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target gene and plays a role in lipid metabolism1Division of Preventive Medicine and Nutrition, Department of Medicine, Columbia University, and 2Departments of Medicine and Neuroscience, Albert Einstein College of Medicine, New York, New York
Submitted 23 May 2008 ; accepted in final form 3 October 2008
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ABSTRACT |
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(PPAR). Moreover, CRBP-III expression was induced in adipose tissue of mice after treatment with the PPAR agonist rosiglitazone. To examine a potential role of CRBP-III in regulating lipid metabolism in vivo, CRBP-III-deficient (C-III-KO) mice were maintained on a high-fat diet (HFD). Hepatic steatosis was decreased in HFD-fed C-III-KO compared with HFD-fed wild-type mice. These differences were partly explained by decreased serum free fatty acid levels and decreased free fatty acid efflux from adipose tissue of C-III-KO mice. In addition, the lack of CRBP-III was associated with reduced food intake, increased respiratory energy ratio, and altered body composition, with decreased adiposity and increased lean body mass. Furthermore, expression of genes involved in mitochondrial fatty acid oxidation in brown adipose tissue was increased in C-III-KO mice, and C-III-KO mice were more cold tolerant than wild-type mice fed an HFD. In summary, we demonstrate that CRBP-III is a PPAR target gene and plays a role in lipid and whole body energy metabolism. adipose tissue; retinoid; liver steatosis; lipolysis; brown adipose tissue; fatty acid-binding protein; intracellular lipid-binding protein; retinol-binding protein-7
10–20% of total retinoids in the body are stored as retinyl ester in adipocytes (52). This storage appears not to merely serve as a passive storage tissue for retinoids. Rather, during insufficient vitamin A intake and low hepatic retinoid levels, adipose tissue stores appear to be readily mobilized (25, 33). Retinoids also act to modulate adipose tissue structure and metabolic function. All-trans retinoic acid (RA) can inhibit adipogenesis by activation of the RA nuclear hormone receptor (RAR) (10, 28). In contrast, activation of the retinoid X receptor (RXR) leads to enhancement of adipogenesis and insulin sensitivity (29). Recently, retinaldehyde (RAL), produced in the adipocytes, was shown to inhibit adipogenesis and, subsequently, the development of diet-induced obesity (55). In addition, data indicate that plasma retinol-binding protein, when secreted from adipose tissue, may act as an adipokine, reducing insulin sensitivity in mice (54). Intracellularly, retinol is the precursor for retinyl ester formation and RA synthesis, with RAL as the intermediate metabolite (5, 30). Similar to fatty acid sequestration by fatty acid-binding proteins in the cytosol, retinol is bound by intracellular cellular retinol-binding proteins (CRBPs) (21, 31). CRBP-I is ubiquitously expressed, with the highest expression in liver, kidney, testes, and eye. CRBP-III and CRBP-I have overlapping expression in heart, muscle, adipose, and mammary tissue (34, 36, 53). CRBP-III binds retinol, and we previously proposed that its function is to facilitate the esterification of retinol to retinyl ester in the mammary gland during lactation (36). Our next goal is to further determine the function of CRBP-III. Our first objective was to analyze the expression of CRBP-III during adipogenesis and, subsequently, determine how its expression may be regulated. We present data that CRBP-III is a peroxisome proliferator-activated receptor- (PPAR) target gene. Since PPAR is essential for adipogenesis and required for maintenance of normal lipid homeostasis (11, 20, 50), the objective of the present study was to understand the role of CRBP-III in lipid metabolism. We pursued our objective by examining CRBP-III-knockout (C-III-KO) mice during the metabolic challenge of a high-fat diet (HFD). |
MATERIALS AND METHODS |
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Isolation of RNA and protein. Total RNA was isolated using RNA-Bee (Tel-Test). Protein extracts were prepared in a buffer composed of Tris-Cl (20 mM, pH 7.5), EDTA (5 mM), EGTA (2 mM), NP-40 (1%), NaF (100 mM), NaVO3 (2 mM), NaP2O7 (10 mM), and proteinase and phosphatase inhibitor cocktails. Western blot analyses were essentially performed as described previously (36, 53). Antibodies for detection of CRBP-III have been described elsewhere (36). Antibodies to detect AMP-activated protein kinase and acetyl-CoA carboxylase (ACC) and their phosphorylation status were obtained from Cell Signaling (Danvers, MA). Separation of brown adipose tissue (BAT) into stromal vascular fraction and adipocytes was accomplished by collagenase digestion before Western blot analysis (53).
