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Am J Physiol Endocrinol Metab 290: E814-E823, 2006. First published November 22, 2005; doi:10.1152/ajpendo.00465.2005
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Lipid metabolism and adipokine levels in fatty acid-binding protein null and transgenic mice

Ann V. Hertzel,1 Lisa Ann Smith,1 Anders H. Berg,2 Gary W. Cline,3 Gerald I. Shulman,3 Philipp E. Scherer,2 and David A. Bernlohr1

1Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota; 2Cell Biology and Medicine, Albert Einstein College of Medicine, Bronx, New York; and 3Internal Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut

Submitted 23 September 2005 ; accepted in final form 17 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fatty acid-binding proteins (FABPs) facilitate the diffusion of fatty acids within cellular cytoplasm. Compared with C57Bl/6J mice maintained on a high-fat diet, adipose-FABP (A-FABP) null mice exhibit increased fat mass, decreased lipolysis, increased muscle glucose oxidation, and attenuated insulin resistance, whereas overexpression of epithelial-FABP (E-FABP) in adipose tissue results in decreased fat mass, increased lipolysis, and potentiated insulin resistance. To identify the mechanisms that underlie these processes, real-time PCR analyses indicate that the expression of hormone-sensitive lipase is reduced, while perilipin A is increased in A-FABP/aP2 null mice relative to E-FABP overexpressing mice. In contrast, de novo lipogenesis and expression of genes encoding lipoprotein lipase, CD36, long-chain acyl-CoA synthetase 5, and diacylglycerol acyltransferase are increased in A-FABP/aP2 null mice relative to E-FABP transgenic animals. Consistent with an increase in de novo lipogenesis, there was an increase in adipose C16:0 and C16:1 acyl-CoA pools. There were no changes in serum free fatty acids between genotypes. Serum levels of resistin were decreased in the E-FABP transgenic mice, whereas serum and tissue adiponectin were increased in A-FABP/aP2 null mice and decreased in E-FABP transgenic animals; leptin expression was unaffected. These results suggest that the balance between lipolysis and lipogenesis in adipocytes is remodeled in the FABP null and transgenic mice and is accompanied by the reprogramming of adipokine expression in fat cells and overall changes in plasma adipokines.

fatty acids; adipocytes; adipokines

OBESITY IS CONSIDERED AN EPIDEMIC affecting a large percentage of the US population (7, 21). Associated with obesity is an increased risk of a variety of pathologies, including Type 2 diabetes, hypertension, and cardiovascular disease, making the metabolic syndrome the most common disorder worldwide (7). Obesity results from the enlargement and expansion in the number of adipocytes that not only store and release excess energy, but also play an active role as an endocrine organ by secreting adipokines that function in regulating energy metabolism in peripheral tissues (4, 25).

Cytosolic fatty acid-binding proteins (FABPs) provide solubility and intracellular trafficking of long-chain fatty acids and other hydrophobic ligands (11, 59). The most abundant FABP in adipose tissue is called the adipocyte-FABP (A-FABP or aP2). Adipose tissue also expresses the epithelial-FABP (E-FABP), albeit to a much lesser extent (12, 23). Within adipose tissue, both FABPs are expressed in adipocytes as well as in macrophages that infiltrate the tissue beds. Within the cellular context, A-FABP/aP2 forms a 1:1 complex with the hormone-sensitive lipase (HSL) on a regulatory docking domain within the NH2-terminal region of the lipase. Such an interaction positions the FABP to bind a product fatty acid and facilitate lipolysis (54, 56).

As a consequence of diet-induced obesity, A-FABP/aP2 and E-FABP are each upregulated approximately fourfold at the protein level, suggesting that one or both of these proteins may play a role in affecting the development of the metabolic syndrome (23, 33). To evaluate the role of the FABPs in the metabolic syndrome, two mouse models have been generated. The first was a knockout of A-FABP/aP2, significantly reducing the amount of total A-FABP (24). Accompanying the loss of A-FABP/aP2 was decreased in vivo and in situ lipolysis rates, as shown using both pharmacological and stable isotope methodologies (3, 12, 51). Importantly, when placed on a high-fat diet, the A-FABP/aP2 null mice exhibit attenuated characteristics of insulin resistance compared with wild-type C57Bl/6J mice (24). When evaluated within a molecular genetic background of obesity (ob/ob mice), the A-FABP/aP2 disruption was protective for the development of insulin resistance (57). Extending the improvement in metabolic properties, when crossed into the apolipoprotein E null mouse and placed on an atherogenic diet, the A-FABP/aP2 null mice exhibited reduced atherogenesis (8, 9, 35). Recent studies using macrophage cell lines derived from wild-type and A-FABP/aP2 null mice reveal that the molecular mechanisms linked to reduced inflammatory responsiveness derive from diminished NF-{kappa}B signaling, leading to lower levels of TNF-{alpha} (24, 36).

