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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Objectives: Tristetraprolin (TTP) family proteins (TTP/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate-rich element-containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3-L1 adipocytes compared to RAW264.7 macrophages and investigated insulin effects on the expression of 43 genes in 3T3-L1 adipocytes.

Methods and Procedures: Insulin was added to mouse 3T3-L1 adipocytes. Relative abundance of mRNA levels was determined by quantitative real-time PCR. TTP and ZFP36L1 proteins were detected by immunoblotting.

Results: Zfp36l1 and Zfp36l2 genes were expressed at eight- to tenfold higher than Ttp in adipocytes. Zfp36l3 mRNA was detected at ∼1% of Ttp mRNA levels in adipocytes and its low level expression was confirmed in RAW cells. Insulin at 10 and 100 nmol/l increased Ttp mRNA levels by five- to sevenfold, but decreased those of Zfp36l3 by 40% in adipocytes after a 30-min treatment. Immunoblotting showed that insulin induced TTP but did not affect ZFP36L1 protein levels in adipocytes. Insulin decreased mRNA levels of Vegf and a number of other genes in adipocytes.

Discussion: Insulin induced Ttp mRNA and protein expression and decreased Vegf mRNA levels in adipocytes. Zfp36l3 mRNA was detected, for the first time, in cells other than mouse placenta and extraembryonic tissues. This study established a basis for the investigation of TTP and VEGF genes in the regulation of obesity and suggested that Vegf mRNA may be a target of TTP in fat cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Obesity, one of the most pervasive public health problems, increases risks of developing conditions such as high blood pressure, type 2 diabetes, heart diseases, stroke, gallbladder disease, as well as breast, prostate, and colon cancers (1,2). It has been recognized that obesity is a chronic disease of appetite regulation and energy metabolism. At the cellular level, obesity is characterized as excesses of fat cell numbers, sizes, and/or fat accumulation in adipocytes. Recent studies suggest that obesity is associated with angiogenesis and low levels of chronic inflammation mediated by adipocytokines, macrophage-derived factors, proinflammatory cytokines, and chemokines, but the mechanisms of how inflammation relates to obesity are not completely understood (1,2,3,4).

Several lines of evidence suggest that tristetraprolin/zinc finger protein 36 (TTP/ZFP36) may be involved in obesity. (A complete list of mRNA abbreviations is presented under Table 1 footnote.) First, Ttp mRNA was induced by insulin in mouse 3T3 fibroblasts overexpressing normal human insulin receptors (HIR3.5 cells) (5). Second, Ttp mRNA and protein are induced by fetal bovine serum and differentiation mixtures during differentiation of preadipocytes (6). Third, Ttp mRNA and protein are induced by cinnamon extract and polyphenols in 3T3-L1 adipocytes (7). Fourth, ZFP36/TTP mRNA levels are four- to fivefold lower in visceral fat of obese people with metabolic syndrome compared to those without metabolic syndrome and the gene is located in regions of linkage for the metabolic syndrome (8). Finally, ZFP36/TTP mRNA levels in visceral adipose tissue in women are negatively correlated with fasting insulin levels, the insulin resistance index, and 2-h postglucose insulinemia, and positively correlated with adiponectinemia, suggesting that ZFP36 gene expression in omental adipose tissue may contribute to partial protection against the development of insulin resistance and diabetes (9).