Quantitative real-time PCR. cDNA was synthesized from total RNA (3 µg) using SuperScript III reverse transcriptase (Invitrogen). The final reaction for the quantitative real-time PCR consisted of 0.5 µM gene-specific primer, 1x SYBR Green PCR Master Mix (Applied Biosystems), and 10 µl of diluted cDNA. The reaction was carried out on a Stratagene cycler. (See supplemental Table S1 in the online version of this article for primer sequences.)
Reporter constructs for CRBP-III promoter and luciferase assays.
A 3.1-kb fragment upstream of the site of transcription was amplified from mouse genomic DNA (C57/Bl6) using a high-fidelity PCR system (Expand, Roche Diagnostics) and cloned into the Zero Blunt TOPO PCR vector (Invitrogen). The fragment was completely sequenced, and the results were compared with the published sequence. The 3.1-kb promoter fragment was cloned into the pGL3 basic vector (Promega) and designated pGL3k, which was used as a template for generation of two shorter promoter constructs, 2.5 kb (pGL2.5k) and 2 kb (pGL2k). A PCR approach was used to introduce mutations of the putative PPAR response elements (PPREs). Putative PPRE1 (mut1), PPRE3 (mut3), or PPRE1 and PPRE3 (mut1 and mut3) were mutated. The primers used to introduce the mutations were 5'-GATCCATAACTGTGTACACAGCCGAAATTTCTGGAG-3 for PPRE1mut and 5'- CTGACTGACTTACACAGCCACAGTTTCCCTCTTACGGG-3' for PPRE3mut. All constructs were sequenced to ensure the correct mutations. 3T3-L1 cells were differentiated in six-well plates and used at day 7 of differentiation. Cells were transfected with 0.2 µg of firefly luciferase DNA constructs (pGL3k, pGL2.5k, or pGL2k). COS-7 cells were plated for 24 h in 12-well plates in complete medium and transfected with firefly luciferase DNA constructs and with pcDNA3.1 alone or equivalent concentrations of pcDNA3.1-PPAR2 and pcDNA3.1-RXR
, which overexpress PPAR2 and RXR, respectively. The Renilla luciferase vector (0.05 µg) was included in all transfection procedures as a control. Transfections were carried out overnight; on the next morning, the medium was replaced with complete medium containing 1 µM rosiglitazone or vehicle (ethanol). After 24 h, the dual luciferase reporter assay (Promega) was performed using a luminometer (TD-20/20, Turner Designs). Results were expressed as relative luciferase activity, with normalization of firefly luciferase activity to Renilla luciferase activity.
EMSA.
Nuclear proteins were isolated from COS-7 cells transiently expressing PPAR2 and RXR using a nuclear protein extraction kit (Pierce). The probes were prepared by annealing oligonucleotide and end-labeling the double-stranded DNA with [-32P]ATP using T4 kinase (Promega). The PPRE of the adipocyte-binding protein aP2 promoter was used as a positive control (50). Oligonucleotides were as follows: aP2-PPRE (5'-GATCTGTGACCTTTGACCTAGTAAG-3', 5'-CTTACTAGGTCAAAGGTCACAGATC-3'), CRBP-III PPRE1 (5'-GAAATTTGGGCAGAGTTCACAGTTA-3', 5'-TAACTGTGAACTCTGCCCAAATTTC-3'), CRBP-III PPRE2 (5'-ATGGATCCCTCAAAGGTCAAGGAAT-3', 5'-ATTCCTTGACCTTTGAGGGATCCAT-3'), and CRBP-III PPRE3 (5'-TCTCCCTTGGAAAAGGTCACATTCA-3', 5'-TGAATGTGACCTTTTCCAAGGGAGA-3'). The DNA-protein binding assays were performed as recommended by the manufacturer (Promega). A supershift assay was performed using PPAR monoclonal (E8, Santa Cruz Biotechnology) or RXR polyclonal (D-20, Santa Cruz Biotechnology) antibody. The binding reaction was incubated with the indicated antibody for 10 min at room temperature before addition of the labeled nucleotides. Electrophoresis of the DNA-protein complexes was performed on 6% DNA retardation gels (Invitrogen), which were placed on Whatman paper and dried using a vacuum gel dryer. The signals were detected using a Storm PhosphorImager (Amersham Biosciences).
