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Endocrinology Vol. 147, No. 9 4517-4524
Copyright © 2006 by The Endocrine Society

Improvement of Insulin Sensitivity after Peroxisome Proliferator-Activated Receptor-{alpha} Agonist Treatment Is Accompanied by Paradoxical Increase of Circulating Resistin Levels

M. M. Haluzik, Z. Lacinova, M. Dolinkova, D. Haluzikova, D. Housa, A. Horinek, Z. Vernerova, T. Kumstyrova and M. Haluzik

Third Department of Medicine (M.M.H., Z.L., M.D., D.Ha., D.Ho., A.H., T.K., M.H.) and Department of Sports Medicine (D.Ha.), First Faculty of Medicine and General University Hospital, Charles University, 128 08 Prague, Czech Republic; Department of Chemistry (M.M.H.), Faculty of Science, University of Ostrava, 70103 Ostrava, Czech Republic; and Department of Pathology (D.Ho., Z.V.), Third Faculty of Medicine and University Hospital Kralovske Vinohrady, Charles University, 100 34 Prague, Czech Republic

Address all correspondence and requests for reprints to: Martin Haluzik, 3 Department of Medicine, 1 Faculty of Medicine, Charles University, U nemocnice 1, 100 34, Prague-2, Czech Republic. E-mail: mhalu{at}lf1.cuni.cz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied the effect of peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) activation on serum concentrations and tissue expression of resistin, adiponectin, and adiponectin receptor-1 and -2 (AdipoR1 and AdipoR2) mRNA in normal mice and mice with insulin resistance induced by lipogenic, simple-carbohydrate diet (LD). Sixteen weeks of LD feeding induced obesity with liver steatosis and increased insulin levels but did not significantly affect circulating adiponectin or resistin. Treatment with PPAR-{alpha} agonist fenofibrate decreased body weight and fat pad weight and ameliorated liver steatosis in LD-fed mice with concomitant reduction in blood glucose, free fatty acid, triglyceride, serum insulin levels, and homeostasis model assessment index values. Euglycemic-hyperinsulinemic clamp demonstrated the development of whole-body and liver insulin resistance in LD-fed mice, which were both normalized by fenofibrate. Fenofibrate treatment markedly increased circulating resistin levels on both diets and adiponectin levels in chow-fed mice only. Fat adiponectin mRNA expression was not affected by fenofibrate treatment. Resistin mRNA expression increased in subcutaneous but not gonadal fat after fenofibrate treatment. In addition to fat, a significant amount of adiponectin mRNA was also expressed in the muscle. This expression markedly increased after fenofibrate treatment in chow- but not in LD-fed mice. Adipose tissue expression of AdipoR1 mRNA was significantly reduced in LD-fed mice and increased after fenofibrate treatment. In conclusion, PPAR-{alpha} activation ameliorated the development of insulin resistance in LD-fed mice despite a major increase in serum resistin levels. This effect could be partially explained by increased AdipoR1 expression in adipose tissue after fenofibrate treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PREVALENCE OF obesity is increasing worldwide and is currently reaching epidemic proportions in most of the developed countries (1). Obesity is accompanied by numerous complications and metabolic abnormalities including heightened risk of insulin resistance, diabetes, and cardiovascular disorders (2, 3). The direct link between obesity and insulin resistance is still only partially understood. Two mechanisms that have been generally accepted to be involved include an excessive ectopic lipid accumulation in nonadipose tissues with subsequent impairment of insulin sensitivity (4) and disturbed endocrine function of adipose tissue (5).

Obesity-induced changes in the endocrine function of adipose tissue have been described in many studies in both experimental models of obesity and in humans. These changes very likely contribute to the development of metabolic complications. Numerous hormones such as TNF-{alpha}, IL-6, plasminogen activator inhibitor-1, and others have been implicated in the development of insulin resistance, diabetes, and/or atherosclerosis in patients with obesity (5, 6). More recently, two more players in the field have been discovered and have drawn major attention as potential links between obesity and insulin resistance: resistin and adiponectin (7, 8, 9, 10, 11). Resistin is a protein hormone produced by both adipocytes and immunocompetent cells. Its circulating levels are increased in most but not all murine models of obesity and insulin resistance (7). Interestingly, its adipose tissue mRNA expression is decreased rather than increased by obesity (12, 13). Resistin administration in mice leads to insulin resistance predominantly in the liver (14). Knockout of the resistin gene impairs liver glucose production (15). Although obesity in mice is relatively consistently accompanied by increased resistin levels, the data in humans are more contradictory, ranging from increased to unchanged resistin levels in subjects with obesity and insulin resistance (16, 17, 18).