The second molecular model evaluating FABP function was the E-FABP transgenic mouse that overexpresses E-FABP in an adipocyte-specific manner utilizing the aP2 promoter (23). In contrast to the A-FABP/aP2 null mouse, the in situ rates of lipolysis, both basal and isoproterenol stimulated, were increased in the E-FABP transgenic mice (12, 23). Importantly, the E-FABP transgenic mice displayed marked insulin resistance compared with wild-type mice, as shown by increased fasting glucose, as well as impaired glucose and insulin tolerance tests (33).

Using stable isotope infusion, the improved insulin sensitivity in A-FABP/aP2 null mice has been mechanistically linked to increased glucose oxidation in muscle and increased fatty acid uptake in the liver (3). These results suggest that fat-derived endocrine factors and/or fatty acid metabolites involved in systemic energy metabolism may also participate in the metabolic properties of the FABP animal models beyond that of altered TNF-{alpha} expression. To that end, we evaluated adipose gene expression using real-time PCR profiling to identify changes in fat cell genes linked to intracellular lipid metabolism and paralleled those changes with alterations in adipokine levels in A-FABP/aP2 null and E-FABP transgenic mice. Herein we report that adipose triglyceride metabolism is altered through changes in the expression of key regulatory genes affecting the balance between lipolysis and lipogenesis and that such alterations are accompanied by changes in serum adipokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Mice used in this study include male C57Bl/6J wild-type, FABP4 null, and FABP5 transgenic mice. A-FABP/aP2 is the product of the FABP4 gene, and E-FABP (also known as KLBP or mal-1) is the product of the FABP5 gene. For consistency throughout this paper, a nomenclature using A-FABP/aP2 null and E-FABP transgenic will be utilized. Mice were weaned at 3 wk of age onto a high-fat diet (Bioserve Industries no. F3282, Frenchtown, NJ; 20% protein, 35.5% fat, 36.3% carbohydrate) and maintained at 70°F on a 12:12-h light-dark cycle and fed ad libitum. At 12 wk of age, animals were euthanized by cervical dislocation, and serum was immediately removed via cardiac puncture. EDTA (5%) was added to the serum in a 1:50 ratio, and the sample was spun at 15,000 rpm for 10 min. The resulting plasma was stored at –80°C before analysis. All procedures were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee.

Isolation and size determination of primary adipocytes. Murine epididymal fat pads were dissected, and primary adipocytes were isolated based on the protocol established for Sprague-Dawley rat adipocytes with minor modifications (1, 23, 49). The fat pads were treated with 1 mg/ml collagenase (Sigma) and 20 mg/ml BSA in Kreb's Ringer-HEPES (KRH) buffer with vigorous shaking for 1 h at 37°C. The floating primary fat cells were collected following a 10-min 2,000 rpm centrifugation at room temperature. The adipocytes were washed three times in KRH, centrifuged as above, diluted, and placed on an improved hemacytometer (Neubauer, American Scientific Products). Digital pictures were taken on an Olympus CK40 camera attached to an inverted microscope, and the cell diameters were measured using hemacytometer lines for calibration.

Lipogenesis. To measure de novo lipogenesis in primary adipocytes, the methods described by Mackall et al. (32) were employed. Briefly, primary adipocytes in KRH, isolated as described, were incubated in triplicate with 5 mM [1-14C]acetate (3 x 105 dpm/µmol) at 37°C for 10, 30, and 60 min. At the indicated times, 100 µl of cells were removed and extracted twice with chloroform-methanol (2:1). The organic layer was dried under nitrogen, and the radioactivity was incorporated into the total lipid pool, quantified by liquid scintillation counting. The rate of acetate incorporation into organically soluble material as a function of time was determined.

Nonesterified fatty acid analysis. Sera fatty acids were measured using a colorimetric nonesterified fatty acid (NEFA) kit (Wako, Richmond, VA), according to the manufacturer's instructions. The results were compared with a linear curve using oleic acid as the standard.