Chromatin immunoprecipitation assay.
Differentiated 3T3-L1 adipocytes were used for the chromatin immunoprecipitation assays. After cross-linking of DNA and proteins in situ, the soluble chromatin was prepared according to the manufacturer's instructions (Upstate), and the DNA was sheared. The protein-chromatin complex was immunoprecipitated with PPAR antibodies, and PCRs that included regions of PPRE1 and PPRE3 were performed.
Animal studies. Three-month-old male wild-type (C57BL/6) mice (Jackson Laboratory, Bar Harbor, ME) were fed a chow diet or a diet supplemented with rosiglitazone (10 mg/kg body wt) for 21 days. Generation of mice lacking CRBP-III (C-III-KO) has previously been reported (36). The mice were backcrossed to the C57BL/6 genetic background for five generations before the experiments. C-III-KO and CRBP-III wild-type mice were fed an HFD providing 60% of calories from fat (Research Diets). The mice were fed the HFD for 20 wk and then killed, the liver was perfused with PBS, and tissues were excised, flash frozen in liquid nitrogen, and stored at –70°C until analysis. Experiments involving mice were performed with the approval of the Institutional Animal Care and Use Committee at Columbia University.
Histology. Liver, white adipose tissue, and BAT were placed in 10% buffered formalin overnight and then embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin-eosin. Liver sections were also fresh frozen in OCT compound, sectioned (7 µm), and stained with oil red O for detection of neutral lipids.
Retinoid measures. Retinol and retinyl ester levels in liver and adipose tissue were determined as previously described (36).
Lipolysis assay of isolated white adipose tissue.
Lipolysis assays were performed essentially as previously described (35, 42). White adipose tissue from HFD-fed wild-type and C-III-KO mice was excised, washed in PBS, and cut into small (50 mg) pieces. The explants were incubated in Krebs-Ringer buffer for 3 h at 37°C in the absence or presence of isoproterenol (10 µM). At the end of the incubation, the plates were placed on ice, and the medium was removed for glycerol and free fatty acid analyses using commercial kits (Sigma Aldrich and Wako). Tissue pieces were washed and saved for protein analysis. For each animal, the assay was performed in quadruplicate.
Hepatic triglyceride levels. Triglycerides were extracted from liver using a Folch extraction. Briefly, livers were homogenized in PBS and incubated in chloroform-methanol (2:1) in a rotator at room temperature for 1 h, and the chloroform phase was dried under a mild stream of nitrogen. The triglycerides were reconstituted in cold isopropanol, and levels were determined using a commercial kit (Thermo Scientific).
Serum parameters. Blood for determination of lipid parameters, glucose, insulin, and adipokine levels was obtained from mice fasted during the day for 6 h (9 AM–3 PM) after 18 wk on the HFD. Serum was obtained after the blood samples had been allowed to clot. Commercially available kits were used to measure serum levels of nonessential fatty acids (Wako), free glycerol (Sigma Aldrich), β-hydroxybutyrate (Stanbio), total insulin (Mercodia), glucose (Invitrogen), and total cholesterol and triglyceride (Thermo Scientific) levels. Serum leptin and resistin levels were determined using a Multiplex Assay (Millipore).
Cold tolerance testing. Cold tolerance was assessed in wild-type and C-III-KO mice fed the HFD for 20 wk by exposure to 4°C for 2 h. Rectal temperature was measured before the cold exposure and then at 1-h intervals during the cold challenge using a thermocouple probe (model IT-23) attached to a thermocouple amplifier (Physitemp BAT-10 Type I, Pharma).