Table 1.  Nucleotide sequences of real-time PCR primers and TaqMan probes
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TTP family proteins are CCCH tandem ZFPs consisting of three members common in mammals (TTP or ZFP36, ZFP36L1 or TIS11B, and ZFP36L2 or TIS11D) and the fourth member in mouse and rat X chromosome but not in humans (ZFP36L3) (10,11). These four proteins are all capable of binding and destabilizing some adenylate uridylate-rich element (ARE)–containing mRNAs including tumor necrosis factor-α (TNF-α) in vitro and in transfected cells (10,11,12). Gene knockout studies have provided evidence for their unique roles in vivo. TTP (ZFP36, TIS11, G0S24, or NUP475) knockout (KO) mice develop a severe inflammatory syndrome (13,14) because the mRNAs encoding TNF-α, granulocyte-macrophage colony-stimulating factor, interleukin-2, immediate-early response 3, and probably others are stabilized in TTP KO mice and in cells derived from them (15,16,17,18). Excessive secretion of these cytokines results in systemic inflammatory responses with erosive arthritis, autoimmunity, and myeloid hyperplasia in the TTP KO mice (13). ZFP36L1 (TIS11B, cMG1, ERF1, BRF1, or Berg36) KO mice develop chorioallantoic fusion defects and embryonic lethality (19). The Zfp36l1 gene in mice is critical for normal fetal-placental development and fetal survival (19), and is required for normal vascularization and post-transcriptionally regulates vascular endothelial growth factor (VEGF) expression (20). Mice with decreased levels of an amino-terminal truncated form of ZFP36L2 (TIS11D, ERF2, or BRF2) exhibit female infertility and disrupted early embryonic development (21). ZFP36L3 was recently identified and appeared to be expressed only in rodents as a placenta-specific protein (11), but the physiological function of ZFP36L3 is unknown. Despite their unique contributions to the regulation of mRNA stability, information is limited on the expression and regulation of TTP family genes in the same types of cells or tissues. In addition, no information is available on expression of ZFP36L3 in cells or tissues other than mouse placenta and extraembryonic tissues.

One of the widely used cell models for obesity research is mouse 3T3-L1 adipocytes (22,23). It can be used for the investigation of the potential roles of TTP family proteins in the regulation of obesity and for the prevention of obesity by plant polyphenols. As an initial step, we investigated the expression and regulation by insulin of TTP family and some related genes in mouse 3T3-L1 adipocytes. Quantitative real-time PCR assays showed that Zfp36l1 and Zfp36l2 mRNAs were the two major forms in adipocytes and that Ttp mRNA is <15% of those of Zfp36l1 and Zfp36l2. Zfp36l3 mRNA was ∼1% of Ttp mRNA levels in adipocytes. Insulin increased Ttp mRNA and TTP protein levels and decreased Vegf mRNA levels in adipocytes. The comparative gene expression of Zfp36l3 was confirmed in mouse RAW264.7 cells.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Cell culture

Mouse 3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were maintained as described (24) in Dulbecco's modified Eagle's medium containing 4,500 mg/l (25 mmol/l) glucose (Gibco BRL, Gaithersburg, MD) supplemented with 10% (vol/vol) fetal bovine serum, 100U/ml penicillin, 100μg/ml streptomycin, and 2 mmol/l l-glutamine (Dulbecco's modified Eagle's medium +). Adipocyte induction was as described with differentiation medium containing the recombinant human insulin expressed in yeast (Sigma Chemical, St Louis, MO), dexamethasone (Sigma), and 1-isobutyl-3-methylxanthine (Sigma) (7). The adipocytes were serum-starved for 4 or 8 h in Dulbecco's modified Eagle's medium before 10 and 100 nmol/l insulin were added to the medium. Cells were collected after 0.5−, 1−, 1.5−, 2−, 3.5−, 4−, and 16-h treatment. Mouse RAW264.7 cells (American Type Culture Collection) were cultured in Eagle's minimum essential medium (Gibco BRL), and were treated with 0.1 μg/ml lipopolysaccharide (Sigma) for various times as described (25).

RNA isolation and cDNA synthesis

Total RNAs were isolated from adipocytes and RAW264.7 macrophages using TRIZOL reagent (Invitrogen, Gaithersburg, MD) as described (7). RNA concentrations and integrity were determined using RNA 6000 Nano Assay Kit and the Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies, Palo Alto, CA) with RNA 6000 Ladder as the standards (Ambion, Austin, TX). Total cDNA synthesis was performed as described (7). The reaction mixture (20μl) contained 5 μg total RNA, 2.4 μg oligo(dT)12–18 primer (Invitrogen), 0.1 μg random primers (Invitrogen), 500 μmol/l deoxyribonucleotide triphosphates, 10 mmol/l DTT, 40 U RNaseOUT (Invitrogen), and 200 U SuperScript II reverse transcriptase (Invitrogen) in 1× first-strand synthesis buffer. The cDNA synthesis reactions were carried out at 42 °C for 50 min.