Long-chain acyl-CoA analysis. Long-chain acyl-CoA (LCACoA) standards (C16:0, C16:1, C17:0, C18:0, C18:1, C18:2) were purchased from Sigma Chemical (St. Louis, MO). Extraction of LCACoAs was carried out using the method previously described (16, 58). Briefly, frozen tissue (50–100 mg) was ground under liquid nitrogen and homogenized in 1 ml of 100 mM KH2PO4 (pH 4.9) and 1 ml 2-propanol. Heptadecanoyl CoA was added as an internal standard. One hundred twenty-five microliters of saturated (NH4)2SO4 and 2 ml of acetonitrile were added to the suspension and then vortexed for 2 min. The emulsion was centrifuged for 10 min at 4,000 rpm, and then the supernatant was diluted with 5 ml of 100 mM KH2PO4 (pH 4.9) for the solid-phase extraction. Before loading, oligonucleotide purification solid-phase columns (Applied Biosystems, Singapore) were conditioned with 5 ml acetonitrile and 2 ml of 25 mM KH2PO4 (pH 4.9). After the samples were loaded, the cartridges were washed with at least 10 ml of distilled water, and then LCACoAs were eluted slowly with 0.5 ml of 60% acetonitrile. The eluent was dried in a speed vac and reconstituted in 100 µl of methanol-H2O for electrospray ionization tandem mass spectrometry analysis performed using a bench top tandem mass spectrometer API3000 (Perkin-Elmer Sciex) interfaced with TurboIonspray ionization source. Mobile phase consists of methanol and H2O with isocratic gradient (50:50). LCACoAs were ionized in negative ionspray mode. Doubly charged ions and corresponding product ions were chosen as transition pairs for each CoA species for selective reaction monitoring quantitation. Calibrations of LCACoAs showed consistent linearity from 0.2 to 20 ng/µl; coefficiency of variance was 2.1–5.5% for all LCACoAs species.

Serum adipokine analysis. Fifty microliters of sera were utilized in ELISA assays. Leptin and resistin were measured by R&D Systems (Minneapolis, MN) in a cytokine multiplex assay using a Luminex instrument. Adiponectin was measured by serial dilution of samples and Western analysis, using a specific antibody, as well as by ELISA analysis (15). Resistin was also measured by a radioimmunoassay from Linco Research (St. Charles, MO), according to the manufacturer's instructions.

Adipose adiponectin analysis. Adipose tissue was homogenized (1 ml/g tissue) in a buffer containing 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% Tween 20, 0.2% Nonidet P-40, 1 mM PMSF, 2 µg/ml pepstatin A, and 10 µg/ml leupeptin. The extract was centrifuged at 12,000 rpm for 10 min, the floating fat cake was removed, and the supernatant was recovered. Adiponectin levels were measured using an adiponectin ELISA from Linco Research.

RNA preparations. Total RNA was isolated from epididymal fat pads in TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer's directions. Briefly, adipose pads were homogenized in TRIzol, and chloroform was added (0.2 ml/ml homogenate), followed by centrifugation at 9,000 rpm at 4°C for 15 min. The supernatant was removed, and an equal volume of cold isopropanol was added and centrifuged again under the same conditions. Pellets were washed in cold 70% ethanol, allowed to dry, and resuspended in TRIzol, and the extraction process was repeated to provide a higher quality RNA preparation. The resultant samples were solubilized in 50 mM sodium phosphate buffer (pH 8.0) and stored at –80°C.

Real-time reverse transcription-PCR. RNA was quantified using a Cary Spectrophotometer, and 3 µg were reverse transcribed in 20 µl using an oligo(dT) primer and SuperScript II enzyme (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Samples were subsequently diluted 1:15, and 5 µl of cDNA were used in each 20-µl real-time PCR reaction using the LightCycler FastStart DNA Master SYBR Green I kit (Roche, Indianapolis, IN), supplemented with 2 mM MgCl2. Samples were amplified using a Roche Light Cycler 3.5. Wild-type cDNA was used for a standard dilution in each run to establish a slope and error. The primer pairs and annealing temperatures are listed in Table 1. Analyses of the results included comparing the threshold crossing (CT) of each sample normalized to that of TATA-binding protein. The changes in the threshold crossing ({Delta}CT) were used to calculate the relative levels of each mRNA from the various adipocyte samples, utilizing the formula Formula (30).


