Fatty acid transport protein-1 mRNA expression in skeletal muscle and in adipose tissue in humans

Christophe Binnert1,*, Heikki A. Koistinen2,*, Geneviève Martin3, Fabrizio Andreelli1, Pertti Ebeling2, Veikko A. Koivisto2, Martine Laville1, Johan Auwerx3, and Hubert Vidal1

1 Institut National de la Santé et de la Recherche Médicale Unité-449 and Centre de Recherche en Nutrition Humaine de Lyon, Faculté de Médecine R.T.H. Laënnec, F-69372 Lyon; 3 Institut de Génétique et Biologie Moléculaire et Cellulaire, 67404 Illkirch, France; and 2 Department of Medicine, Division of Geriatrics, Helsinki University Central Hospital, 00290 Helsinki, Finland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fatty acid transporter protein (FATP)-1 mRNA expression was investigated in skeletal muscle and in subcutaneous abdominal adipose tissue of 17 healthy lean, 13 nondiabetic obese, and 16 obese type 2 diabetic subjects. In muscle, FATP-1 mRNA levels were higher in lean women than in lean men (2.2 ± 0.1 vs. 0.6 ± 0.2 amol/µg total RNA, P < 0.01). FATP-1 mRNA expression was decreased in skeletal muscle in obese women both in nondiabetic and in type 2 diabetic patients (P < 0.02 vs. lean women in both groups), and in all women there was a negative correlation with basal FATP-1 mRNA level and body mass index (r = -0.74, P < 0.02). In men, FATP-1 mRNA was expressed at similar levels in the three groups both in skeletal muscle (0.6 ± 0.2, 0.6 ± 0.2, and 0.8 ± 0.2 amol/µg total RNA in lean, obese, and type 2 diabetic male subjects) and in adipose tissue (0.9 ± 0.2 amol/µg total RNA in the 3 groups). Insulin infusion (3 h) reduced FATP-1 mRNA levels in muscle in lean women but not in lean men. Insulin did not affect FATP-1 mRNA expression in skeletal muscle in obese nondiabetic or in type 2 diabetic subjects nor in subcutaneous adipose tissue in any of the three groups. These data show a gender-related difference in the expression of the fatty acid transporter FATP-1 in skeletal muscle of lean individuals and suggest that changes in FATP-1 expression may not contribute to a large extent to the alterations in fatty acid uptake in obesity and/or type 2 diabetes.

fatty acids; insulin; insulin resistance; obesity; type 2 diabetes mellitus; hyperinsulinemic clamp; reverse transcription-polymerase chain reaction; messenger ribonucleic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SEVERAL STUDIES HAVE DEMONSTRATED that the transport of long-chain fatty acids across the plasma membrane of the cells is, at least in part, a saturable process mediated by specific translocators (3, 32, 33). However, this view of fatty acid uptake being fundamentally a protein facilitated process has also been challenged by arguing that fatty acid uptake occurs principally by diffusion (16). Nevertheless, three groups of putative fatty acid transport proteins have been identified so far and characterized: 1) the fatty acid translocase (FAT), which is an 88-kDa plasma membrane protein with sequence similarity to the human CD36 cell surface antigen (1), 2) the plasma membrane fatty acid binding protein (FABP-pm), which is biochemically closely related to the mitochondrial glutamic-oxaloacetic aminotransferase (6), and 3) the family of fatty acid transport proteins (FATP) recently characterized in mice and humans (17, 31). At least five different FATP cDNAs have been identified (17). Mouse FATP-1 was first cloned by Schaffer and Lodish (31) and recently also in humans (Martin and Auwerx, unpublished observation). Mouse and human FATP-1 cDNAs share 80% of identity, and sequence analysis predicts a 63-kDa membrane protein with six transmembrane domains. FATP-1 is the only member of the FATP family that is significantly expressed in muscle (17). Implication of FATP-1 in fatty acid transport is supported by the overexpression of the mouse protein in cultured fibroblasts, which results in a marked increase in long-chain fatty acid uptake (31). In addition, FATP-1 mRNA was found to be expressed at noticeable levels in mouse tissues known to use fatty acids as energy fuel or to actively metabolize them, such as adipose tissue, heart, and skeletal muscle (31). Moreover, the expression of FATP-1 is increased during fasting (24), a situation associated with enhanced fatty acid mobilization and utilization.