Body composition analysis. Body composition was measured by MRI using an EchoMRI (Echo Medical Systems).
Metabolic studies. Animals were individually housed in metabolic chambers maintained at 20–22°C on a 12:12-h light-dark cycle, with lights on at 7 AM. Metabolic measurements (oxygen consumption, food intake, and locomotor activity) were obtained continuously using a Comprehensive Lab Animal Monitoring System (Columbus Instruments) open-circuit indirect calorimetry system. Metabolic data were collected over 5 consecutive days following 3 days of adaptation to the metabolic cages.
Statistical analysis. Values are means ± SE. Statistical differences were determined using Student's t-test. Statistical significance was considered at P < 0.05.
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RESULTS |
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in vitro and in vivo.
We previously showed that, in white adipose tissue, CRBP-III is expressed in the stromal vascular fraction and in adipocytes (53). To further understand the role of CRBP-III in adipocytes, we differentiated 3T3-L1 preadipocytes and analyzed gene expression at different stages of adipocyte differentiation. CRBP-III mRNA was detected 4 days and protein 7 days after the start of differentiation, suggesting posttranscriptional modification (Fig. 1, A and B). On the basis of the time of induction of CRBP-III expression, we hypothesized that CRBP-III could be regulated by PPAR. After treatment of differentiated adipocytes with the PPAR agonist rosiglitazone, CRBP-III expression levels were significantly increased compared with those measured in untreated cells (Fig. 1, A and B). Next, to test whether PPAR also regulates CRBP-III expression in vivo, we fed wild-type mice a chow diet alone or a chow diet enriched with the PPAR agonist rosiglitazone. CRBP-III expression levels were increased in white adipose tissue of mice fed the rosiglitazone-enriched diet compared with mice fed the unsupplemented chow diet (Fig. 1C). As a comparison, expression of aP2, a well-known target of PPAR (49), also increased in mice fed the rosiglitazone-enriched diet (Fig. 1C).
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elicits its transcriptional effect through binding as a heterodimer with RXR to specific PPREs in the noncoding sequences of genes. We cloned different-sized promoter fragments upstream of the transcriptional start site to understand whether a PPRE is present in the CRBP-III promoter. All promoter constructs (3, 2.5, and 2 kb) showed basal activity in fully differentiated 3T3-L1 adipocytes (Fig. 1D). However, treatment with rosiglitazone increased relative luciferase activity up to threefold over basal activity for a promoter fragment including the region 2.5 and 3 kb upstream of the transcriptional start site (Fig. 1D). In contrast, for the shorter (2-kb) promoter construct, no change relative to basal activity was observed after rosiglitazone treatment. Similar data were obtained when the promoter constructs were cotransfected with PPAR and RXR into COS-7 cells, where treatment with rosiglitazone only increased luciferase activity for the 3- and 2.5-kb constructs (see supplemental Fig. S1A). Taken together, these data indicate that a putative PPRE is present in a 500-bp region between 2.0 and 2.5 kb upstream of the transcriptional start site. Analyzing the DNA sequence of this 500-bp region, we identified three putative PPRE sites that contained the PPRE consensus sequence (22).
On the basis of EMSAs, PPAR and RXR bind to two PPRE sites (PPRE1 and PPRE3), forming a single DNA-protein complex (Fig. 1E). In contrast, there was no specific band for PPRE2. Binding of the labeled PPRE to the nuclear protein appears to be specific, inasmuch as it can be competed off with excess of specific competitor, but not with nonspecific DNA (Fig. 1E). When antibodies against PPAR or RXR were used, the DNA-protein complex supershifted to a higher molecular weight, further indicating that PPAR and RXR are binding to the specific DNA region in PPRE1 and PPRE3 (see supplemental Fig. S1B). Furthermore, on the basis of chromatin immunoprecipitation assay, PPAR binds to the PPRE region including PPRE1 and PPRE3 in vivo (Fig. 1F). Because PPRE1 and PPRE3 are within close proximity (132 bp apart), we were unable to discern whether PPAR binds to PPRE1 or PPRE3 or both. Therefore, we generated promoter constructs that contained mutations in one PPRE alone or in both. Mutations in either PPRE alone lead to a decreased response to rosiglitazone treatment. Importantly, the response to rosiglitazone was abolished only when both PPREs were mutated (Fig. 1G). Taken together, these data demonstrate that PPAR agonists regulate CRBP-III expression in vivo and in vitro through two PPREs present in the promoter of CRBP-III.