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  		Table 1. Primers and annealing temperatures for real-time RT-PCR

 
Statistical analyses. All values are expressed as the means ± SE, unless otherwise noted. In some cases, statistical significance was determined by an unpaired, two-tailed Student's t-test. ANOVA was performed using Proc GLM using a least squares difference of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adipose biology is intimately linked to whole body insulin sensitivity (10, 41). Two major adipocentric regulatory themes have been implicated in this process: 1) lipolytically derived free fatty acid (FFA) metabolites and their effects on muscle and liver glucose metabolism, and 2) adipokine secretion from adipocytes and resident macrophages. While the inflammatory NF-{kappa}B signaling in macrophages from the A-FABP/aP2 null mice has been implicated (36), far less is known about adipokine synthesis and secretion from adipocytes in the FABP animal models. To that end, the increase in insulin sensitivity of the A-FABP/aP2 null mice and the decrease in insulin sensitivity in the E-FABP transgenic mice were evaluated in terms of altered lipid homeostasis and adipokine secretion.

The expression of adipose FABPs was evaluated using quantitative real-time PCR in the animals maintained under a high-fat feeding regimen (31, 48). Wild-type adipose contains high levels of A-FABP/aP2 mRNA and to a much lesser extent E-FABP mRNA. In A-FABP/aP2 null animals, the levels of E-FABP mRNA are upregulated 12.4-fold compared with the wild type. In the E-FABP transgenic animals, adipose E-FABP mRNA is upregulated 1,210-fold compared with the wild type, whereas A-FABP/aP2 mRNA is reduced to 70% of that of wild type (Table 2). Similar results were obtained using Northern blotting (results not shown). At the protein level, the adipose expression of the FABPs was determined using immunochemical titration using mono-specific antibodies. E-FABP expression in the A-FABP/aP2 null mice is increased twofold but still is only ~25% that of A-FABP/aP2 in wild-type adipose tissue. In the E-FABP transgenic mice, A-FABP/aP2 level is reduced by 50% (23, 33), while the E-FABP level is increased 10-fold, resulting in the total FABP level in transgenic mice being increased ~1.5-fold (Fig. 1A).


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  		Table 2. Quantitative RT-PCR of epididymal adipose tissue

 

Figure 1
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  		Fig. 1. Characteristics of adipose tissue in fatty acid-binding protein (FABP) mouse models. A: expression of FABPs in adipose tissue from the mice on a high-fat diet. Quantitation of FABPs was performed by Western analyses using specific antibodies for adipocyte (A)-FABP/aP2 and epithelial (E)-FABP, with comparison to protein standard curves generated by purified A-FABP/aP2 and E-FABP. B: adipose depot weights. The mass of various adipose depots from 12-wk-old high-fat-fed mice (n ≥ 6) is plotted as the means ± SE. Open bars indicate adipose from A-FABP/aP2 null mice; filled bars are from wild-type C57Bl/6J mice; gray bars are from E-FABP transgenic mice. *Student's t-test indicated significant differences within a particular depot with P < 0.05. C: adipocyte cell size. Adipocytes were collagenase digested and their diameters measured. A minimum of 200 cells were measured and plotted. Each triangle indicates the diameter of a single cell. Numbers above the plots are the mean size ± SE for each genotype.

 
The sizes of various subcutaneous (gluteal, inguinal/dorsolumbar, interscapular/brown adipose, and axillary) and visceral (epididymal and perirenal/retroperitoneal) depots were determined (Fig. 1B). No statistical differences were seen in the adipose from E-FABP transgenic mice compared with the wild type (n = 6–10 mice), although there was a trend toward reduced total adipose tissue. When the results were extended to an analysis of 60 mice and focused on epididymal fat pads, a small but statistically significant decrease in the E-FABP transgenic mice was determined. In contrast, the A-FABP/aP2 null mice have an increased adipose mass in all depots measured. This is consistent with previous work documenting the decrease in lipolysis in high-fat-fed A-FABP/aP2 null mice (3, 12, 51). As such, in contrast to convention, in the FABP animal models, increased adipose mass is associated with attenuated insulin resistance, whereas decreased adipose mass is associated with enhanced insulin resistance.

Next we evaluated adipocyte size, because of the negative correlation of size with insulin sensitivity (5, 38). The majority of adipocytes from all three genotypes had diameters in a similar range; however, a small percentage of adipocytes from the E-FABP transgenic animals was considerably larger (Fig. 1C). The extent to which these cells contribute to the insulin resistance of the E-FABP transgenic animals is not known. This result was also demonstrated in E-FABP transgenic mice fed a chow diet; ~5% of the adipocytes were considerably larger than the rest (23). Therefore, high-fat feeding of the E-FABP transgenic mice had no effect on the percentage of cells with an enlarged size.