Real-time PCR analysis

The procedures were similar to those described (7). Real-time PCR primers and TaqMan probes were designed using Primer Express software (Applied Biosystems, Foster City, CA) and were synthesized by Biosearch Technologies (Navato, CA). The mRNA names, GenBank accession numbers, amplicon sizes, and the sequences (5′ to 3′) of the forward primers, TaqMan probes (TET-BHQ1) and reverse primers, respectively, are described in Table 1. The TaqMan reaction mixture (25 μl) contained 25 ng of total RNA-derived cDNAs, 200 nmol/l each of the forward primer, reverse primer, and TaqMan probe, and 12.5 μl of 2× Absolute QPCR Mix (ABgene House, Epson, Surrey). The reactions were performed in 96-well plates in an ABI Prism 7700 real-time PCR instrument (Applied Biosystems, Foster City, CA). The thermal cycle conditions were as follows: 2 min at 50 °C and 15 min at 95 °C, followed by 50 cycles at 95 °C for 15 s and 60 °C for 60 s each cycle. Fluorescence signals measured during amplification were considered positive if the fluorescence intensity was >20-fold greater than the s.d. of the baseline fluorescence (26). The ΔΔCT method of relative quantification was used to determine the fold change in expression as described (7).

Cell extracts and protein concentration determination

Cell extracts were prepared as described (25). In brief, washed adipocytes were lysed in a buffer containing 50 mmol/l NaH2PO4, pH 7.6, 250 mmol/l NaCl, 50 mmol/l NaF, 0.5% Nonidet P-40, 1 mmol/l phenylmethylsulfonyl fluoride, and 0.2% (vol/vol) of protease inhibitor cocktails (104 mmol/l AEBSF, 0.08 mmol/l aprotinin, 2 mmol/l leupeptin, 4 mmol/l bestatin, 1.5 mmol/l pepstatin A, and 1.4 mmol/l E-64) (Sigma). The lysate was centrifuged at 10,000g for 10 min at 4 °C. The 10,000 g supernatant was stored at −20 °C. Protein concentrations were determined using the Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratory, Hercules, CA) following NaOH treatment of the samples as described (7). Bovine serum albumin (Bio-Rad) was used as the protein standard.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting

The procedures were performed essentially as described (27). Briefly, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in TTBS buffer, and successively incubated in buffers containing the primary antibodies and the secondary antibodies for 4 h. Proteins on the immunoblots were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) followed by imaging with BioChemi Image Acquisition and Analysis System (UVP BioImaging Systems; UVP, Upland, CA). The primary antibodies were anti-MBP-mTTP serum (25) and anti-MBP-ZFP36L1 serum (28) raised against the recombinant full-length mouse TTP or ZFP36L1 fused to Escherichia coli maltose-binding protein. The secondary antibodies were affinity-purified goat anti-rabbit IgG (H + L) horseradish peroxidase conjugate with human IgG absorbed (Bio-Rad).

Statistical analyses

The data were analyzed by SigmaStat 3.1 software (Systat Software, Point Richmond, CA) using one-way ANOVA. Multiple comparisons were performed using Duncan or Tukey's Multiple Range Test. Values with different lower case letters displayed above the columns of the figures are significantly different at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Characterization of mouse 3T3-L1 adipocytes

Mouse 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium. Adipocytes were induced with a differentiation medium containing insulin, dexamethasone, and 1-isobutyl-3-methylxanthine. Microscopic observation indicated that ∼90% of the cells accumulated lipid drops (indication of differentiation from preadipocytes to adipocytes) (Figure 1a). Real-time PCR assays showed that these adipocytes contained higher levels of adiponectin and lower levels of leptin mRNAs compared with the expression of Rpl32 mRNA levels (Figure 1b). Adiponectin and leptin are two of the most abundant adipocytokines produced by adipocytes (4,29) and leptin gene expression is lower in cultured 3T3-L1 adipocytes (22). These results suggested that 3T3-L1 adipocytes were differentiated and suitable for further analysis.

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Figure 1. Relative expression of adipocyte-specific markers in mouse 3T3-L1 adipocytes. (a) Differentiated mouse 3T3-L1 adipocytes before treatments. (b) Detection of adiponectin and leptin mRNAs in the differentiated adipocytes. Adipoq, adiponectin; Lep, leptin; Rpl32, ribosomal protein L32.