Enhanced delivery of fatty acids into the cells can induce insulin resistance of glucose metabolism (28). Insulin resistance is a common feature of obesity and type 2 diabetes mellitus, two complex metabolic diseases, the causes of which are not yet clearly understood. Obesity and type 2 diabetes are characterized by altered lipid metabolism associated with increased levels of circulating triglycerides and nonesterified fatty acids (NEFA; see Refs. 13 and 29). The basal rate of total body lipid oxidation is generally increased in these pathologies (13). However, the postabsorptive NEFA uptake across the leg is reduced, and there is also a tendency for a decreased postabsorptive lipid oxidation in the leg with increasing visceral obesity in healthy women (12). In type 2 diabetes, the uptake of long-chain fatty acids and lipid oxidation in skeletal muscle are reduced in fasting conditions (19). It is not known whether a defective regulation of the expression or activity of the fatty acid transporters contributes to these alterations. A possible role of FATP-1 in fatty acid uptake is suggested by the recent observation that the thiazolidinedione BRL-49653 modifies the expression levels of FATP-1 in rodents (25). Thiazolidinediones are a new class of insulin-sensitizing agents that improve the circulating lipid profile and reduce insulin resistance in experimental animals and in type 2 diabetic patients (30). BRL-49653 promotes a robust induction of FATP-1 gene expression in rat adipose tissue and, to a lesser extent, in muscle in vivo (25). Furthermore, this effect is associated with an increase in fatty acid uptake in 3T3-L1 preadipocytes in vitro (25).

The FATP gene has recently been demonstrated to be expressed in human skeletal muscle (10). At present, however, few data are available regarding the expression of FATP-1 in human tissues in normal and pathophysiological conditions such as obesity and type 2 diabetes. Fasting increases FATP-1 gene expression in mouse adipose tissue, whereas refeeding restores the basal levels (24), indicating that the nutritional status modulates FATP-1 expression in vivo. Because insulin is a negative regulator of FATP-1 gene expression in differentiated 3T3-L1 adipocytes (24), the regulation of FATP-1 expression by fasting and refeeding may be a consequence of changes in ambient insulin levels. However, the direct effect of insulin on FATP-1 expression has never been investigated in vivo.

The present study was undertaken to examine whether obesity and/or type 2 diabetes mellitus are associated with altered gene expression of FATP-1 in skeletal muscle and in subcutaneous adipose tissue and to investigate the acute effect of insulin on FATP-1 mRNA expression in healthy lean, nondiabetic obese, and type 2 diabetic subjects.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. We studied 25 subjects (10 lean, 6 obese, and 9 type 2 diabetic subjects) recruited in Lyon, France, and 21 male subjects (7 lean, 7 obese, and 7 type 2 diabetic subjects) recruited in Helsinki, Finland. Skeletal muscle FATP-1 expression was investigated in the French subjects, whereas the expression in subcutaneous adipose tissue was studied in the Finnish subjects. The characteristics of the subjects are presented in Table 1. None of the healthy lean subjects had a familial or personal history of diabetes. None of the obese subjects had impaired glucose tolerance, as assessed by an oral glucose tolerance test. All participants gave their written consent after being informed of the nature, purpose, and possible risks of the study. The experimental protocols were approved by the Ethical Committees of the Hospices Civils de Lyon (Lyon, France) and the Helsinki University Central Hospital (Helsinki, Finland).

                              
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Table 1.   Subject characteristics

Euglycemic-hyperinsulinemic clamp. To investigate insulin action on FATP-1 mRNA expression, all subjects were studied using the euglycemic-hyperinsulinemic clamp technique after an overnight fast. The clamp lasted for 3 h in Lyon and for 4 h in Helsinki. The clamps were performed as previously described, either in Lyon with the 25 French subjects (23) or in Helsinki with the 21 Finnish subjects (14). Similar rates of insulin infusion (450 and 432 pmol · m body surface area-2 · min-1, in Lyon and Helsinki, respectively) were used in both Hospital Centers. Serum insulin concentration was determined by commercial RIAs (14, 23). Plasma glucose levels were measured by hexokinase assay in Lyon (23) and by the glucose oxidase method in Helsinki (14). To estimate glucose and lipid oxidation rates, respiratory exchange measurements were performed in the basal state and during the final 30 min of the clamp period using a flow-through canopy gas analyzer system (Deltatrac Metabolic Monitor; Datex, Helsinki, Finland; see Refs. 14 and 23).

