance; thiazolidinedione; mice


    INTRODUCTION
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma (PPARgamma ) (15). PPARgamma 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 PPARgamma 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 PPARgamma to further elucidate the physiological role of PPARgamma in glucose homeostasis (homozygous PPARgamma -null animals were not viable). Paradoxically, PPARgamma +/- 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 PPARgamma 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 PPARgamma +/- 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 PPARgamma +/- mice are protected from high-fat diet-induced insulin resistance. To address these questions, we measured insulin action in PPARgamma +/- 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 PPARgamma +/- mice compared with WT littermates under these various conditions.


    RESEARCH DESIGN AND METHODS
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Mice carrying the PPARgamma -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 (PPARgamma +/+) and PPARgamma +/- 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. PPARgamma +/- 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 PPARgamma +/- 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.


    RESULTS
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Our studies were performed with WT and PPARgamma -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 PPARgamma deficiency: comparison of 2-, 4-, and 8-mo-old mice. We have previously shown (18) that, in animals 8 mo of age, heterozygous PPARgamma +/- 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 PPARgamma +/- 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.

Body and epididymal fat pad weights steadily increased with age, and PPARgamma +/- mice were not different from WT mice at all three ages, as seen in Table 1. However, epididymal fat cell size (1,810 ± 180 vs. 2,240 ± 190 µm2, P < 0.05) was less in the PPARgamma +/- group compared with WT. The fasting plasma glucose concentrations of the PPARgamma +/- mice were comparable to those of the WT mice and did not change from 2 to 8 mo of age. The fasting insulin concentrations were similar at 2 and 4 mo of age, but at 8 mo of age, the insulin level of the WT mice was significantly higher than that of the PPARgamma +/- mice (0.31 ± 0.07 vs. 0.45 ± 0.05 ng/ml, P < 0.05). We also measured in vivo insulin sensitivity in PPARgamma +/- and WT animals at 2, 4, and 8 mo of age. For comparison purposes, insulin sensitivity of the animals at the three different ages is expressed as the exogenous glucose infusion rate (Glcinf) necessary to maintain euglycemia, and the results are presented in Fig. 1. As can be seen in WT animals, there was a marked and progressive decline in insulin-stimulated Glcinf going from 2 to 4 to 8 mo of age, resulting in a fall from 85.2 ± 8.1 to 32.7 ± 2.8 mg · kg-1 · min-1 from 2 to 8 mo. In the PPARgamma +/- animals, insulin sensitivity was comparable to controls at 2 and 4 mo of age, but the decline in insulin sensitivity going from 4 to 8 mo of age is substantially less, such that, by 8 mo of age, the PPARgamma +/- animals are more insulin sensitive than WT controls (47.3 ± 5.3 vs. 32.7 ± 2.8 mg · kg-1 · min-1, P < 0.01). These data indicate that heterozygosity for the PPARgamma receptor provides partial protection from the age-related physiological insulin resistance that occurs in mice.

                              
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Table 1.   Evaluation of mice at 2, 4, and 8 mo of age



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Fig. 1.   Steady-state exogenous glucose infusion rate during hyperinsulinemic euglycemic glucose clamp experiments as an indication of insulin sensitivity in wild-type (WT) and heterozygous peroxisome proliferator-activated receptor-gamma -deficient (PPARgamma +/-) mice at 2, 4, and 8 mo of age (n = 7-9) fed a regular chow diet. * Significantly different from WT group.

Effect of TZD treatment on 8-mo-old WT and PPARgamma +/- mice. The PPARgamma 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 PPARgamma +/- mice compared with WT littermates. Accordingly, chow-fed 8-mo-old WT and PPARgamma +/- animals were given troglitazone for 4 wk or not, and various measurements were made in these groups of animals.

Figure 2 shows the effects of TZD treatment on body weight, adipose tissue, free fatty acids (FFAs), and leptin levels in WT and PPARgamma +/- animals. Body weight was the same in all animal groups under all conditions. In WT animals, fat pad weight was unaffected by TZD treatment. However, after TZD treatment, fat pad weight increased in the PPARgamma +/- animals and was now more comparable to values seen in WT littermates. The average fat cell size in the epididymal fat pad was smaller in untreated PPARgamma +/- animals compared with WT littermates (P < 0.05) and was significantly increased after TZD treatment to values comparable to those of controls. Fat cell size was unaffected by TZD treatment in WT littermates. FFA and serum leptin levels were not significantly different among the groups.


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Fig. 2.   Body weight, fat pad weight, epididymal fat cell size, and circulating free fatty acid (FFA) and leptin levels in 8-mo-old WT and PPARgamma +/- mice (n = 8-11) fed a chow diet ± troglitazone [a thiazolidinedione (TZD)]. * Significantly different from WT group; # significantly different from chow-fed group.