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Expression profiles of TTP family mRNA levels

There was little information about the relative abundance of TTP family mRNA levels in the same cells or tissues (10) and Zfp36l3 mRNA was only detected previously in mouse placenta and extraembryonic tissues (11). Quantitative real-time PCR analysis indicated that Ttp mRNA levels were <15% of Zfp36l1 or Zfp36l2 mRNA levels in adipocytes (Table 2). Zfp36l3 mRNA was consistently detected in adipocytes, although its expression levels were only 0.7% of those of Ttp mRNA (Table 2).

Table 2.  Relative levels of Ttp/Zfp36 family mRNAs in mouse 3T3-L1 adipocytes and RAW264.7 macrophages
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To confirm the expression of Zfp36l3 and assess the relative levels of TTP family mRNAs in other cell lines, we analyzed TTP family gene expression in mouse RAW264.7 cells. Zfp36l2 mRNA was more abundant than the other three mRNAs in RAW264.7 cells. Zfp36l1, Zfp36l2, and Zfp36l3 mRNA levels were ∼47, 398, and 1.2% of those of Ttp, respectively (Table 2). Zfp36l2 mRNA levels were similar in the two different cell types. Ttp and Zfp36l3 mRNA levels in RAW cells were approximately three- and fourfold of those in adipocytes, respectively. In contrast, Zfp36l1 mRNA levels in adipocytes were about tenfold higher than those in RAW cells (Table 2).

Insulin increases TTP mRNA and TTP protein levels

Insulin was shown to induce Ttp mRNA levels in HIR 3.5 preadipocytes (5), but its effects on adipocytes are unknown. Therefore, mouse 3T3-L1 adipocytes were treated with 0, 10, and 100 nmol/l insulin for 0.5, 1, 1.5, 2, and 4 h following serum starvation for 4 h and treated for 16 h following serum starvation for 8 h. Quantitative real-time PCR analysis indicated that Ttp mRNA was rapidly induced in the cells by 10 and 100 nmol/l insulin treatment, with a 0.5 h induction resulted in approximately five- and sevenfold over the control, respectively (Figure 2a). Ttp mRNA levels then declined but were still twofold over the control after 1 h induction (Figure 2a). However, Ttp mRNA levels were decreased in cells treated with 10 nmol/l insulin for 1.5, 2, and 4 h and were similar between the control and the cells treated with 10 nmol/l for 16 h or treated with 100 nmol/l insulin for 1.5, 2, 4, or 16 h (Figure 2a).

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Figure 2. Effect of insulin on tristetraprolin (TTP) mRNA, TTP protein, and ZFP36L1 protein levels in mouse 3T3-L1 adipocytes. (a) Real-time PCR assay. Total RNAs were isolated from 3T3-L1 adipocytes following treatment with the control or 10 and 100 nmol/l insulin for 0.5, 1, 1.5, 2, and 4 h after serum starvation for 4 h or treatment for 16 h after serum starvation for 8 h. The RNAs were reversely transcribed into cDNAs. Twenty-five nanograms of RNA-derived cDNAs were used for quantitative real-time PCR assays. The ΔΔCT method of relative quantification was used to determine the fold change in expression. The results represent the means and the s.d. from four determinations. Values with different lower case letters displayed above the columns of the figure are significantly different at P < 0.05. (b) Immunoblotting. Proteins in the 10,000 g supernatants of 3T3-L1 adipocytes were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. TTP was detected by immunoblotting with anti-MBP-mTTP and anti-MBP-ZFP36L1 antibodies. Each lane was loaded with 100 µg of protein. Lanes 1 and 4, control; lanes 2 and 5, insulin (10 nmol/l); lanes 3 and 6, insulin (100 nmol/l).

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Immunoblotting results showed that TTP protein was barely detectable in untreated cells (Figure 2b, lane 1), but it was significantly induced by 10 and 100 nmol/l of insulin treatment for 3 h (Figure 2b, lanes 2–3). In contrast, ZFP36L1 protein levels were not significantly affected by insulin treatment under the same conditions (Figure 2b, lanes 4–6).