Muscle and adipose tissue biopsies. Skeletal muscle biopsies were taken under local anesthesia (2% lidocaine without epinephrine) before and at the end of the hyperinsulinemic clamp period in the 25 French subjects by percutaneous biopsies of the vastus lateralis muscle using a Weil Blakesley plier (22). The average size of the muscle biopsies was 51 ± 4 mg wet wt (n = 50) with no difference between samples from lean, obese, and type 2 diabetic subjects or before and after clamp. A contamination of the muscle biopsies by fat cells is unlikely, since we determined the expression of leptin mRNA in these muscle samples by RT-competitive PCR (5), and it was found to be below the detection limit of this assay (data not shown).

Abdominal subcutaneous adipose tissue was aspirated from the periumbilical area through a 2.3-mm (14 gauge) needle under local anesthesia (1% lidocaine without epinephrine), before and at the end of the hyperinsulinemic clamp period in the 21 Finnish subjects. The mean size of tissue that was used for total RNA preparation was 144 ± 5 mg wet wt (n = 42), without significant difference between groups or before and after clamp. Tissue samples were frozen in liquid nitrogen and were stored at -80°C until analysis.

Total RNA preparation. Tissue samples were pulverized in liquid nitrogen, and total RNA was prepared according to a modified procedure of Chomczynski and Sacchi (11) for muscle samples and using the RNeasy total RNA kit from Qiagen (Courtaboeuf, France) for adipose tissue. Average yields of total RNA were 26 ± 3 µg/100 mg (wet wt) of muscle and 1.6 ± 0.1 µg/100 mg (wet wt) of adipose tissue and were not significantly different in tissues from lean, obese, and type 2 diabetic subjects, before or after the clamp. All RNA preparations were done in the Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 449 laboratory in Lyon, France. Total RNA solutions were stored at -80°C until quantification of FATP-1 mRNA.

Quantification of FATP-1 mRNA. Human FATP-1 mRNA was quantified by RT-competitive PCR, which consists of the coamplification of target cDNA with known amounts of a specific DNA competitor molecule added in the same PCR tube (5). A homologous FATP-1 competitor DNA molecule was constructed by deleting 26 bp from a human FATP-1 cDNA fragment (pBShFATP, from nucleotide +423 to +1961, which corresponds to the translation termination signal, and 150 bp of 3'-untranslated region; Martin and Auwerx, unpublished observations; Fig. 1) using a two-step PCR overlap extension method and high-fidelity pfu DNA polymerase (Stratagene, La Jolla, CA). The construction of the competitor is depicted in Fig. 1. The FATP-1 competitor fragment (360 bp) was subcloned (pGEM-T Vector System; Promega), and the plasmid (pCompFATP) was purified, carefully quantified, and stored at -20°C. Working solutions (20 amol/µl to 10-3 amol/µl) were prepared by serial dilutions in 10 mM Tris · HCl (pH 8.3) and 1 mM EDTA buffer.


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Fig. 1.   Construction of the fatty acid transport protein (FATP)-1 competitor. Representation of the portion of the FATP-1 cDNA and location of the primers used to construct the homologous competitor by two-step PCR overlap extension method. The deleted FATP-1 fragment was subcloned into the pGEM-T plasmid. h, Human; S, sense; AS, antisense; Del, deletion; UTR, untranslated region; TGA, stop codon.