In contrast to resistin and most of other adipose tissue-derived hormones, adiponectin levels are decreased in both rodent models and patients with obesity, insulin resistance, and/or atherosclerosis (19, 20, 21, 22, 23). Numerous studies suggested that its deficiency may play a causal role in the development of these diseases. In support of this hypothesis, adiponectin-knockout mice display modest to moderate insulin resistance and accelerated atherosclerosis, and adiponectin administration normalizes this phenotype (24, 25).

The changes of endocrine function of adipose tissue thus may be involved in both the etiopathogenesis of insulin resistance and in the mechanism of action of different insulin-sensitizing drugs. We and others have demonstrated that peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) activation increased insulin sensitivity in both the obese and lipoatrophic mice with severe insulin resistance and diabetes (26, 27, 28). The mechanism of its action is only partially understood. Here we focused on the possible involvement of the two adipose-tissue-derived hormones in the mechanism of insulin-sensitizing action of PPAR-{alpha} agonists in mice with diet-induced insulin resistance. To this end, we studied their changes on both mRNA and circulating levels and assessed insulin sensitivity by means of euglycemic-hyperinsulinemic clamp.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male C57BL/6J mice were purchased from Velaz (Czech Republic). Animals were maintained on a 12-h light (0600–1800 h), 12-h dark cycle and fed either standard chow diet or lipogenic, simple carbohydrate diet (AIN-93G; Dyets, Bethlehem, PA) for 16 wk followed by 14 d administration of fenofibrate (100 mg/kg) in the food. The fenofibrate dose was chosen based on its previously reported effects on metabolic parameters in mice (29, 30). Experiments were performed using male mice at 22 wk of age. The blood glucose, serum insulin, adiponectin, and resistin concentrations measurements were repeated in two independent sets of animals, with results similar to those described below.

Livers were fixed in 4% paraformaldehyde and processed for histological study. Liver sections were stained by Oil-red O staining. All experiments were performed in accordance with the 1 Faculty of Medicine institutional guidelines and with the approval of the Animal Care and Use Committee of the 1 Faculty of Medicine, Charles University in Prague.

Hyperinsulinemic-euglycemic clamp
The clamp procedure was described in detail previously (31, 32). In brief, catheters were implanted under ketamine and xylazine anesthesia. The SILASTIC brand catheter (inner diameter 0.30 mm, outer diameter 0.64 mm, no. 508-001; Dow Corning, Midland, MI), filled with heparin solution (100 USP U/ml in 0.9% NaCl), was inserted via a right lateral neck incision, advanced into the superior vena cava via the right internal jugular vein, and sutured in place, a procedure adapted from MacLeod and Shapiro (33). The distal end of the catheter was knotted, tunneled sc, exteriorized first at the dorsal cervical midline, and then further tunneled sc and exteriorized in the dorsal midline, 2 cm above the tail. A silk suture was fastened around the catheter at the neck site. The clamps were then performed 4–5 d later after complete recovery of the animals from the operation.

