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© 2008 American Society for Nutrition J. Nutr. 138:833-840, May 2008

Biochemical, Molecular, and Genetic Mechanisms

Cinnamon Polyphenol Extract Affects Immune Responses by Regulating Anti- and Proinflammatory and Glucose Transporter Gene Expression in Mouse Macrophages1–3,

Heping Cao*, Joseph F. Urban, Jr and Richard A. Anderson

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705-2350

* To whom correspondence should be addressed. E-mail: heping.cao{at}ars.usda.gov; peacetd2003{at}yahoo.com.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Tristetraprolin (TTP/zinc finger protein 36) family proteins have antiinflammatory effects by destabilizing proinflammatory mRNA. TTP expression is reduced in fats of obese people with metabolic syndrome and brains of suicide victims and is induced by insulin and cinnamon polyphenol extract (CPE) in adipocytes, by lipopolysaccharide (LPS) in macrophages, and by green tea polyphenol extract in rats. CPE was reported to improve immune function against microorganisms, but the mechanism is unknown. This study tested the hypothesis that CPE regulates immune function involving genes encoding TTP, proinflammatory cytokines, and glucose transporter (GLUT) families and compared the effects of CPE to those of insulin and LPS in mouse RAW264.7 macrophages. CPE increased TTP mRNA and protein levels, but its effects were less than LPS. CPE (100 mg/L, 0.5–4 h) increased TTP and tumor necrosis factor (TNF) mRNA levels by up to 2- and 6-fold that of the control, respectively, and the base level of TTP was 6-fold that of TNF. LPS (0.1 mg/L, 4 h) increased TTP, TNF, granulocyte-macrophage colony-stimulating factor, cyclooxgenase-2, and interleukin 6 mRNA levels by 39–1868 fold. CPE and LPS increased GLUT1 expression (the major GLUT form in macrophages) to 3- and 2-fold that of the control, respectively. Insulin (100 nmol/L, 0.5–4 h) did not exhibit major effects on the expression of these genes. CPE increased TTP expression more rapidly than those of proinflammatory cytokines and the net increases of TTP mRNA levels were larger than those of proinflammatory cytokines. These results suggest that CPE can affect immune responses by regulating anti- and proinflammatory and GLUT gene expression.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Tristetraprolin [TTP4/zinc finger protein 36 (ZFP36)/tetradecanoyl phorbol acetate (TPA)-induced sequence 11 (TIS11)/ G0/G1 switch gene 24 (G0S24)/nuclear protein 475 (NUP475)] family proteins regulate gene expression at the post-transcriptional level. TTP family proteins consist of 3 well-known members in mammals [ZFP36 or TTP, ZFP36L (ZFP36-like)-1 or TIS11B, and ZFP36L2 or TIS11D] and the 4th member in mouse and rat but not in humans (ZFP36L3) (1,2). TTP is the product of the immediate-early response gene Zfp36 in the mouse and ZFP36 in humans (3–5). It binds to AU-rich elements in some mRNA and destabilizes those transcripts encoding proteins, such as tumor necrosis factor-{alpha}(TNF{alpha}) (6–9), granulocyte-macrophage colony-stimulating factor/colony-stimulating factor 2 (GM-CSF/CSF2) (10,11), and cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX2/PTGS2) (12). The mRNA encoding TNF{alpha} and GM-CSF are stabilized in TTP knockout mice and in cells derived from them (8,10). Excessive levels of these cytokines in TTP knockout mice results in a severe systemic inflammatory syndrome, including arthritis, autoimmunity, and myeloid hyperplasia (13,14). Up-regulation of TTP reduces inflammatory responses in macrophages (15). These lines of evidence support the conclusion that TTP is an antiinflammatory protein.