For the assay of FATP-1 mRNA, the RT reaction was performed from 0.2 µg of tissue total RNA with 2.5 units of thermostable RT (Tth; Promega, Charbonières, France), in the presence of 15 pmol of FATP-1 specific antisense primer (5'-AGCCCATAGATGAGAC ACTG-3', nucleotides +908 to +927 of the human FATP-1 cDNA relative to the translation start site). The conditions of the reaction have been described in detail previously (5). For PCR amplification, the RT medium was added to a PCR master mix (10 mM Tris · HCl, pH 8.3, 100 mM KCl, 0.75 mM EGTA, and 5% glycerol) containing 0.2 mM deoxynucleoside triphosphates, 5 units of Taq polymerase (Life Technologies, Cergy Pontoise, France), 30 pmol of FATP-1 antisense primer, and 45 pmol of FATP-1 sense primer (5'-CTTTGGAGGAGAAATGGTGG-3', +541 to +560). FATP sense primer was 5'-labeled with the CY-5 fluorescent dye (Eurogentec, Seraing, Belgium). The final volume was 100 µl, and four aliquots of 20 µl were transferred to 0.5-ml microtubes containing 5 µl of a defined working solution of the FATP-1 DNA competitor. After 120 s at 94°C, the PCR mixtures were subjected to 40 cycles of PCR amplification with a cycle profile including denaturation for 60 s at 94°C, hybridization for 60 s at 58°C, and elongation for 60 s at 72°C. The PCR products were separated and analyzed by PAGE (6%) on a ALFexpress DNA sequencer (Pharmacia, Uppsala, Sweden) using the Fragment Manager software (Pharmacia; see Refs. 4 and 27). The length of the PCR products was 386 bp for the natural human FATP-1 and 360 bp for the competitor. The absence of contamination with genomic DNA was checked by omitting RT in the reaction.

To accurately determine the effect of insulin, total RNA of the two muscle (or adipose tissue) biopsies from the same individual (before and after clamp) were prepared simultaneously, and the assays of the target mRNAs were always made in the same run of PCR, with the same working solutions of competitor. All quantifications of FATP-1 mRNA were performed in the INSERM U449 laboratory in Lyon, France.

Preparation of FATP-1 RNA by in vitro transcription. A 594-nucleotides-long RNA fragment of human FATP-1 was synthetised by in vitro transcription (Riboprobe System; Promega) from the pBShFATP plasmid that was linearized by digestion using the restriction enzyme Bgl II (+1,017). After the elimination of the plasmid, FATP-1 RNA was purified with the RNeasy kit (Qiagen), quantified by absorbance measurement, and diluted in water.

Statistical analysis. All data are presented as means ± SE. Between-group comparisons were done using the Kruskal-Wallis one-way ANOVA followed by the Mann-Whitney U-test when the ANOVA indicated significant difference. Wilcoxon's test for paired values was used when comparing mRNA levels before and after clamp. Correlation coefficients were calculated using Spearman's test. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RT-competitive PCR assay of human FATP-1 mRNA. The quantification of FATP-1 mRNA relies on the addition of known amounts of a specific competitor DNA molecule in the PCR tubes to standardize the amplification process. Figure 2A shows a typical assay of FATP-1 mRNA in skeletal muscle RNA (0.2 µg) obtained from a lean subject. After a specific RT reaction, the single-strand cDNA was amplified with four different concentrations of FATP-1 competitor. The PCR products of the four reactions were separated in polyacrylamide gel (6%) and analyzed. The ratio of the pic area of the competitor on the target was then plotted against the initial amounts of FATP-1 competitor added to the PCR (data not shown). The amount of FATP-1 mRNA was 1.7 amol/µg total RNA in this example. To validate the RT-competitive PCR assay, different amounts (0.5-25 amol) of in vitro synthetized FATP-1 RNA were quantified. Figure 2B shows the obtained dose-response curve. The linearity (r = 1.00) and the slope (0.90) demonstrate that the RT-competitive PCR assay developed in this work is quantitative over the range of concentrations tested.


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Fig. 2.   Example of an analysis of the PCR products and validation of the FATP-1 mRNA assay. After the RT reaction from 0.2 µg of total RNA from skeletal muscle of a lean subject, the single-strand cDNA was amplified with 4 different concentrations of FATP-1 cDNA competitor. The PCR products were separated on 6% PAGE. For the validation of the RT-competitive PCR assay, known amounts of in vitro synthetized FATP-1 RNA (see MATERIALS AND METHODS for further details) were quantified by RT-competitive PCR. The linearity (r = 1.00) and the slope (0.9) of the obtained dose-response curve demonstrate that the assay is quantitative.