Clamps began at 1000 h and were performed in mice fasted for 4 h. Mice were placed into a restrainer (552-BSRR; Plas-Labs, Lansing, MI), and the catheter was externalized. The tip of the tail was cut before the start of the first infusion, and all subsequent blood drawings were carried out using this site. Blood was collected into heparinized micro-hematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 sec to obtain plasma. Basal endogenous glucose production was estimated by continuously infusing [3-3H]glucose (3 µCi bolus, then 0.02 µCi/min, 740 GBq/mmol; NET 331C; NEN Life Science Products, Boston, MA) for 2 h. Samples for determination of plasma [3-3H]glucose concentration were taken after 90 and 115 min of basal infusion. Basal glucose and insulin concentrations were measured in the sample taken 90 min after the start of basal infusion. After 120 min of basal [3-3H]glucose infusion, the hyperinsulinemic-euglycemic clamp was begun with a prime continuous infusion of human insulin (bolus 300 mU/kg over 3 min, then 2.5 mU/kg·min; Humulin R; Eli Lilly, Indianapolis, IN). Plasma glucose was measured at 15-min intervals, and 20% glucose was infused at a rate that was adjusted to keep the plasma glucose at approximately 6.0 mmol/liter. Insulin-stimulated whole-body glucose uptake was measured using a prime continuous infusion of [3-3H]glucose (0.1 µCi/min) throughout the clamps.

Blood samples (20 µl) were withdrawn at 80, 85, 90, 100, 110, and 120 min after the start of the insulin infusion for the measurement of plasma 3H. Clamp insulin levels were measured in 5 µl of plasma from the 110-min point. All infusions were performed using a microdialysis pump (model CMA 102; CMA/Microdialysis, Acton, MA). Hamilton Gastight syringes (10 µl; Hamilton Co., Reno, NV) were used for the bolus injections. After 120 min of insulin infusion, animals were euthanized with a ketamine/xylazine solution. The determination of plasma [3-3H]glucose concentrations were performed as described previously (34).

Calculations
Basal endogenous glucose production was calculated as the ratio of the preclamp [3-3H]glucose infusion rate (dpm/min) to the specific activity of the plasma glucose (mean of the values in the 90 and 120 min of basal preclamp period in dpm/µmol). Clamp whole-body glucose uptake was calculated as the ratio of the [3-3H]glucose infusion rate (dpm/min) to the specific activity of plasma glucose (dpm/µmol) during the last 30 min of the clamp (mean of the 90- to 120-min samples). Clamp endogenous glucose production was determined by subtracting the average glucose infusion rate in the last 30 min of clamp from the whole-body glucose uptake.

Biochemical and hormonal assays
Glucose was measured using a Glucometer Elite (Bayer, Elkhart, IN). Insulin and adiponectin (SRI-13K and MADP-60HK, respectively; Linco Research, St. Charles, MO), resistin (RD293016100; BioVendor, Czech Republic), triglycerides (337-B; Sigma Chemical Co., St. Louis, MO), and nonesterified fatty acids (13831175; Roche Molecular Biochemicals, Indianapolis, IN) were quantitated with the indicated kits. Homeostasis model assessment (HOMA) index was calculated using the following formula: fasting serum insulin (U/ml) x fasting serum glucose (mmol/liter)/22.5.

RNA extraction and analysis
Tissues for RNA analysis were quickly dissected, immediately frozen in liquid nitrogen, and stored at –70 C until further processing. Total RNA was extracted from adipose, muscle, and liver tissues, respectively, after homogenization with an ULTRA-TURRAX T 18 basic (IKA Werke GmbH, Staufen, Germany) using RNeasy Lipid Tissue Mini Kit for adipose tissue or RNeasy Mini Kit for liver tissue (QIAGEN GmbH, Hilden, Germany).

All samples had a 260/280-nm absorbance ratio above 1.8. The integrity of the RNA was checked by visualization of 18S and 28S ribosomal bands on 1% agarose gel with ethidium bromide.

One microgram of total RNA was used for the RT reaction to synthesize the first-strand cDNA using the oligo(dT)18 primers following the instructions of the RevertAid First Strand cDNA synthesis kit (Fermentas, Lithuania).

Adiponectin, resistin, and adiponectin receptor (AdipoR) gene expression were quantified on an ABI PRISM 7000 real-time PCR cycler (Applied Biosystems, Foster City, CA), using commercially available TaqMan Universal PCR Master Mix, NO AmpErase UNG, and TaqMan Gene Expression Assays (Applied Biosystems) for adiponectin (Mm00456425 m1), resistin (Mm00445641 m1), AdipoR1 (Mm01291334 mH), and AdipoR2 (Mm008159 m1).