Previous work has demonstrated that the loss of a FABP is inversely correlated with the intracellular levels of FFAs (12); A-FABP/aP2 null mice exhibit increased FFA, whereas E-FABP transgenic mice exhibit reduced cellular FFA levels. FFAs or their metabolites have been implicated as regulatory molecules, controlling the expression of a number of genes of intermediary metabolism (37, 44). To begin to understand the changes in adipose tissue resulting from altered FABP expression, real-time RT-PCR was utilized to identify differentially regulated adipose genes. Given the association of A-FABP/aP2 with HSL (54, 56), we focused on enzymes and proteins that reside at the lipid droplet surface that are thought to play a pivotal role in lipolysis and/or lipid droplet formation (Table 2). HSL mRNA was reduced in the A-FABP/aP2 null adipose tissue compared with the wild-type or E-FABP transgenic adipose tissue. The mRNA levels for adipose triglyceride lipase (ATGL)/desnutrin paralleled that for HSL, a trend toward reduction in the A-FABP/aP2 null adipose and increase in the E-FABP transgenic adipose. The messenger RNA for perilipin A, a lipid droplet protein intimately involved in lipolysis that may serve as a scaffold for droplet-associated regulatory proteins, was reduced in the E-FABP transgenic adipose, consistent with the increase in lipolysis seen in the E-FABP transgenic adipose (23). The messenger RNA for another lipid droplet-associated protein S3-12 was elevated in the E-FABP transgenic adipose compared with the A-FABP/aP2 null adipose, while Tip47 levels were unaltered.

To parallel changes in gene expression whose products are linked to lipolysis, we carried out real-time RT-PCR analysis of genes involved in lipogenesis. Such an evaluation revealed that acetyl-CoA carboxylase 1 mRNA levels were elevated in the A-FABP/aP2 null adipose tissue compared with wild-type adipose (Table 2). When comparing A-FABP/aP2 null to E-FABP transgenic adipose, modest increases in long-chain acyl-CoA synthetase (ACSL) 5, CD36, diacylglycerol acyltransferase, and lipoprotein lipase mRNAs were also detected. Other genes that were not affected included ACSL1, fatty acid transporter protein-1, fatty acid transporter protein-4, GLUT-1, GLUT-4, glycerol-3-phosphate acyltransferase, steroyl-CoA desaturase-1, peroxisome proliferator-activated receptor (PPAR){gamma}, and sterol responsive element binding protein-1 (Table 2). In contrast, ACSL4 was elevated in E-FABP transgenic adipose compared with wild-type adipose. The overall trend observed is that genes encoding enzymes linked to lipolysis are reduced, and those linked to lipogenesis are increased, in the A-FABP/aP2 null vs. the E-FABP transgenic adipose tissue. This is consistent with the increase in adipose mass in A-FABP/aP2 null mice and the decrease in adipose mass in the E-FABP transgenic animals (Fig. 1B). The expression of a macrophage marker, F4/80 (26), was unaltered in the A-FABP/aP2 null and decreased in the E-FABP transgenic adipose tissue. These results indicate that the number of infiltrating macrophages in the adipose tissue is not likely to be a plausible explanation for the altered insulin sensitivities in the FABP mouse models.

Previous experiments have measured the rates of lipolysis in adipose tissue from the FABP mouse models (3, 12, 51), whereas few have been directed at measuring the rates of lipogenesis (53). To extend the studies on lipolysis to de novo lipogenesis in A-FABP/aP2 null with E-FABP transgenic animals, we evaluated the conversion of [14C]acetate into lipid in primary adipocytes from both FABP mouse models. Consistent with the real-time PCR measurements of genes controlling lipid flux, there was an increase in de novo lipogenesis in the A-FABP/aP2 null adipocytes compared with the wild-type or E-FABP transgenic adipocytes (Fig. 2), consistent with the increased adiposity in the A-FABP/aP2 null animals. These results are similar to the data from the Fried and Storch laboratories demonstrating an increase in basal rates of glucose conversion to lipids in the A-FABP/aP2 null mice (53).


Figure 2
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  		Fig. 2. Rates of de novo lipogenesis from the FABP mouse models. Primary adipocytes were incubated with [14C]acetate, and the rate of incorporation into organically extractable lipids was determined. Values are expressed as a pmol incorporated·min–1·mg protein–1. *Student's t-test indicated significant difference with wild type (P < 0.05). N = 3.