FATP-1 mRNA levels in muscle and in adipose tissue in humans. Figure 3 shows the abundances of FATP-1 mRNA in skeletal muscle (French subjects; A) and in abdominal subcutaneous adipose tissue (Finnish subjects; B) in lean, nondiabetic obese, or obese type 2 diabetic patients. An approximately twofold reduction in the mean values of FATP-1 mRNA levels was observed in muscle from both the obese nondiabetic and the diabetic subjects (1.4 ± 0.3 vs. 0.8 ± 0.1 and 0.7 ± 0.1 amol/µg total RNA in lean vs. obese and type 2 diabetic subjects, respectively), but the differences were not statistically significant. When the male and female subjects were analyzed separately (Fig. 3), a marked gender-related difference in the expression of FATP-1 mRNA was observed in the lean subjects. The mRNA levels of FATP-1 were significantly higher in the lean women than in the lean men (2.2 ± 0.1 vs. 0.6 ± 0.2 amol/µg total RNA in female vs. male subjects, P < 0.01). This difference was not observed in the groups of nondiabetic and diabetic obese subjects. The mRNA levels of FATP-1 were significantly higher in the lean women than in the obese nondiabetic (0.9 ± 0.2 amol/µg total RNA, P < 0.02) and type 2 diabetic women (0.6 ± 0.03 amol/µg total RNA, P < 0.02), whereas there was no difference in FATP-1 mRNA expression levels between the obese nondiabetic and type 2 diabetic women (Fig. 3). A significant negative correlation (r = -0.74, P < 0.02) was found between FATP-1 mRNA levels in skeletal muscle and the body mass index (BMI) of the female, but not of the male, subjects (Fig. 4). In men, FATP-1 mRNA was expressed at similar levels in the three groups both in skeletal muscle (0.6 ± 0.2, 0.6 ± 0.2, and 0.8 ± 0.2 amol/µg total RNA in lean, obese, and type 2 diabetic male subjects) and in subcutaneous adipose tissue (0.9 ± 0.2, 0.9 ± 0.2, and 0.9 ± 0.2 amol/µg total RNA in lean, obese, and type 2 diabetic subjects, respectively, not significant; Fig. 3).


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Fig. 3.   Basal levels of FATP-1 mRNA in skeletal muscle or in subcutaneous adipose tissue from lean, obese nondiabetic, and type 2 diabetic subjects. All skeletal muscle samples were from French subjects (A), and all adipose tissue samples were from Finnish subjects (B). open circle , Men; , women. The expression of FATP-1 mRNA in lean women is higher than in lean men (P < 0.01) or in obese nondiabetic or type 2 diabetic women (P < 0.02 vs. both groups).



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Fig. 4.   Correlation between muscle FATP-1 mRNA expression and body mass index (BMI). The basal expression of FATP-1 mRNA in skeletal muscle correlates negatively with BMI in the females ( and straight line, r = -0.74, P < 0.02) but not in the males (open circle  and broken line, P = 0.627).

When the whole population was considered (n = 25), no significant correlations were found between basal FATP-1 mRNA levels in skeletal muscle and any of the parameters shown in Table 1. When the female subjects were analyzed separately (n = 13), in addition to the correlation with the BMI (Fig. 4), muscle FATP mRNA levels correlated negatively with fasting NEFA concentrations (r = -0.73, P < 0.02) and positively with insulin-stimulated glucose disposal rates (r = 0.58, P < 0.05). In multiple linear regression analysis with glucose disposal rate, BMI, and plasma NEFA concentrations as independent determinants, BMI was independently associated with FATP-1 mRNA expression (partial correlation coefficient -0.71, P = 0.002), whereas the associations of NEFA and glucose disposal rate with FATP-1 mRNA were not significant after BMI was accounted for. Finally, no significant correlations were found between FATP-1 mRNA levels in adipose tissue and any of the parameters shown in Table 1 (data not shown).