PCRs for each gene were performed in different wells with the same batch of TaqMan Universal PCR Master Mix to reduce variation in its efficiency, and all samples were run at least in duplicate. The increase in fluorescence was measured in real time, and data were obtained as threshold cycle (CT) values. To compensate for variations in input RNA amounts and efficiency of RT, ß2-microglobulin (TaqMan Gene Expression Assay Mm00437762 m1) was used as an endogenous reference, and results were normalized to these values. Relative expression of respective genes was calculated using the formula 2–{Delta}{Delta}(CT cytokine – CT B2M) (see user bulletin no. 2, ABI Prism 7700 Sequence Detection System, Applied Biosystems).

Western blot analysis
Adipose tissue adiponectin, AdipoR1 and -2, and resistin levels were determined by Western blot analysis. The removed deeply frozen adipose tissue was homogenized in 2x NOVEX Tris-glycine SDS sample buffer (Invitrogen, Inc., Carlsbad, CA) and boiled for 5 min, and than the homogenates were centrifuged at 10,000 x g for 3 min. The fat cake was removed, and adipose tissue extracts were used for Western blot analysis. The concentration of total protein amount was measured spectrophotometrically. Aliquots of the tissue extracts (~20 µg protein) prepared in 1x SDS sample buffer were incubated for 5 min at 100 C. Denatured proteins were separated by SDS-PAGE and then blotted to S&S NC nitrocellulose membrane (Schleicher & Schuell BioScience, Inc., Keene, NH) using Mini TransBlott*Cells (Bio-Rad Laboratories, Hercules, CA). After visualization by Ponceau S staining (Sigma-Aldrich, St. Louis, MO), membranes were blocked with 5% nonfat dried milk in PBS (wt/vol) with 0.1% Tween 20 (vol/vol) at room temperature for 1 h. The membranes were incubated with a 1:1000 dilution of antimouse adiponectin rabbit polyclonal antibody Acrp30 N-20 (sc-17044-R; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:200 dilution of antimouse AdipoR1 (41–65) serum (Phoenix Pharmaceuticals, Inc., Belmont, CA), 1:200 dilution of antimouse AdipoR2 (374–386) serum (Phoenix Pharmaceuticals), and 1:500 antimouse resistin polyclonal antibody (Alexis Corp., Lausen, Switzerland), respectively, at 4 C overnight in the wet chamber. All primary antibodies were diluted in 5% nonfat dried milk in PBS with 0.1% Tween 20. After washing, the filters were incubated with horseradish peroxidase-conjugated donkey antirabbit IgG antibody (Amersham Biosciences, Piscataway, NJ) 1:5000 diluted in PBS with 0.1% Tween 20. An ECL Plus Western blotting detection kit (Amersham) was used for detection following the manufacturer’s protocol. The relative changes in adiponectin, AdipoR1 and -2, and resistin were evaluated semiquantitatively.

Statistical analysis
Data are expressed means ± SE. Statistical significance between the groups was determined with SigmaStat, version 3.00 (SPSS Inc., Chicago, IL) using two-way ANOVA or t test as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biometric and biochemical parameters
Biometric and biochemical parameters are shown in Table 1Go. Lipogenic, simple-carbohydrate diet (LD) markedly increased body weight, gonadal and sc fat pad, and brown adipose tissue weight (Table 1Go). This increase was completely abolished by fenofibrate treatment. Liver weights were not significantly affected by the diet and increased markedly after fenofibrate treatment. Histological examination of the livers with Oil Red O staining revealed increased lipid content in LD-fed mice relative to the control group. Fenofibrate treatment significantly reduced liver lipid content in LD-fed mice (data not shown).


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  		TABLE 1. Metabolic and anatomical parameters of 22-wk-old male C57BL/6J mice fed chow or LD for 16 wk

 
Plasma glucose levels tended to be higher in mice fed the LD diet (P = 0.08). Fenofibrate treatment significantly reduced blood glucose levels in mice fed both normal chow and LD. Feeding with LD increased insulin levels by more than 2-fold, indicating the development of insulin resistance. This increase was significantly blunted by fenofibrate treatment. HOMA index values were significantly increased in LD-fed mice relative to all other groups. Fenofibrate treatment significantly decreased HOMA index in both chow-fed and LD-fed mice. Neither serum triglyceride nor free fatty acids were affected by the diet. Fenofibrate treatment significantly reduced both triglyceride and free fatty acid levels in mice on both diets.