Insulin regulates TTP homologue mRNA levels

In contrast to Ttp mRNA profiles, real-time PCR analysis showed that Zfp36l1 mRNA levels were slightly but significantly decreased by insulin treatment after 0.5, 1.5, and 2 h, although the 1, 4, and 16 h treatment resulted in similar mRNA levels (Figure 3a). Zfp36l2 mRNA levels were also decreased after 0.5 h insulin treatment (Figure 3b). Zfp36l3 mRNA was decreased by 40% in adipocytes after 0.5 h treatment with 10 and 100 nmol/l of insulin (Figure 3c). Similar decreases of Zfp36l3 mRNA levels in adipocytes by insulin treatments were observed after longer treatment but returned to the control levels after 4 and 16 h treatment (Figure 3c).

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Figure 3. Effect of insulin on Zfp36l1/Tis11b, Zfp36l2/Tis11d, and Zfp36l3 mRNA levels in mouse 3T3-L1 adipocytes. RNA isolation, cDNA synthesis, real-time PCR assays, and statistical analyses were described in Figure 2 legend. (a) Zfp36l1/Tis11b mRNA, (b) Zfp36l2/Tis11d mRNA, (c) Zfp36l3 mRNA.

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Insulin regulates TTP family protein-targeted mRNA levels

To test the functional consequences of insulin-elevated TTP expression levels in mouse 3T3-L1 adipocytes, we analyzed the levels of Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Vegf mRNAs in insulin-treated adipocytes. Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Vegf mRNAs are shown to be the targets of TTP family proteins by in vivo and/or in vitro experiments. Quantitative real-time PCR assays indicated that Vegfa mRNA levels were decreased by ∼30–50% by 10 and 100 nmol/l insulin treatments for 0.5–4 h after 4 h serum starvation (Figure 4a). Vegfa mRNA levels were also significantly decreased by 100 nmol/l insulin treatment for 16 h, although its level was slightly increased in adipocytes treated with 10 nmol/l insulin under the same conditions (Figure 4a). Vegfb mRNA levels were also significantly decreased by the same treatments (Figure 4b). Csf2/Gm-csf mRNA levels were significantly decreased by 100 nmol/l insulin treatment for 30 min or 10 nmol/l insulin treatment for 120 min, but the levels were not significantly affected by either 10 nmol/l insulin treatment for 30 min or 100 nmol/l insulin treatment for 120 min (Table 3). However, Csf2/Gm-csf mRNA levels in adipocytes were significantly increased by 100 nmol/l insulin treatment for 4 and 16 h treatment (data not shown). Ptgs/Cox2 mRNA levels were not significantly affected by insulin treatment for 30 min but were significantly increased by insulin treatment for 120 and 210 min (Table 3). Tnf mRNA levels were too low to be reliably quantified (Table 3).

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Figure 4. Effect of insulin on vascular endothelial growth factor (VEGF) mRNA levels in mouse 3T3-L1 adipocytes. RNA isolation, cDNA synthesis, real-time PCR assays, and statistical analyses were described in Figure 2 legend. (a) Vegfa mRNA, (b) Vegfb mRNA.

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Table 3.  Effects of insulin on mRNA levels of 36 genes encoding proinflammatory family, Glut/Slc2a family, and insulin signaling pathway components in mouse 3T3-L1 adipocytes
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Insulin regulates adipocytokine mRNA levels

The expression and insulin regulation of adiponectin, leptin, and leptin receptor genes were analyzed by real-time PCR (Table 3). Adiponectin mRNA levels were significantly decreased up to 30% by insulin treatment for 30 min, but they were not significantly affected by insulin treatment for 120 and 210 min (Table 3). Leptin mRNA levels were also significantly decreased by ∼40% by insulin treatment for 30 min, but they were significantly increased by two- to fourfold by insulin treatment for 120 min (Table 3). Leptin receptor mRNA levels were not significantly affected by insulin treatment (Table 3).

Insulin regulates the expression of genes involved in insulin signaling pathway

The expression and insulin regulation of 20 genes involved in glucose uptake and insulin signaling were analyzed by real-time PCR (Table 3). Insulin treatment for 30 min decreased all but Ins1, Ins2, Pik3r1, Slc2a1/Glut1, and Sos1 mRNA levels. Insulin treatment for 120 min increased Slc2a1/Glut1, Ins1, and Pik3r1 mRNA levels but decreased Irs1, Irs2, Gys1, Igf2r, and Slc2a3/Glut3 mRNA levels in adipocytes (Table 3). Insulin treatment for 210 min increased Pik3r1 but decreased Igf1r, Igf2, Igf2r, Insr, Irs1, Irs2, and Slc2a4/Glut4 mRNA levels (Table 3). Slc2a1/Glut1 mRNA levels were increased but those of Slc2a4/Glut4 and Insr were decreased by insulin in adipocytes after 4 and 16 h treatment (data not shown). Pik3cb, Shc1, and Slc2a2/Glut2 mRNA levels were too low to be reliably quantified (Table 3).