Agents that induce TTP gene expression may have potential therapeutic value for the prevention and/or treatment of inflammation-related diseases. TTP mRNA and protein levels are induced by a number of agents, including growth factors (3,4), cytokines [TNF{alpha}, GM-CSF, and interferon-{gamma} (IFN{gamma})] (4,5,8,15), zinc (16), and plant nutritional products (cinnamon and green tea) (17,18). However, TTP gene expression is also induced by tumor promoters (3,5), bacterial endotoxin lipopolysaccharide (LPS) (8,19), and viral infection (20). The fact that most of these agents also increase the expression levels of proinflammatory cytokines such as TNF{alpha} in the same cells and/or tissues (8) may limit the therapeutic potential of these agents. Therefore, it is important to search for other agents with the potential to favor antiinflammatory and reduce proinflammatory gene expression.

Common spices (cinnamon, cloves, turmeric, and bay leaves) display insulin-like activity in vitro (21). Polyphenolic polymers from an aqueous extract of commercial cinnamon increase glucose metabolism in an epididymal fat cell assay (22). Cinnamon polyphenol extract (CPE) has other potential benefits for human health. For example, CPE was reported to exhibit activities against microorganisms (23), suggesting a potential role of CPE in regulating immune function; but the mechanism is unknown. Because CPE induces antiinflammatory TTP gene expression in mouse 3T3-L1 adipocytes (17), TTP is potentially involved in CPE-mediated immune regulation.

Mouse RAW264.7 macrophages are widely used as a cell model for inflammation research. TTP gene expression has been investigated extensively using this cell line (19,24). TTP protein was rapidly induced by LPS and accumulated in the cytosol of RAW264.7 cells (19). However, whether LPS also regulates the expression of the 3 TTP homologs in RAW264.7 macrophages is not known. In addition, glucose is the dominant metabolic substrate critically important for the host response to injury and infection (25,26). Glucose uptake is facilitated by glucose transporter (GLUT) family proteins in mammalian cells and GLUT1 is the major form in macrophages (27). We therefore utilized this model to evaluate the antiinflammation properties of CPE.

The objective of this study was to test the hypothesis that CPE regulates immune function involving genes encoding TTP, proinflammatory cytokines, and GLUT families, and compare the effects of CPE to those of insulin and LPS in RAW264.7 macrophages.


   Materials and Methods
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 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials and reagents. Mouse RAW264.7 macrophages were from American Type Culture Collection. DMEM was from Gibco BRL. Protein Assay Dye Reagent Concentrate and bovine serum albumin were from Bio-Rad. LPS and recombinant human insulin expressed in yeast were from Sigma. TRIzol reagent and SuperScript II RT were from Invitrogen. RNA 6000 Ladder was from Ambion. PCR primers and TaqMan probes were designed using Primer Express software (Applied Biosystems) and synthesized by Biosearch Technologies. Absolute QPCR mix was from ABgene House.

    CPE. Water-soluble CPE was prepared as described previously (17,22). Briefly, ground cinnamon (Cinnamomum burmannii) was suspended in 0.1 mol/L acetic acid and autoclaved for 15 min at 15 psi. The supernatant was mixed with ethanol and refrigerated overnight followed by filtration through glass wool and Whatman no. 1 filter paper. The ethanol was removed by rotoevaporation and the remaining solution was freeze-dried. The dried CPE was reconstituted at 100 g/L in 100% dimethylsulfoxide (DMSO). The compositions of CPE mixture were analyzed by HPLC using a Symmetry Prep C18 column and were similar to those reported (22,23).

    Cell culture and cell extracts. Mouse RAW264.7 macrophages were maintained as described (19) at 37°C in a humidified incubator with 5% CO2 in DMEM containing 4500 mg/L (25 mmol/L) glucose supplemented with 10% (v:v) fetal bovine serum, 0.1 million units/L penicillin, 100 mg/L streptomycin, and 2 mmol/L L-glutamine. RAW cells were treated with 100 mg/L CPE (corresponding to 0.1% DMSO in the culture medium), 100 nmol/L insulin, or 0.1 mg/L LPS for 0, 15, 30, 45, 60, 90, 120, 180, and 240 min. The selection of the doses of CPE, insulin, and LPS was based on previous studies showing effective stimulation of TTP expression in adipocytes (17,28) and macrophages (19). Cell extracts were prepared as described (19). Protein concentrations in the 10,000-g supernatant were determined with modifications using the Protein Assay Dye Reagent Concentrate following NaOH treatment of the samples (7). Bovine serum albumin was used as the protein standard.