The changes in insulin sensitivity as measured by euglycemic-hyperinsulinemic clamp
At the basal state (after 4 h fasting), plasma glucose was significantly decreased in the chow-fed fenofibrate-treated group relative to the chow-fed group, whereas it did not differ among the rest of the groups studied (Table 2Go). Plasma insulin levels were significantly higher in LD-fed mice relative to all other groups and did not differ among other groups studied (Table 2Go). Basal endogenous glucose production did not significantly differ among the groups studied (data not shown).


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  		TABLE 2. Metabolic parameters during basal (4-h fasted) period and hyperinsulinemic-euglycemic clamp of 22-wk-old male C57BL/6J mice fed chow or LD for 16 wk

 
Figure 1Go shows the influence of LD feeding and fenofibrate treatment on the whole-body glucose uptake and clamp endogenous glucose production. Whole-body glucose uptake under euglycemic-hyperinsulinemic clamp conditions is mostly a measure of muscle insulin sensitivity. This variable was markedly decreased in LD-fed mice, indicating the development of muscle insulin resistance. Fenofibrate treatment of LD-fed mice restored normal whole-body glucose uptake.


Figure 1
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  		FIG. 1. Whole-body glucose uptake and clamp endogenous glucose production in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Values are mean ± SE with n = 6 per group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice; {wedge}, P < 0.05 for fenofibrate-treated vs. untreated mice fed LD.

 
Endogenous glucose production during the clamp reflects liver insulin sensitivity. LD feeding induced liver insulin resistance as evidenced by significantly higher endogenous glucose production in LD-fed mice relative to the control group. Fenofibrate treatment significantly decreased endogenous glucose production in the mice fed both normal chow and LD relative to untreated groups fed the respective diets.

Circulating adiponectin and resistin levels
Figure 2Go shows the changes in serum adiponectin and resistin levels. Serum adiponectin levels were not significantly affected by the diet. Fenofibrate treatment increased serum adiponectin levels in chow-fed group but not in the LD-fed group.


Figure 2
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  		FIG. 2. Circulating adiponectin and resistin levels in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Samples are from nonfasting mice anesthetized with ketamine/xylazine and bled between 0900 and 1200 h. Values are mean ± SE with n = 6 per group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice; {wedge}, P < 0.05 for fenofibrate-treated vs. untreated mice fed LD.

 
Serum resistin levels tended to be lower in mice fed LD (P = 0.07). Fenofibrate treatment increased serum resistin levels almost 2-fold in both the chow-fed and LD-fed mice.

No statistically significant relationship was found between serum resistin or adiponectin levels and insulin levels or HOMA index, respectively (data not shown).

Adiponectin, resistin, and AdipoR tissue mRNA and protein expression
In gonadal adipose tissue, adiponectin expression was reduced by LD feeding and tended to be increased by fenofibrate treatment (Fig. 3Go). The same was true for adiponectin mRNA expression in sc adipose tissue (data not shown).


Figure 3
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  		FIG. 3. Gonadal adipose tissue and muscle adiponectin mRNA expression in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Samples are from nonfasting mice anesthetized with ketamine/xylazine and bled between 0900 and 1200 h. Values are mean ± SE with n = 6 per group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice.

 
A significant amount of adiponectin mRNA expression was detectable not only in adipose but also in muscle tissue. Muscle adiponectin expression was markedly up-regulated by fenofibrate in chow-fed mice, whereas this effect was completely abolished in LD-fed mice (Fig. 3Go). The presence of adiponectin protein in muscle fibers from gastrocnemius muscle was independently confirmed by immunohistochemistry and indirect immunofluorescence with antibody Acrp30 N-20 (Santa Cruz Biotechnology) (data not shown).