Insulin regulates the expression of other inflammation-related genes

Expression of the other 10 selected genes in adipocytes was analyzed by real-time PCR (Table 3). Elavl1/Hur/Hua gene codes for HuR, an mRNA stabilizing protein. Its expression was significantly decreased up to 25% by 30 min treatment but was increased to ∼45% after 120 and 210 min treatments (Table 3). Il6 mRNA levels were shown a similar pattern as those of Hur. Crp and Ifng mRNA levels were significantly increased after 30 min treatment with 10 nmol/l insulin but were decreased by insulin at 100 nmol/l for 30 min (Table 3). Csf3/G-csf and Serpine1/Pai1 mRNA levels were increased by insulin treatments for 30–120 min (Table 3). Il12b mRNA levels were not affected by insulin under most of the experimental conditions and Il1a mRNA levels were too low to be reliably quantified (Table 3). Finally, App and Tau mRNA levels were decreased by insulin treatments for 30 min in the adipocytes (Table 3).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Obesity is recognized as a chronic disease resulting from excess accumulation of fats in adipocytes. Recent studies suggest that obesity is associated with chronic inflammation mediated by adipocytokines and proinflammatory cytokines (1,3,4,30). Evidence suggests that anti-inflammatory mRNA destabilizing protein TTP/ZFP36 may be involved in obesity development, because Ttp mRNA is induced by insulin in mouse 3T3 fibroblasts (5), by fetal bovine serum and differentiation mixtures during differentiation of preadipocytes (6), and by cinnamon extract and polyphenols in 3T3-L1 adipocytes (7). Furthermore, Ttp mRNA levels are decreased in visceral fat of obese people with metabolic syndrome and the Ttp gene is linked to metabolic syndrome (8). Finally, Ttp mRNA levels in visceral adipose tissue of women are negatively correlated with fasting insulin levels, the insulin resistance index, and 2-h postglucose insulinemia, and positively correlated with adiponectinemia (9). We are interested in the roles of TTP family proteins in the regulation of obesity. As an initial step, we investigated the expression and regulation by insulin of 43 genes including TTP family and some related genes in mouse 3T3-L1 adipocytes, a widely used cell model for obesity research. Our results demonstrated that insulin increased Ttp mRNA and TTP protein levels and decreased Vegf mRNA levels in adipocytes, and that Zfp36l3 expression was detected in mouse 3T3-L1 adipocytes and RAW264.7 cells.

TTP is the product of the immediate-early response gene Zfp36 in the mouse (ZFP36 in humans) (5). TTP binds to ARE in some mRNAs and destabilizes those transcripts encoding proteins such as TNF-α (15,27,31,32), granulocyte-macrophage colony-stimulating factor (16), immediate-early response 3 (18), and VEGF (33). The mRNA binding activity of TTP is zinc dependent (27,31) and is regulated by post-translational phosphorylation (31,34,36). Ttp mRNA and/or TTP protein levels are increased in mammalian cells by a wide range of agents including growth factors (insulin, insulin-like growth factor I, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, and fetal calf serum) (5,37), cytokines (TNF-α, granulocyte-macrophage colony-stimulating factor, and interferon-γ) (15,37,39), zinc (40), cinnamon extract and polyphenols (7), green tea extract (41), tumor promoters (phorbol 12-myristate 13-acetate and tetradecanoyl phorbol acetate) (5,38), bacterial endotoxin lipopolysaccharide (25), and viral infection (42).