OGTTs were performed on these TZD-treated and untreated groups, and the results are presented in Fig. 3. As can be seen, basal and postchallenge glucose levels were the same in both groups of animals with and without TZD administration. However, both basal and postchallenge insulin levels were decreased in the untreated PPARgamma +/- animals compared with WT littermates, but after TZD treatment insulin levels increased to values comparable to those observed in WT mice.


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Fig. 3.   Glucose and insulin levels during oral glucose tolerance tests (OGTTs) in 8-mo-old WT and PPARgamma +/- mice (n = 7-11) fed a chow diet ± troglitazone. * Significantly different from WT group.

The expected effect of TZD treatment is to increase insulin sensitivity. However, the increase in insulinemia during the OGTTs in the TZD-treated PPARgamma +/- animals suggests the opposite, i.e., a TZD-induced decrease in insulin sensitivity. To directly assess this possibility, we conducted a series of hyperinsulinemic euglycemic glucose clamps in the various animal study groups, and these data are summarized in Fig. 4. TZD treatment had no effect on insulin action in WT littermates, and these findings are consistent with previous studies demonstrating that various TZDs are without effect on insulin sensitivity in normal animals (4, 5) and humans (19, 28). In the PPARgamma +/- animals, GDR values are 34% higher in untreated animals compared with WT littermates at 8 mo, and TZD treatment leads to a significant reduction in GDR. Thus, after the period of drug treatment, the PPARgamma +/- animals are less insulin sensitive than the untreated group, and the GDR values in the treated PPARgamma +/- animals are now comparable to those seen in WT littermates. Thus a 50% genetic deletion of the PPARgamma receptor led to the unexpected finding of enhanced, rather than diminished, insulin sensitivity in chow-fed untreated animals, and, in keeping with this counterintuitive finding, TZD treatment has the paradoxical effect of reducing insulin sensitivity in these animals, bringing insulin-stimulated glucose uptake back to control values.


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Fig. 4.   Glucose disposal rate and hepatic glucose production in 8-mo-old WT and PPARgamma +/- mice (n = 8-11) fed a chow diet ± troglitazone. * Significantly different from WT group; # significantly different from chow-fed group.

Similar results were seen when hepatic insulin sensitivity was assessed (Fig. 4). In WT animals, the insulin infusion led to a 55% inhibition of HGP during the glucose clamp study, and this was unaffected by TZD treatment. In the PPARgamma +/- animals, HGP suppression by insulin was greater in the untreated animals compared with WT controls, and TZD treatment led to a blunting of this aspect of insulin action so that HGP suppression was now comparable to WT controls.

Effect of PPARgamma deficiency on high-fat diet-induced insulin resistance. In 8-mo-old PPARgamma +/- 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 PPARgamma +/- and WT animals fed a high-fat diet are equally insulin resistant. As seen in Fig. 7, basal glucose turnover in the PPARgamma +/- 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 PPARgamma +/- 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 PPARgamma +/- and WT mice are equally insulin resistant on a high-fat diet.


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Fig. 5.   Body weight, fat pad weight, epididymal fat cell size, and circulating FFA and leptin levels in 8-mo-old WT and PPARgamma +/- mice (n = 6-11) fed a high-fat diet ± troglitazone. The chow-fed group is included from Fig. 2 for comparison purposes. * Significantly different from WT group; # significantly different from chow-fed group; &significantly different from high-fat diet-fed group.



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Fig. 6.   Glucose and insulin levels during OGTTs in 8-mo-old WT and PPARgamma +/- mice (n = 7-11) fed a high-fat diet ± troglitazone. # Significantly different from non-TZD-treated group.



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Fig. 7.   GDR and HGP in 8-mo-old WT and PPARgamma +/- mice (n = 6-11) fed a high-fat diet ± troglitazone. The chow-fed group is included from Fig. 4 for comparison purposes. * Significantly different from WT group; # significantly different from chow-fed group; & significantly different from high-fat diet-fed group.

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 PPARgamma +/- 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 PPARgamma +/-) 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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin-sensitizing thiazolidinediones are high-affinity ligands for the PPARgamma 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 PPARgamma 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 PPARgamma 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 PPARgamma 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 PPARgamma +/- and WT mice were similar and progressively declined by 4 and 8 mo of age. The rate of decline of the PPARgamma +/- mice, however, was less than that for the WT mice, such that by 8 mo of age, insulin sensitivity of the PPARgamma +/- mice was greater than in WT mice.

A more detailed examination was conducted in the 8-mo-old mice, and the current findings are consistent with those of our previous study (18). The heightened insulin sensitivity seen in the PPARgamma +/- mice occurred in peripheral tissue (muscle and fat tissue) and in the liver, as reflected by the greater insulin stimulation of GDR and suppression of HGP, respectively. Furthermore, fat cell size in the PPARgamma +/- mice was smaller than in the WT littermates, and smaller fat cells have been associated with insulin sensitivity (20).