Resistin mRNA expression in gonadal adipose tissue was reduced by LD feeding and was not significantly affected by fenofibrate treatment (Fig. 4Go). On the contrary, resistin mRNA expression in sc adipose tissue was significantly increased by fenofibrate treatment on both diets.


Figure 4
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  		FIG. 4. Gonadal and sc adipose tissue and liver resistin mRNA expression in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Samples are from nonfasting mice anesthetized with ketamine/xylazine and bled between 0900 and 1200 h. Values are mean ± SE with n = 6 per group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice; {wedge}, P < 0.05 for fenofibrate-treated vs. untreated mice fed LD.

 
Resistin mRNA expression was also detectable in the liver (albeit in much lower quantities than in adipose tissue). Its mRNA levels were significantly reduced by LD feeding and up-regulated by fenofibrate treatment on both diets (Fig. 4Go).

Gonadal fat AdipoR1 expression was reduced by LD feeding, and fenofibrate normalized its mRNA levels (Fig. 5Go). The same was true for AdipoR1 mRNA expression in sc adipose tissue (data not shown).


Figure 5
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  		FIG. 5. Gonadal adipose tissue, muscle, and liver AdipoR1 expression in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Samples are from nonfasting mice anesthetized with ketamine/xylazine and bled between 0900 and 1200 h. Values are mean ± SE with n = 6 per group for chow and chow + fenofibrate group and n = 12 per group for lipogenic and lipogenic + fenofibrate group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice; {wedge}, P < 0.05 for fenofibrate-treated vs. untreated mice fed LD.

 
In contrast, muscle and liver AdipoR1 mRNA expression in LD-fed mice did not differ from the chow-fed group. In muscle, AdipoR1 mRNA expression was decreased after fenofibrate treatment in chow-fed but not LD-fed animals.

No significant changes in fat AdipoR2 mRNA expression were found in the groups studied, although it tended to follow the same pattern as AdipoR1 (Fig. 6Go). In contrast, both muscle and liver AdipoR2 expression was increased by fenofibrate treatment in chow-fed but not LD-fed mice (Fig. 6Go).


Figure 6
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  		FIG. 6. Gonadal adipose tissue, muscle, and liver AdipoR2 expression in 22-wk-old male C57BL/6J mice fed chow or LD with or without fenofibrate. Samples are from nonfasting mice anesthetized with ketamine/xylazine and bled between 0900 and 1200 h. Values are mean ± SE with n = 6 per group. Statistical significance is from two-way ANOVA: *, P < 0.05 vs. untreated chow group; +, P < 0.05 vs. fenofibrate-treated chow-fed mice.

 
The protein expression of adiponectin, resistin, AdipoR1, and AdipoR2 measured by Western blot analysis followed the same pattern as mRNA expression described above (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most important finding of this study is that PPAR-{alpha} activation by fenofibrate markedly increased serum resistin levels with simultaneous improvement of insulin sensitivity. This finding is in disagreement with previously published papers linking increased resistin levels to liver insulin resistance. In the original paper, resistin administration was found to worsen insulin sensitivity in vivo, whereas treatment with antiresistin antibody had the opposite effect (7). In another study, acute resistin infusion induced liver insulin insensitivity as measured by euglycemic-hyperinsulinemic clamp (14). Furthermore, chronic adenovirus-mediated resistin overexpression worsened insulin sensitivity in both rats and mice (35, 36). Finally, the most compelling evidence of resistin’s role in the regulation of glucose homeostasis comes from the phenotype of resistin-knockout mice. These animals display decreased fasting glucose levels as a result of impaired liver gluconeogenesis as evidenced by decreased expression of gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6 phosphatase (15).

Here we found that improvement of insulin sensitivity as documented by a decrease of HOMA index was accompanied by increased circulating resistin levels. It has to be noted that resistin concentrations were not measured in the same set of animals that underwent euglycemic-hyperinsulinemic clamp. Therefore, we were not able to assess directly the relationship between insulin sensitivity in respective tissues and circulating resistin levels. To our best knowledge, there are no published studies focusing directly on the effect of PPAR-{alpha} on resistin concentrations. However, two studies reported the relationship between resistin mRNA expression in adipose tissue and PPAR-{alpha} activation. Fukui and Motojima (37) found decreased resistin mRNA expression in white adipose tissue of PPAR-{alpha} null mice, indicating some role of this receptor in the regulation of resistin expression. Jove et al. (38) reported that 8 wk of fenofibrate treatment in patients with dyslipidemia increased resistin mRNA expression in omental white adipose tissue. Thus, PPAR-{alpha} appears to have a regulatory role in the resistin mRNA expression, albeit the mechanism of its action remains unclear.