Among the 43 genes whose expression levels were evaluated by real-time PCR, Ttp gene expression was the most significantly increased by insulin treatment in mouse 3T3-L1 adipocytes. Insulin at physiological concentrations (10 and 100 nmol/l) increased Ttp mRNA levels up to sevenfold with a 30-min treatment and that resulted in TTP protein induction. Because Ttp gene expression is directly related to obesity (8,9), the above results suggest potential beneficial effects of acute treatment with insulin in improving obesity and related diseases. The five- to sevenfold increases of Ttp mRNA levels in adipocytes treated with 10–100 nmol/l insulin for 30 min in our study is much larger than any of those reported values generated by microarray analysis of 3T3-L1 adipocytes (43). In that study, the expression of 3,718 nonredundant genes evaluated from 10,117 probes and 38 genes measured by real-time PCR assays from 3T3-L1 adipocytes treated with 1,000 nmol/l insulin for 2 days were not increased by >2-fold by microarray analysis and >1.5-fold by real-time PCR assays (43). The inability to detect larger than twofold changes in gene expression by insulin was probably due to the extended length of insulin treatment, because our study showed a rapid decline of Ttp mRNA levels to baseline levels after 90 min of induction. It is also possible that high level induction is due to the concentration of glucose (25 mmol/l) used in our culture medium, because the study by Wang et al. (43) reported that the insulin effect was more pronounced with higher glucose (15.75 mmol/l) than lower glucose (4.5 mmol/l) (43). Insulin induced Ttp mRNA rapidly and transiently in adipocytes. The pattern of insulin induction in adipocytes reported here is similar to that reported in a previous study using HIR3.5 preadipocytes (5). This transient induction pattern is different from the sustained induction pattern of Ttp mRNA or TTP protein through cinnamon polyphenolic extract in 3T3-L1 adipocytes (7) or through lipopolysaccharide in RAW264.7 cells (25). In addition, our results contrast with one study that reported that Zfp36l1, but not Ttp, is induced by insulin and insulin-like growth factor I in rat intestinal epithelial (RIE1) cell lines (44). This difference is probably due to the differential response of Ttp family gene expression in the two cell types.

The other important finding reported here was that insulin significantly decreased Vegf mRNA levels in mouse 3T3-L1 adipocytes. VEGF is a mitogenic and angiogenic factor involved in tumor progression, collateral vessel formation in ischemic tissues, inflammation, as well as in the development of diabetic retinopathy (45,46). VEGF is also a key mediator of adipogenesis in obesity (47) and its binding to VEGF receptor is inhibited by plant polyphenols, such as green tea catechin (48). Plasma VEGF levels in obese mice and in athymic mice implanted with 3T3-L1 adipocytes in visceral fat are significantly higher than control and related to adiposity (49). Plasma VEGF levels in patients with type 2 diabetes treated with insulin are 16% less than those treated with diet alone, and Vegf mRNA levels in 3T3-L1 adipocytes are increased by troglitazone and rosiglitazone, two antidiabetic TZD compounds shown to cause weight gain and edema (50). The decreases of Vegf mRNA levels in 3T3-L1 adipocytes treated with 10 and 100 nmol/l insulin reported here are in agreement with the study of Emoto et al. (50), but contrast with two reports showing increases of Vegf mRNA levels in freshly isolated rat white adipocytes treated with 100 nmol/l insulin for 4–24 h (51) and freshly prepared human adipocytes treated with 10 nmol/l insulin for 48 h (52). Vegf expression was also reported to be increased in the retinae of diabetic rats by acute intensive insulin therapy (53). It was also documented that Vegf mRNA levels in NIH3T3 fibroblasts are increased by sixfold with 100 nmol/l insulin treatment for 4–24 h and this effect is inhibited by PI3K inhibitor wortmannin; however, they did not report if insulin also induced Vegf expression in adipocytes under their experimental conditions (54). Another study showed that Vegf expression in human retinal epithelial cells is due to insulin activation of transcription factor hypoxia-inducible factor 1 through a PI3K/target-of-rapamycin-dependent pathway (55). These differences on insulin-regulated Vegf gene expression among the different studies may be due to cell types, insulin treatments, or methods used. For example, a number of inflammatory factors are induced in freshly isolated primary adipose cells from mouse epididymal fat pads (56). As discussed above, different length and concentrations of insulin treatments also resulted in different patterns of gene expression between this study and the previous study (43).