    SDS-PAGE and immunoblotting. SDS-PAGE and immunoblotting were performed as described (6) using 10% SDS-PAGE and nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and successively incubated in buffers containing the primary antibodies (1:1000 dilution) for 4 h and the secondary antibodies (1:10,000 dilution) for 1.5 h. Proteins on the immunoblots were detected using SuperSignal West Pico Chemiluminescent substrate (Pierce) followed by imaging with BioChemi Image Acquisition and Analysis System (UVP BioImaging Systems). The primary antibodies were anti-maltose binding protein-TTP and anti-MBP-ZFP36L1 sera raised against recombinant Escherichia coli maltose-binding protein fused to the full-length mouse TTP (19) or ZFP36L1/TIS11B (29). The secondary antibodies were affinity-purified goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate with human IgG absorbed (Bio-Rad).

    RNA extraction and real-time PCR analysis. Total RNA were isolated from mouse RAW264.7 cells treated with DMSO, CPE, insulin, or LPS using TRIzol reagent (Invitrogen). RNA concentrations and integrity were determined using RNA 6000 Nano Assay kit and the Bioanalyzer 2100 (Agilent Technologies) with RNA 6000 Ladder as the standards. The cDNA were synthesized from total RNA using SuperScript II RT as described (17). The gene 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 Supplemental Table 1. TaqMan reaction mixtures and the thermal cycle conditions were identical to those described (17). PCR were performed in 96-well plates in an ABI Prism 7700 real-time PCR instrument (Applied Biosystems). The {Delta}{Delta}cycle of threshold (CT) method of relative quantification was used to determine the fold-change in expression (17).

    Statistical analyses. The real-time PCR data from the control, CPE, and insulin treatments with or without those from LPS treatment were analyzed separately by SigmaStat 3.1 software (Systat Software) using ANOVA or ANOVA on ranks. Multiple comparisons were performed with the Student-Newman-Keuls method. Differences were considered significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Relative expression levels of TTP family, GLUT family, and other selected genes in mouse RAW264.7 macrophages. Quantitative real-time PCR was used to evaluate the relative expression levels of TTP family genes (Ttp/Zfp36, Zfp36l1, Zfp36l2, and Zfp36l3) and other selected genes in mouse RAW264.7 cells (Table 1). ZFP36L2 mRNA was the most abundant molecule among the tested mRNA and its levels were >4-fold that of TTP in RAW264.7 cells. TTP mRNA levels (the 4th most abundant among those tested targets) were ~2- to 100-fold those of ZFP36L1 and ZFP36L3 (Table 1). Hu antigen R/embryonic lethal, abnormal vision-like 1 (HuR/ELAVL1) mRNA levels were the 2nd most abundant and were ~2-fold that of TTP. Vascular endothelial growth factor (VEGF) B mRNA levels were 19% less than those of TTP. TTP mRNA levels were ~6- to 10,000-fold those of TNF, GM-CSF, COX2, VEGFA, interleukin (IL) 6, and IFN{gamma} in untreated RAW cells (Table 1).


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  		TABLE 1 Relative levels of TTP family, GLUT family, and other selected mRNA in untreated mouse RAW264.7 macrophages12

 
We used PCR assays to evaluate GLUT family, insulin receptor (INSR), and glycogen synthase kinase 3-β (GSK3B) mRNA levels in macrophages. GLUT1 mRNA was the 3rd most abundant among those tested, was the most abundant form of GLUT family in macrophages, and was 8- and 1300-fold those of GLUT3 and GLUT4 mRNA, respectively (Table 1). GLUT2 mRNA was undetectable by the PCR assay with 50 cycles (Table 1). INSR and GSK3B mRNA were detected in RAW macrophages, although their levels were much less than those of GLUT1 (Table 1).