Another important question raised by our results is the source of increased circulating resistin levels after fenofibrate treatment. In our study, we found a significant increase of resistin mRNA expression only in sc but not in gonadal adipose tissue. Interestingly, we also found a 2-fold increase of resistin expression in the liver after fenofibrate treatment. It has to be noted that the overall liver resistin mRNA expression was markedly lower than that in white adipose tissue. Finally, circulating immunocompetent cells such as monocytes or macrophages were found to produce resistin (39, 40), and the possibility of its involvement in fenofibrate-induced resistin production has to be taken into account.

As described in detail above, increased resistin levels are normally linked to insulin resistance, whereas in our study they were paralleled by improved insulin sensitivity. It thus appears that the insulin-resistance-inducing effect of resistin was overcome by other mechanisms in our study. The first possible mechanism lies in the reduction of ectopic lipid content in nonadipose tissues caused by increased fatty acid oxidation induced by PPAR-{alpha} agonists. Histological examination of the livers revealed that this may have been the case. Another, although rather speculative, possibility is that increased expression of AdipoR1 in white adipose tissue could have led to enhanced adiponectin action in adipocytes and by an indirect mechanism affect liver insulin sensitivity. Our finding of increased fat AdipoR1 mRNA expression is in agreement with recently published work of Tsuchida et al. (41), who found that PPAR-{alpha} activation markedly increased both AdipoR1 and AdipoR2 in the adipose tissue. Finally, it has been demonstrated that resistin, similarly to adiponectin, circulates in several isoforms of different molecular weight with potentially variable effects on insulin sensitivity (42). Thus, the increase of total resistin levels may not necessarily reflect its ultimate effect on liver insulin sensitivity.

In contrast to resistin, circulating adiponectin levels were increased by fenofibrate treatment in chow-fed mice but not in LD-fed animals in our study. Although no major increase in adipose tissue adiponectin mRNA expression was detected in this group, we found a significant amount of adiponectin mRNA to be expressed in muscle tissue of fenofibrate-treated chow-fed mice. The possibility that muscle can under certain conditions express adiponectin has been demonstrated previously (43). It remains to be determined how important this production is with respect to overall circulating adiponectin levels. Heightened muscle adiponectin expression was accompanied by decreased AdipoR1 and increased AdipoR2 mRNA expression. Thus, adiponectin receptors in the muscle appear to be affected by circulating adiponectin levels differently from those in the adipose tissue and liver, respectively.

In summary, we demonstrated that fenofibrate treatment ameliorated the development of diet-induced insulin resistance despite an increase in serum resistin levels. The exact mechanism of a PPAR-{alpha}-induced increase of resistin mRNA expression/serum concentration was not addressed in this study and remains to be elucidated. The overall positive effect of PPAR-{alpha} activation on insulin sensitivity may be in addition to reduced ectopic lipid content in nonadipose tissue mediated by heightened adiponectin signaling caused by increased AdipoR1 expression in the adipose tissue and possibly by other yet unexplained mechanisms.


   Footnotes
 
This work was supported by Internal Grant Agency of Ministry of Health of Czech Republic Grant No. 7429-3 and Research Project MSM 0021620814 (to M.H.).

Disclosure of potential conflicts of interest: M.M.H., Z.L., M.D., D.Ho., D.Ha., A.H., Z.V., T.K., and M.H. have nothing to declare.

First Published Online June 1, 2006

Abbreviations: AdipoR, Adiponectin receptor; HOMA, homeostasis model assessment; LD, lipogenic, simple-carbohydrate diet; PPAR-{alpha}, peroxisome proliferator-activated receptor-{alpha}.

Received December 20, 2005.

Accepted for publication May 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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