Effect of insulin on FATP-1 mRNA expression levels. Figure 5A shows that 3 h of a hyperinsulinemic clamp resulted in a significant decrease in FATP-1 mRNA levels in skeletal muscle of the five lean women (-35 ± 10%, P < 0.05). In contrast, insulin infusion did not affect FATP-1 mRNA levels in muscle from the lean males. FATP-1 mRNA levels did not change during the hyperinsulinemic clamp in the muscle of obese nondiabetic or type 2 diabetic patients. In subcutaneous adipose tissue, FATP-1 mRNA levels were not modified by 4 h of hyperinsulinemia in any of the groups (Fig. 5B).


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Fig. 5.   Regulation of FATP-1 mRNA expression by insulin. A: acute 180 min, insulin infusion decreases the expression of FATP-1 mRNA in skeletal muscle in lean women (, P < 0.05) but not in lean men (open circle ) or in obese nondiabetic or type 2 diabetic subjects. B: insulin infusion for 240 min does not affect the expression of FATP-1 mRNA in subcutaneous adipose tissue in lean, obese nondiabetic, or type 2 diabetic male subjects. All skeletal muscle samples were from French subjects, and all adipose tissue samples were from Finnish subjects.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FATP-1 is a member of the family of putative transporters of fatty acids across the plasma membrane that also includes FAT and FABP-pm (3, 1, 6, 31-33). In addition, the cytosolic fatty acid-binding protein (FABPc) has been also proposed to participate in the uptake of fatty acids by facilitating their diffusion to the aqueous cytoplasm (15). Physiological situations characterized by increased fatty acid utilization have been associated with changes in the expression of some of these transporter systems. For example, FABPc protein content increased during caloric restriction (20) and FABP-pm protein content increased during endurance training in human skeletal muscle (21). In rodents, it was recently reported that the contractile activity increased fatty acid uptake and induced the expression of FAT both in red and in white muscles (8). Fasting increased FATP-1 mRNA expression in mouse adipose tissue, whereas refeeding restored the basal levels (24), indicating that the nutritional status can modulate FATP-1 expression. It was also shown that the expression of FATP-1, FAT, and FABP-pm was about fivefold greater in red than in white muscle (9). In adipose tissue, a recent report showed an ~40% reduction of the levels of FATP-1 mRNA in the ob/ob mice, associated with an increased expression of FAT and FABP-pm mRNAs (26). In contrast, Berk et al. (7) have observed an increased expression of the three transporter mRNAs in adipocytes of the obese Zucker fa/fa rats. Transcripts for these three putative long-chain fatty acid transporter genes have recently been demonstrated to be present in human skeletal muscle (10). However, in human tissues, a quantitative comparison of the expression of the different putative transporters has never been done, neither at the mRNA nor at the protein level. Therefore, the relative contribution of the different transporters to fatty acid uptake in vivo is presently unknown. At present, there is also controversy as to whether fatty acid uptake in cells occurs by a protein-mediated fashion or by free diffusion through the lipids of the cell membranes (2, 16). Moreover, the exact physiological function of the putative fatty acid transport proteins is not known (16). For example, FATP-1 may also function as a very long chain fatty acyl-CoA synthetase (2).

We report in this work the first quantitative determination of FATP-1 expression in human tissues. FATP-1 mRNA was expressed at relatively low levels in human skeletal muscle and abdominal subcutaneous adipose tissue (~1 amol that corresponds to ~6 × 105 molecules of FATP-1 mRNA/µg of total RNA, which represents 10- to 20-fold lower expression levels than for the glucose transporter GLUT-4 in skeletal muscle; see Ref. 22). Therefore, a highly sensitive method like the RT-competitive PCR assay (5) developed in this work is required for accurate and reliable measurements.