Hepatic steatosis is reduced in HFD-fed C-III-KO mice.
PPAR is essential for normal adipogenesis and critically required for maintaining normal glucose and lipid homeostasis (11, 20, 50). Since CRBP-III is regulated by PPAR in adipose tissue, we sought to understand its role in regulating lipid homeostasis by studying mice with a gene-targeted deletion of CRBP-III (C-III-KO) (36).
Since excess dietary fat leads to an expansion of adipose tissue and, often, to perturbations in energy metabolism, we examined the effects of diet-induced obesity in C-III-KO compared with wild-type mice. Weight gain in HFD-fed animals was not different between the two groups (Fig. 2A). In addition, serum glucose and insulin levels did not differ between HFD-fed wild-type and C-III-KO mice (Table 1). Next, we examined liver, white adipose tissue, and BAT for differences between wild-type and C-III-KO mice. As anticipated, livers from HFD-fed wild-type mice contained abundant, large lipid droplets (Fig. 2B). In contrast, livers from C-III-KO mice contained significantly fewer lipid droplets (Fig. 2B). In agreement with these observations, hepatic triglyceride concentrations were significantly lower in C-III-KO than wild-type mice (Fig. 2C). Hepatic retinyl ester levels were not different between the two groups (Fig. 2D).
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and acyl-coenzyme A dehydrogenase, long-chain). Similarly, in the liver, phosphorylation of AMP-activated kinase and its downstream target ACC were not different between HFD-fed C-III-KO and wild-type mice (Table 2). These data imply that there was no apparent change in fatty acid synthesis or oxidation in the liver of HFD-fed C-III-KO mice that would account for the decreased hepatic lipid accumulation.
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Maintenance of BAT function in HFD-fed C-III-KO mice.
CRBP-III is also highly expressed in BAT, in addition to white adipose tissue (see supplemental Fig. S3). Therefore, we examined this tissue following HFD consumption. Consistent with previous reports, morphology of BAT from HFD-fed wild-type mice was altered, with large unilocular lipid droplets and only a few multilocular lipid droplets characteristic of brown adipocytes (Fig. 5A). In contrast, BAT from C-III-KO mice is characterized by multilocular lipid droplets and contained only a few interspersed large lipid droplets (Fig. 5A). We next determined whether these changes were also reflected in altered gene expression between wild-type and C-III-KO mice. We found that genes involved in mitochondrial oxidative metabolism were expressed in higher levels in BAT from C-III-KO than wild-type mice. Expression of genes involved in oxidative phosphorylation (cytochrome c and cytochrome c oxidase II and VIII) and the citric acid cycle (aconitase) was significantly higher in BAT from C-III-KO than wild-type mice (Fig. 5B). In addition, PPAR levels and the expression of its downstream target genes for fatty acid oxidation (medium- and long-chain acyl-CoA dehydrogenase) were significantly higher in BAT from C-III-KO than wild-type mice (Fig. 5C). There was a trend for an increase in uncoupling protein-1 (UCP-1) expression levels in BAT of C-III-KO mice, but the difference did not reach statistical significance: 0.86 ± 0.07 and 1.40 ± 0.18 (SE) in wild-type and C-III-KO, respectively (P = 0.06, n = 4 each). To understand the functional implications of these differences for thermogenesis, we subjected mice to a 2-h cold challenge. C-III-KO mice were significantly more tolerant to the cold than were wild-type mice (Fig. 5D). Taken together, these results suggest that BAT from C-III-KO mice is phenotypically and functionally conserved during HFD feeding, in contrast to BAT from wild-type mice. This conclusion is further underscored by the fact that thermogenesis may be more preserved in C-III-KO than wild-type mice during a cold challenge.