 
Of the various etiologies linked to the development of insulin resistance in the obese, elevated serum NEFAs (6, 42) have received considerable attention. Mechanistically, elevated serum FFAs can lead to an increase in lipid availability (suggested to be acyl-CoAs) in peripheral tissues and inhibition of the normal insulin-signaling cascade. Previous studies have demonstrated that, in situ, adipocytes from A-FABP/aP2 null mice exhibit reduced basal and stimulated lipolysis, whereas adipocytes from E-FABP transgenic mice have increased lipolysis (12, 23, 51). Additional results using stable isotope infusion into high-fat-fed A-FABP/aP2 null mice revealed that lipolysis is also significantly decreased in vivo (3). To determine whether the changes in muscle and/or liver metabolism could be linked to the level of serum FFAs, the level of total NEFA was evaluated in each animal genotype when maintained on a high-fat diet. NEFAs were quantified from sera, either on morning blood draws (without a fast) or after a 6-h daytime fast. No differences in the quantity of serum fatty acids were detected in either of the high-fat-fed FABP mouse models (Fig. 3). These results demonstrate that, although the rates of lipolysis are altered in the FABP mice models, serum NEFAs were maintained similar to wild type, indicating a compensatory regulation of lipid disposal at peripheral tissues such as liver and/or muscle (3).


Figure 3
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  		Fig. 3. Serum free fatty acids from the FABP mouse models. Nonesterified fatty acids (NEFAs) were measured for each genotype (n ≥ 10). Values are expressed as means ± SE. Open bars represent free fatty acids from blood draws in the morning; filled bars indicate free fatty acids from blood draws after a 6-h morning fast. Student's t-test across the genotypes indicated no significant differences with P < 0.05.

 
Intracellular FFAs are metabolized in an ATP-dependent manner to LCACoAs. An increase in muscle acyl-CoA pools has been shown to correlate with insulin resistance (22, 55) and is likely to involve the serine phosphorylation of insulin receptor substrate-1 by PKC{theta} (58). Therefore, the levels of LCACoAs were quantified in adipose, muscle, and liver tissues from high-fat-fed mice. Strikingly, no statistically significant differences were seen in muscle or liver tissue when comparing A-FABP/aP2 null, wild-type, or E-FABP transgenic mice (Table 3). Consistent with increased de novo lipogenesis, C16:0 and C16:1 acyl-CoA pools were elevated in adipose of the A-FABP/aP2 null mice compared with the wild-type or E-FABP transgenic animals.


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  		Table 3. Tissue acyl-CoA levels for various lipid classes

 
An alternate adipocentric mechanism linked to control of systemic insulin sensitivity is through the secretion of adipokines (4, 25). As such, serum protein levels of three adipokines implicated in insulin sensitivity were measured (Fig. 4). Serum leptin concentrations were similar in all genotypes (Fig. 4A), and, although there is a trend toward reduced leptin in the E-FABP transgenic mice, statistical significance was not reached after measuring samples from 12 individual mice. Resistin, an adipokine implicated in the development of insulin resistance in the liver, was unchanged between the A-FABP/aP2 null animals and wild-type C57Bl/6J mice and was actually reduced by 30% in the E-FABP transgenic animals (Fig. 4B). Therefore, resistin levels do not correlate to insulin resistance in these animal models. In contrast to leptin and resistin, adiponectin was the only measured adipokine that changed in both FABP models in a manner consistent with the development of the metabolic syndrome. Levels of adiponectin were elevated in the A-FABP/aP2 null mice (125%) and were decreased (53%) in the E-FABP transgenic mice, compared with the wild-type mice (Fig. 4C). This is consistent with the insulin sensitivities of the FABP mouse models (24, 33), since adiponectin has been shown to play a significant role as an insulin sensitizer (4, 28, 34, 41). Importantly, the oligomeric state of adiponectin, important for the overall action of the protein (40), was unaltered (data not shown). To determine whether the change in serum adiponectin arises from altered expression within adipose, adiponectin was quantified within visceral adipose depots. As shown in Fig. 4D, visceral adipose adiponectin paralleled serum adiponectin, implying a mechanism of altered expression. However, this does not rule out the possibility that there are changes in secretion from adipocytes or altered clearance rates in peripheral tissues. Serum protein levels of IL-6 and TNF-{alpha} were below reliable quantification but may also contribute to overall insulin sensitivity. Interestingly, the levels of adiponectin mRNA did not parallel changes in adiponectin protein (Table 4). Furthermore, although the levels of resistin mRNA correlated with the metabolic characteristics of the animal genotypes (decreased expression in the A-FABP/aP2 null, increased expression in the E-FABP transgenic), they did not correlate with serum protein levels. Leptin mRNA levels paralleled the adiposity of the animals, and a similar trend (although without statistical significance, n = 12) was observed at the protein level.