    CPE and LPS but not insulin increased antiinflammatory TTP mRNA and protein levels. Antiinflammatory TTP expression is induced by CPE and insulin in mouse 3T3-L1 adipocytes (17,28) and by LPS in RAW264.7 macrophages (19). We compared the effects of CPE, insulin, and LPS on TTP gene expression in macrophages, a well-established cell type for inflammatory response. RT-PCR analysis showed that CPE rapidly increased TTP mRNA levels in mouse RAW264.7 cells. TTP mRNA levels in cells treated with 100 mg/LCPE for 30–240 min were ~50–100% greater than those in the corresponding controls (Table 2). Insulin did not exhibit any major effect on TTP mRNA levels in RAW cells, except that its levels were slightly lower when cells were treated for 120 min (Table 2). LPS had a much larger effect on TTP gene expression in RAW cells. TTP mRNA levels in cells treated with 0.1 mg/L LPS for 30–240 min were 9- to 39-fold of the controls (Table 2).


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  		TABLE 2 Effects of CPE, insulin, and LPS on TTP family, VEGF, and HuR mRNA levels in mouse RAW264.7 macrophages12

 
Immunoblotting was used to investigate if CPE could induce TTP protein levels in RAW cells. TTP increased in cells treated with 100 mg/L CPE for 90–180 min (Fig. 1, lanes 6–8). Under similar blotting and exposure conditions, LPS increased TTP protein levels in RAW cells with much earlier and greater magnitude than CPE induction (Fig. 1, lanes 9–14). However, TTP protein levels were below detection in cells treated with insulin for the same length of time (data not shown).


Figure 1
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  		FIGURE 1  CPE and LPS but not insulin increased TTP protein levels in mouse RAW264.7 macrophages. Protein extracts were isolated from RAW cells treated with DMSO control (0.1%), insulin (100 nmol/L), CPE (100 mg/L), and LPS (0.1 mg/L) for various times. Proteins in the 10,000-g supernatants were separated by 10% SDS-PAGE. TTP was detected by immunoblotting with anti-MBP-TTP serum. Each lane was loaded with 100 µg of protein. Lane 1, DMSO control; lanes 2–8, CPE treatment; lanes 9–14, LPS treatment. Insulin did not affect TTP protein levels in the same cells (data not shown).

 
    CPE, LPS, and insulin effects on TTP homologs, VEGF, and HuR gene expression. TTP homologs (ZFP36L1, ZFP36L2, and ZFP36L3) are also capable of destabilizing proinflammatory cytokine mRNA molecules in transfected cells (1). We therefore analyzed the effects of CPE, LPS, and insulin on the regulation of these 3 TTP homologs in RAW cells. The effects of CPE on the expression of TTP homologs in RAW cells were generally small. CPE decreased ZFP36L1 mRNA levels by ~30% in cells treated for 30 and 240 min (Table 2). ZFP36L2 mRNA levels were decreased by 20% in cells treated for 30 min (Table 2). ZFP36L3 mRNA levels were also slightly decreased in RAW cells after 120- and 240-min treatment (Table 2). LPS did not affect the expression of ZFP36L1 gene but decreased mRNA levels of ZFP36L2 in cells treated for 120 and 240 min and those of ZFP36L3 gene in cells treated for 120 min (Table 2). Insulin did not affect the expression of the 3 TTP homologs in RAW cells (Table 2). ZFP36L1 protein was barely detected in RAW cells treated with the 3 agents (data not shown).

We also analyzed the expression of 2 additional classes of genes in RAW cells:. HuR/ELAVL1 (an mRNA-stabilizing protein with distinct but overlapping binding specificity with TTP) (30) and VEGF (a proangiogenic cytokine as a target of TTP family proteins) (31). CPE increased VEGFA mRNA levels only in cells treated for 240 min, but LPS and insulin did not affect VEGFA gene expression (Table 2). CPE, LPS, and insulin all decreased VEGFB mRNA levels in cells treated for 120 min, but LPS also decreased in those treated for 240 min (Table 2). CPE, LPS, and insulin did not affect the levels of mRNAs encoding HuR in RAW cells (data not shown).