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DISCUSSION |
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target gene and plays a role in lipid homeostasis under conditions of diet-induced obesity.
CRBP-III is a small cytosolic protein belonging to the intracellular lipid-binding protein family, which, in addition to the CRBPs (I, II, III), includes cellular RA binding proteins (CRABP I and CRABP II) and fatty acid-binding proteins, such as aP2 (4, 31). CRBPs bind retinol, which can be metabolized intracellularly to RAL and RA, the latter being the major active retinoid (5, 31, 34). In addition, retinol can be esterified to retinyl ester for storage (5, 34). It is thought that the primary role of CRBP-I and CRBP-III is facilitatation of the esterification of retinol to retinyl ester catalyzed by lecithin:retinol acyltransferase (16, 36). Absence of CRBP-I leads to a significant reduction of hepatic retinyl ester storage (16). Similarly, the lack of CRBP-III leads to decreased retinol esterification during lactation (36). In the present study, we expanded the possible roles of CRBP-III. We show that the expression of CRBP-III can be directly modulated by PPAR agonists. In this context, we elucidated that the promoter of CRBP-III harbors functional PPREs for binding of PPAR in vitro and in vivo. PPAR, through its transcriptional regulation of a plethora of genes, is essential for normal adipose tissue development and systemic energy homeostasis (2, 19, 23, 37, 50).
Therefore, to understand the potential role of CRBP-III, we examined gene-targeted mice lacking CRBP-III during diet-induced obesity. Overall, the lack of CRBP-III has distinct effects on 1) whole body energy balance, 2) the oxidative machinery in BAT, and 3) serum free fatty acid levels and white adipose tissue lipid metabolism.
CRBP-III and whole body energy metabolism. In the present study, the lack of CRBP-III appears to affect whole body energy balance. Specifically, lack of CRBP-III resulted in a reduction in food intake and a nonsignificant trend toward reduced energy expenditure and oxygen consumption. Body weight was similar between C-III-KO and wild-type mice fed the HFD, suggesting that decreased food intake in C-III-KO mice may be balanced by decreased energy expenditure. However, fat mass was significantly reduced in C-III-KO compared with wild-type mice. This decrease in fat mass was offset by an increase in lean body mass, resulting in no net change in total mass relative to that of wild-type mice. The increased lean body mass would be predicted to increase the metabolic load in C-III-KO mice, which would, in turn, mitigate against a more significant reduction in oxygen consumption and energy expenditure.
The small, but significant, increase in RER in C-III-KO mice indicates increased carbohydrate, rather than fat, oxidation. This interpretation is consistent with our observation that expression of genes important in fatty acid oxidation was unchanged in skeletal muscle of C-III-KO compared with wild-type mice. Overall, the lack of CRBP-III induces a distinctive profile of behavioral and metabolic consequences, consisting of a change in nutrient partitioning, reduced feeding, decreased fat mass, and increased lean mass, resulting in an overall maintenance of body weight.
CRBP-III and BAT. BAT is characterized by multilocular lipid droplets and a high density of mitochondria in mice fed a chow diet. The central function of BAT is oxidation of fatty acids and dissipation of energy in the form of heat as part of adaptive nonshivering thermogenesis to maintain body temperature in small mammals (7). On the basis of our data, C-III-KO mice likely maintained a better oxidative capacity, as reflected in higher expression of key genes involved in fatty acid oxidation and oxidative phosphorylation in BAT from C-III-KO than wild-type mice fed the HFD. In addition, C-III-KO mice were able to maintain body temperature during a cold challenge, indicating preserved thermogenesis in BAT of C-III-KO compared with wild-type mice. On the basis of this observation, lower body weights of C-III-KO than wild-type mice fed the HFD may be have been expected. In addition to the protection against cold, BAT has been suggested to play an important role in energy metabolism, but a direct role in body weight regulation remains controversial (26). Ablation of BAT or inactivation of all three β-adrenergic receptors rendered mice thermogenically inactive and susceptible to diet-induced obesity (3, 18, 27). In contrast, other mouse models, including inactivation of UCP-1 or failure to induce UCP-1, render chow- or HFD-fed mice cold sensitive but not obese (12, 46–48). Several studies indicate that interscapular BAT may not play a direct role in regulating body weight but, rather, that diet-induced thermogenesis may be mediated by brown adipocytes or increased expression of BAT-specific genes in tissues such as skeletal muscle or white adipose tissue (1, 9, 26, 48). In the present mouse model, we may have a more preserved BAT, but it is not contributing to the phenotype, and/or its contribution may be blunted.