Vegf mRNA is destabilized by TTP, TIS11B (ZFP36L1), and TIS11D (ZFP36L2) in intact cells (57) and is stabilized in TIS11B KO mice (20). It was reported recently that TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on Vegf mRNA because TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice (33). Whether insulin-induced Vegf mRNA reduction reported here is directly mediated by insulin-induced TTP in the 3T3-L1 adipocytes requires further detailed analysis. The increases of Ttp and decreases of Vegf gene expression by insulin in adipocytes suggest a potential role of insulin in obesity prevention and care by restricting blood supply to adipose tissue (4), possibly through TTP protein-mediated Vegf mRNA reduction. Proof of this hypothesis requires further investigation.

Since the initial discovery that Ttp gene expression was induced by insulin and other mitogens, a number of other agents have been shown to increase Ttp mRNA and protein levels in mammalian cells. However, little is known about the concurrent regulation of the expression of the three homologs of Ttp in the same cell type. In this study, we analyzed the expression profile of Ttp family members in both 3T3-L1 adipocytes and RAW264.7 macrophages and their regulation by insulin in mouse 3T3-L1 adipocytes. Several points of results are worth of discussion. First, Zfp36l3 expression was detected in mouse adipocytes and RAW264.7 macrophages. Its mRNA levels, however, were ∼1% of Ttp mRNA levels and even much less than those of Zfp36l1 or Zfp36l2 mRNA levels in both cell types. Zfp36l3 mRNA was previously identified by northern blot and as a placenta-specific protein only in mice (11). We reported that Zfp36l3 mRNA was undetectable in rat liver and muscle (41). Sequence similar to Zfp36l3 protein has been reported in GenBank for Pan troglodytes (chimpanzee), suggesting that Zfp36l3 may be present in multiple species and is differentially expressed in different cell types. The physiological function of ZFP36L3 requires further investigation. Second, the expression patterns of Ttp family genes in the two mouse cell lines were different from that in rat liver and muscle. Zfp36l1 and Zfp36l2 mRNAs are the major forms in adipocytes, but Zfp36l2 mRNA is the major form in RAW cells. Ttp and Zfp36l1 mRNAs are the two major forms in the liver and muscle, and Ttp, Zfp36l1, and Zfp36l2 mRNA levels are more abundant in liver than those in muscle, while Zfp36l3 mRNA levels are below detection (41). In contrast, the expression profiles of Ttp family mRNAs in uninduced cultured human monocytes are similar (58). Third, insulin regulation of Ttp family gene expression is different among its members in adipocytes. Insulin induced Ttp mRNA rapidly and transiently in adipocytes, while Zfp36l1 mRNA levels were slightly decreased. This contrasts with two previous reports showing the induction of both Ttp and Zfp36l1 mRNAs by insulin in RIE1 cells (44) and by lipopolysaccharide in human monocytes (58). Zfp36l3 gene expression was only modestly regulated by insulin in adipocytes.

In summary, baseline Ttp mRNA levels are much less than those of Zfp36l1 and Zfp36l2 in both adipocytes and RAW cells, and insulin increased Ttp gene expression rapidly and transiently but decreased Vegf mRNA levels in adipocytes. Zfp36l3 gene expression was detected for the first time in cells other than in mouse placenta and extraembroynic tissues, although mRNA levels were ∼1% of Ttp. This study established a foundation for the investigation of Ttp/Zfp36 family and Vegf genes in the regulation of obesity in an adipocyte cell model system in vitro, and suggested that reduced Vegf gene expression is related to obesity prevention by insulin. These studies could link the prevention of obesity and type 2 diabetes by cinnamon polyphenols and green tea that increase Ttp mRNA levels in 3T3-L1 adipocytes (7), macrophages (59), and rats (41) to regulation of Vegf mRNA stability.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

We appreciate Perry J. Blackshear (National Institute of Environmental Health Sciences, National Institutes of Health) for his generous permission of using the antibodies H.C. produced in his laboratory, and Andrew Greenberg and Mohsen Meydani (US Department of Agriculture-Agriculture Research Service Human Nutrition Research Center on Aging at Tufts University) for stimulating discussion. We thank Meghan Kelly and Noella Bryden for technical assistance and Allison Yates for helpful comments on the manuscript. This work was supported in part by US Department of Agriculture-Agriculture Research Service Human Nutrition Research Program. A preliminary report of this study was presented at the Experimental Biology 2007 in Washington, DC, on 28 April to 2 May 2007 and the Obesity Society 2007 Annual Meeting in New Orleans, LA, on 20–24 October.

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  1. Top of page
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  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES
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