Figure 4
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  		Fig. 4. Serum adipokines and adipose adiponectin from the FABP mouse models. A: serum leptin; B: serum resistin; C: serum adiponectin; D: adiponectin in visceral adipose depots. Values are expressed as total micrograms adiponectin in epididymal plus perirenal adipose depots. Experiments were performed in duplicate with n = 8–12 for the serum samples and n = 5 for the adipose tissue samples. Values are expressed as means ± SE. *Student's t-test indicated significant differences from wild type with P < 0.05. Open bars indicate A-FABP/aP2 null mice; solid bars indicate wild-type C57Bl/6J; shaded bars indicate E-FABP transgenic mice.

 

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  		Table 4. Adipokine mRNA expression in FABP animal models using quantitative RT-PCR analysis

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Obesity is associated with various metabolic pathophysiologies and is central to the development of the metabolic syndrome involving a cluster of insulin resistance, dyslipidemia (low HDL cholesterol; high triglycerides), and hypertension (50). Associated with the metabolic syndrome is an increased risk of type 2 diabetes and cardiovascular disease, and ~95% of people with type 2 diabetes are obese (19). The mouse FABP models provide insight into how intracellular FFA metabolism may influence adipose metabolism and, in turn, whole body insulin sensitivity.

The Randle cycle, an increase in FFAs in serum leading to increased fat oxidation, preferentially over glucose oxidation (46, 47), implies that changes in adipose lipolysis may be a major contributing factor in obesity-linked insulin resistance. Additionally, the effect of chronic exposure of elevated serum fatty acids on the beta-cell is a reduction in the glucose-stimulated insulin secretion (6, 60). Insulin resistance in the liver leads to increased gluconeogenesis, which further exacerbates the increase in serum glucose and decrease in insulin sensitivity (29, 52). Although this mechanism of insulin resistance is well documented, in the FABP mice models there were no changes in serum fatty acids, despite altered fasting blood glucose levels (Fig. 2), suggesting that mechanisms independent of serum FFAs are linked to insulin resistance. Consistent with this, there were no changes in muscle or liver acyl-CoA pools, as has been observed for other murine models of insulin resistance linked to fatty acid metabolism (20, 27, 43).

Within adipose tissue, the FABP mouse models have altered lipid metabolism, including misregulated lipolysis and lipogenesis. Analyses of these pathways indicate that genes (HSL, and a trend for ATGL) whose protein products are linked to lipolysis are downregulated in the A-FABP/aP2 null relative to the E-FABP transgenic adipose tissue. Also promoting the increased lipolysis in the E-FABP transgenic adipose is the decreased perilipin A expression. In contrast, multiple genes involved in lipogenesis (ACSL5, CD36, diacylglycerol acyltransferase, lipoprotein lipase) are upregulated in the A-FABP/aP2 null compared with the E-FABP transgenic adipose tissue. S3–12 mRNA expression was decreased in the A-FABP/aP2 null adipose relative to expression in the E-FABP transgenic mice; protein levels were not assessed. The mechanism(s) responsible for the coordinate reprogramming of gene expression in response to the loss or gain of a FABP remains obscure but may be linked to the activity of PPAR{gamma}. The accumulation of intracellular FFAs in A-FABP/aP2 null mice or decrease FFAs in E-FABP transgenic mice may alter the availability of endogenous PPAR{gamma} ligands and, in turn, affect the expression of target genes. However, global changes in all PPAR{gamma}-regulated genes were not observed, suggesting that only a subset of genes encoding critical metabolic proteins is regulated. Some of the statistically significant changes in mRNA were modest and thus are not easy to interpret physiologically. Future studies on protein expression from these lipolytic and lipogenic genes are necessary to determine whether these changes in mRNA result in similar effects on protein abundance.