We found that muscle FATP-1 mRNA levels are higher in women than men in the group of lean subjects. There were no significant differences with respect to age, BMI, fasting plasma glucose, NEFA, or insulin concentrations or in basal glucose or lipid oxidation rates, or insulin sensitivity between women and men in these lean subjects (data not shown), suggesting that the observed gender difference in FATP-1 mRNA expression may be due to the effect of sex steroids. Increased expression levels of FATP-1 mRNA in skeletal muscle of female subjects may suggest that lean women could utilize lipids to a greater extent than lean men. A higher utilization of fatty acids by women has been observed indeed, but only during exercise (18, 34) and not in the resting state or in the postexercise period (18). Thus the apparent gender difference in FATP-1 expression observed in the basal state in lean subjects may not be of functional consequence. In women, skeletal muscle FATP-1 mRNA levels are reduced in proportion to the degree of obesity in nondiabetic obese and in type 2 diabetic subjects. It is thus interesting to underscore the possible relationship between this obesity-associated reduction in the expression of FATP-1 and the decreased postabsorptive utilization of NEFA in the skeletal muscle of obese women reported by Colberg et al. (12). However, the correlation between obesity and FATP-1 mRNA expression was not observed in male subjects, neither in skeletal muscle nor in subcutaneous adipose tissue. FATP-1 mRNA levels were similar in lean, nondiabetic obese, and type 2 diabetic male subjects, and in skeletal muscle they corresponded to the levels observed in the groups of obese nondiabetic and diabetic women. Therefore, changes in FATP-1 expression may probably not explain or participate to a large extent in the alterations of fatty acid metabolism in type 2 diabetes mellitus and/or in obesity in humans.

Insulin has been shown to negatively regulate FATP-1 gene expression in a murine adipocyte cell line (24). In our study, acute hyperinsulinemia reduced FATP-1 mRNA levels in skeletal muscle in lean women but not in lean men. In addition, there was no effect of insulin on FATP-1 mRNA in adipose tissue or in skeletal muscle of the obese nondiabetic and diabetic subjects. Thus insulin is not a major regulator of FATP-1 gene expression in humans. However, we cannot exclude the possibility that a longer exposure to insulin than 4 h might be required to observe an effect of insulin on FATP-1 mRNA.

It is important to note that, in our study, FATP-1 was investigated at the mRNA level only, and extrapolation of these data to the physiological role of this transporter should be done with caution. Unfortunately the limited amount of tissue available prevented the analysis of FATP-1 protein. In addition to FATP-1, possible modifications in the expression of the other transporter proteins cannot be excluded in obesity and/or type 2 diabetes. Furthermore, because the thiazolidinedione antidiabetic agents increase FATP-1 expression in rodent tissues (25), FATP-1 may be an interesting target for the pharmacological therapy of pathologies with altered fatty acid utilization. Further studies are thus warranted to understand the physiological role of FATP-1 in vivo and to test the possibility of modulating its expression to control fatty acid uptake and utilization in human tissues.

In conclusion, FATP-1 mRNA expression is decreased in skeletal muscle in obese women. This reduction is observed in female obese nondiabetic and in type 2 diabetic subjects. In addition, acute insulin infusion decreases FATP-1 mRNA levels in muscle in lean but not in obese women. In male subjects, in contrast, FATP-1 mRNA is expressed at similar levels both in skeletal muscle and in subcutaneous adipose tissue in lean, nondiabetic obese, and type 2 diabetic patients. Insulin does not affect FATP-1 mRNA levels in the tissues of male subjects. These results, together with the fact that FATP-1 mRNA expression is relatively low in human tissues, suggest that changes in FATP-1 expression do not play a crucial role in the altered metabolism of fatty acids in type 2 diabetes or in obesity.


    ACKNOWLEDGEMENTS

The expert technical assistance of E. Koivisto, T. Kyöstiö-Renvall, C. Urbain-Pellissier, and N. Vega is deeply appreciated.


    FOOTNOTES

* C. Binnert and H. A. Koistinen contributed equally to this work.

This study was financially supported by grants from the Finnish Academy of Science, Finnish Cultural Foundation, Emil Aaltonen Foundation, Jalmari and Rauha Ahokas Foundation, Helsingin Sanomat Centennial Foundation, Research Foundation of Orion Corporation, Finnish Medical Foundation, Maud Kuistila Foundation, Novo-Nordisk Foundation, the Centre International de la Charcuterie, and by an Institut National de la Santé et de la Recherche Médicale research grant (Progres no. 4P020D).

Current address for V. A. Koivisto: Lilly Research Laboratories, D-22419 Hamburg, Germany.

Address for reprint requests and other correspondence: H. Vidal, INSERM U-449, Faculté de Médecine R.T.H. Laënnec, F-69372 Lyon, France (E-mail: vidal{at}laennec.univ-lyon1.fr).

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.

Received 6 March 2000; accepted in final form 15 June 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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