The present studies show that PPARgamma receptor-deficient animals have greater insulin sensitivity than WT controls but that this effect occurs only in the postmature state. In other words, the PPARgamma +/- animals are relatively protected from the normal physiological decrease in insulin sensitivity, which occurs when mice age from 2 to 8 mo. Although the mechanism(s) of this "developmental" insulin resistance is not fully understood, in addition to simple aging, as mice get older they become more obese and less physically active, and a similar series of events occurs in humans. Thus relative PPARgamma deficiency apparently mitigates these physiological causes of insulin resistance, raising the possibility that a therapeutic maneuver that could produce the same effect as PPARgamma deficiency might be of clinical value in the treatment of insulin resistance.

TZDs are traditionally used to increase insulin sensitivity. In normal WT mice, TZD treatment had no effect on insulin action, consistent with previous studies demonstrating that various TZDs are without effect on insulin sensitivity in normal animals and humans (4, 5, 19, 28). In the PPARgamma +/- animals, however, TZD treatment significantly lowered GDR and impaired insulin's suppressive effects on HGP (increased HGP values during the clamp study) compared with untreated animals. Thus, after the period of drug treatment, the PPARgamma +/- animals are less insulin sensitive than the untreated group, and the GDR and HGP values in the treated PPARgamma +/- animals are now comparable to those seen in WT littermates. Thus a 50% genetic deletion of the PPARgamma receptor led to the unexpected finding of enhanced, rather than diminished, insulin sensitivity in chow-fed animals, and, in keeping with this counterintuitive finding, TZD treatment reduced insulin sensitivity in these animals, bringing insulin-stimulated glucose uptake back to control values. Troglitazone also increased fat pad weight and fat cell size in PPARgamma +/- animals, and this may contribute to the decreased insulin sensitivity.

PPARgamma 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 PPARgamma deficiency confers protection against age-related insulin resistance, it did not protect against high-fat diet-induced insulin resistance. Thus the PPARgamma +/- 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 PPARgamma +/- 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 PPARgamma +/- mice on the high-fat diet.

It has been shown that leptin administration can increase insulin sensitivity in vivo in rodents (8, 24); however, at first approximation, our data do not seem to support this idea. Thus the major directional changes in leptin levels due to TZD treatment and high-fat feeding were concordant between the WT and PPARgamma +/- groups. Leptin levels increased with high-fat feeding in parallel with the onset of insulin resistance and decreased after TZD treatment, and these effects go in the opposite direction in changes in insulin sensitivity. However, it is important to note that, in vivo, a major site of action of leptin is the central nervous system (CNS), and intracerebroventricular administration of leptin has been shown to increase insulin sensitivity (8). Clearly, we did not measure cerebrospinal fluid levels of leptin in our studies; therefore, concentrations of leptin that could exert central effects or CNS leptin sensitivity are unknown in these animals.

The results of our high-fat feeding studies differ from the work of Kubota et al. (13), who showed that PPARgamma receptor-deficient mice did not display adipocyte hypertrophy and appeared to be protected from insulin resistance while on a high-fat diet. The reasons for the discrepancy between the two studies are not clear, but several possibilities exist. For example, the physiological response of mice to a diet high in fat is highly dependent on the background strain of the animal. In our studies, heterozygous PPARgamma offspring were backcrossed eight times onto the C57BL/6J strain background and for practical purposes can be considered a pure strain. The purity of the mice used in the study by Kubota et al. is not specified. It is also possible that the composition of the respective high-fat diets is a factor. In the present study, the fat in the diet came predominately from partially hydrogenated vegetable oil as opposed to the polyunsaturated safflower oil used in the study by Kubota et al. Although saturated fats tend to cause a greater degree of insulin resistance than unsaturated fat, it has been reported that both result in a substantial impairment of insulin action (26, 27). However, the latter studies were conducted in normal animals, whereas the present experiments and those of Kubota et al. were in genetically altered animals. These findings raise the possibility that there may be more than one mechanism by which a diet high in fat leads to insulin resistance. In the present study, PPARgamma +/- mice fed saturated fat responded like WT mice and developed obesity and insulin resistance, suggesting that the mechanism by which this occurred was intact in PPARgamma +/- mice. Conversely, the PPARgamma +/- mice fed unsaturated fat in the study of Kubota et al. were protected from adipocyte hypertrophy and insulin resistance, suggesting that the mechanism by which unsaturated fat induces insulin resistance may involve PPARgamma receptors. It is possible that different kinds of fat diets influence production of endogenous PPARgamma ligands, and the interaction of these ligands with PPARgamma receptors may be quantitatively or qualitatively different in the presence of receptor deficiency.


    ACKNOWLEDGEMENTS

We thank Michael C. Nelson for excellent mouse colony management and genotyping.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 284(3):E618-E626