Consistent with a reprogramming of lipid metabolism, the A-FABP/aP2 null mice have increased incorporation of acetate into lipid pools. However, utilizing acetate incorporation could potentially bias the results by providing only one carbon source for the production of lipids. Interestingly, this finding is similar to that from the Fried and Storch laboratories demonstrating an increase in basal rates of glucose conversion to lipids in the A-FABP/aP2 null mice (53). Furthermore, in vivo evidence of elevated C16:0 and C16:1 acyl-CoA pools in adipose (which are derived from de novo lipogenesis) supports this conclusion. The increase in lipogenesis, along with the decrease in lipolysis, may explain the increased adiposity seen in the A-FABP/aP2 null mice fed a high-fat diet. It is not surprising that alteration in the levels of protein(s) that bind fatty acids may influence pathways involved in the production and mobilization of its substrate.

Adipocytes secrete several proteins that impact whole body insulin sensitivity, including leptin, adiponectin, resistin, cellular retinal binding protein-4, TNF-{alpha}, and IL-6 (4, 25). Of these, this report describes serum adiponectin levels paralleling changes in insulin sensitivity in the FABP models: elevated in the A-FABP/aP2 null animals and decreased in the E-FABP transgenic mice. It is not known whether the modest increase in adiponectin in the A-FABP/aP2 null mice functionally accounts for the overall increased insulin sensitivity. Resistin levels were reduced in the E-FABP transgenic mice. Since resistin is thought to play a causative role in insulin resistance, this result is inconsistent in providing a mechanism that results in insulin resistance in the E-FABP transgenic mice. Two other adipokines implicated in altering insulin resistance are TNF-{alpha} and IL-6; however, levels of both were below accurate detection. In a previous study, TNF-{alpha} mRNA levels were reduced in the A-FABP/aP2 null adipose (24). In this study, there was only a trend for reduced levels of TNF-{alpha} mRNA in the A-FABP/aP2 null adipose, whereas TNF-{alpha} mRNA levels were reduced in the E-FABP transgenic adipose. If protein levels are consistent with the mRNA levels, this again is inconsistent as a mechanism to explain the increase in insulin resistance. Overall, of the adipokines measured, the metabolic syndrome seen in the FABP mouse models correlates most closely with serum adiponectin levels.

The change in serum adiponectin protein levels was not mirrored by changes in mRNA levels. Several recent studies have also reported discrepancies between mRNA levels and serum concentrations of a number of adipocyte-derived proteins. Combs et al. (13) demonstrated that adipose tissue mRNA levels for adiponectin do not differ between males and females, even though the serum adiponectin levels show a pronounced sexual dimorphism. This may be caused, at least in part, by an extensive posttranscriptional and posttranslational control of protein secretion and degradation within the secretory pathway (14). Similarly, sharp discrepancies have been reported for resistin mRNA and protein levels as well, where insulin resistance causes a decrease in resistin mRNA levels, but an increase in plasma levels of resistin (45). In addition to adiponectin and resistin, this inconsistency has also been demonstrated for leptin (2, 17, 18, 39).

In summary, this report demonstrates that the A-FABP/aP2 null and E-FABP transgenic animals exhibit reprogrammed expression of genes encoding enzymes of lipogenesis and lipolysis. These changes result in altered rates of lipolysis and lipogenesis that, in turn, lead to changes in adipose mass associated with the animal models. The altered lipid homeostasis does not result in changes in serum FFA or acyl-CoAs, suggesting that other factors, potentially adipokines, may be responsible for the characteristics of attenuated or potentiated insulin resistance. While TNF-{alpha} has previously been implicated as a causal agent in this process, this study demonstrates that the expression of other adipokines, most notably adiponectin, is also altered in the animal models. The molecular events that link FABP activity within the adipocyte (and/or macrophage) to control of adipokine expression remain to be determined.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funds from National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-053189 (D. A. Bernlohr), DK-40936 (G. I. Shulman), and DK-59635 (G. I. Shulman); a Distinguished Clinical Scientist Award from the American Diabetes Association (G. I. Shulman); the National Institutes of Health supported Minnesota Obesity Center's Pilot and Feasibility Grant (A. V. Hertzel); as well as the Minnesota Supercomputing Institute (D. A. Bernlohr).


   ACKNOWLEDGMENTS
 
We thank members of the Bernlohr laboratory for helpful suggestions and comments during the preparation of this manuscript. We thank Josh Andersland for initial work on quantifying fatty acids and Maura Romanshek for work on quantification of FABPs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Bernlohr, Dept. of Biochemistry, Molecular Biology, and Biophysics, Univ. of Minnesota, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455 (e-mail: bernl001{at}umn.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.


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