CRBP-III and lipolysis. Significantly lower serum levels of free fatty acids and glycerol in C-III-KO than wild-type mice may have contributed to lower hepatic triglyceride accumulation in C-III-KO than wild-type mice. In addition to fatty acid reesterification to triglyceride in the liver, free fatty acids are used for ketogenesis, leading to increased ketone body levels in the circulation, which are significantly increased in states of insulin resistance and diabetes (15). Serum levels of ketone bodies were significantly lower in C-III-KO than wild-type mice, consistent with the notion that fatty acid flux to the liver is reduced.
Hepatic steatosis during obesity and insulin resistance are consequences of increased rates of lipogenesis in the liver and increased uptake of nonesterified fatty acids released by adipose tissue (6, 17, 24, 44). Excess triglyceride stored as cytosolic lipid droplets in hepatocytes contributes to the development of fatty liver and insulin resistance (6, 40, 41, 45). Since we found no evidence that changes in hepatic lipid synthesis or oxidation contributed to differences in hepatic steatosis, significantly lower serum free fatty acid levels in C-III-KO mice may offer an explanation for the lower hepatic triglyceride accumulation. Serum free fatty acid levels, especially under postabsorptive conditions, are mostly a function of fatty acid release from adipose tissue (13). During insulin resistance, white adipose tissue lipolysis is increased, leading to a rise in the release of free fatty acids and glycerol into the circulation. Consistent with decreased serum fatty acids in C-III-KO mice, in vitro lipolysis assays of adipose tissue from C-III-KO mice indicated diminished efflux of glycerol and free fatty acids from adipose tissue compared with wild-type mice in the basal state.
CRBP-III and PPAR.
Given our findings that CRBP-III is a direct target of PPAR activation, it is surprising that CRBP-III deficiency resulted in attenuation of hepatic steatosis and reduction of free fatty release from the adipose tissue. PPAR agonists, such as thiazolidinediones, have been shown to decrease fatty acid levels in the circulation (32, 39, 51). In addition, adipose-specific deficiency of PPAR abolished this effect of PPAR activation on serum free fatty acids (19). The mechanism that leads to decreased free fatty acid efflux from adipose tissue of C-III-KO mice remains to be determined. It is unlikely that CRBP-III regulates PPAR action, inasmuch as expression of PPAR target genes was not altered in the absence of CRBP-III.
CRBP-III has been known to play a role in the mammary gland by facilitating retinol esterification during lactation (36). On the basis of this observation, we initially hypothesized that CRBP-III may have a similar role maintaining retinyl ester levels in adipose tissue. Therefore, CRBP-III would have an important role in keeping retinoid in the storage form, i.e., retinyl ester, and would effectively lower the amount of RA available for nuclear receptor activation. However, this is unlikely, given the similar levels of retinol and retinyl ester in adipose tissue from C-III-KO and wild-type mice, indicating that the actions of CRBP-III may be independent of its binding to retinol and may also be independent of retinoid metabolism.
Taken together, CRBP-III deficiency confers a protection for multiple pathological aspects in diet-induced obesity, resulting in reduced fatty liver and serum free fatty acid levels and decreased food intake and adiposity.
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FOOTNOTES |
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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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O2) of HFD-fed WT and C-III-KO mice. Values are means ± SE (n = 4 for each genotype). D: respiratory exchange ratio (RER) was significantly higher for C-III-KO than WT mice during light and dark phases. Values are means ± SE (n = 4 for each genotype). E: activity levels were not different between WT and C-III-KO mice. Ambulatory and rearing activity were measured and combined to express total activity. Values are means ± SE (n = 4 for each genotype). *P < 0.05 vs. WT.