    CPE and LPS but not insulin increased TTP-targeted proinflammatory cytokine mRNA levels. TTP is a mRNA-destabilizing factor for a number of immediate-early response genes, such as TNF and GM-CSF genes (1). Therefore, selected proinflammatory cytokine gene expression was investigated in RAW264.7 macrophages after treatment with CPE, LPS, and insulin. TNF mRNA levels in RAW cells treated with 100 mg/L CPE for 30–240 min were 120–620% of those in the untreated controls (Table 3). Unlike CPE, insulin did not exhibit any effect on TNF mRNA levels in RAW cells (Table 3). LPS possessed a much stronger effect than CPE on TNF gene expression in RAW cells. TNF mRNA levels in cells treated with 0.1 mg/L LPS for 30–240 min were 11- to 49-fold of the controls (Table 3).


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  		TABLE 3 Effects of CPE, insulin, and LPS on TNF, GM-CSF, COX2, IL6, and IFN{gamma} mRNA levels in mouse RAW264.7 macrophages12

 
Both CPE and insulin did not affect GM-CSF mRNA levels in RAW macrophages (Table 3). LPS stimulation on GM-CSF gene expression was slow but with profound effects in RAW cells. GM-CSF mRNA levels in cells treated with 0.1 mg/L LPS for 60–240 min were 5- to 1260-fold of the controls (Table 3).

CPE increased COX2 and IL6 mRNA levels in RAW cells. CPE (100 mg/L) treatment for 60–240 min increased COX2 and IL6 mRNA levels by ~200–340% and 160–350% of the controls, respectively (Table 3). These increases were significant when the data from the control, CPE, and insulin treatments were analyzed separately (data not shown) but were not significant if the data from LPS treatment were included in the multiple comparison statistical analyses (Table 3). Insulin (100 nmol/L, 0.5–4 h) did not affect the expression of COX2 or IL6 gene (Table 3). COX2 and IL6 gene expression in RAW cells was stimulated by LPS more than by CPE. The mRNA levels of COX2 and IL6 in RAW cells treated with 0.1 mg/L LPS for 60–240 min were 24- to 532-fold and 24- to 1868-fold of those in the control cells, respectively (Table 3).

    CPE and LPS but not insulin increased GLUT1 mRNA levels. Glucose is the major metabolic substrate critically important for host immunity (25,26). Glucose uptake in mammalian cells is facilitated by GLUT family proteins (27). Because none of the gene expression was apparently affected by insulin in the above PCR assays, we therefore analyzed the effects of insulin on the regulation of the GLUT family and related gene expression in RAW cells compared with those of CPE and LPS. Insulin did not affect the expression of GLUT family genes (Table 4). However, both CPE and LPS increased GLUT1 mRNA levels (the major GLUT mRNA in RAW cells; refer to Table 1) in RAW cells after 120 min treatment (Table 4). After 240 min treatment, CPE and LPS increased GLUT1 mRNA levels in RAW cells by more than 3- and 2-fold those of the control, respectively (Table 4). CPE did not affect the mRNA levels of GLUT3 (Table 4) but increased GLUT4 mRNA levels by >2-fold after 240 min treatment (Table 4). However, LPS decreased the mRNA levels of GLUT3 and GLUT4 (Table 4) in RAW cells.


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  		TABLE 4 Effects of CPE, insulin, and LPS on GLUT family, INSR, and GSK3B mRNA levels in mouse RAW264.7 macrophages12

 
INSR and GSK3B were selected to determine whether these 3 agents affected the components in the insulin signaling pathway. Insulin increased INSR mRNA levels in cells treated for 240 min (Table 4). CPE decreased INSR mRNA levels only in cells treated for 120 min, but LPS decreased those cells treated for 120 and 240 min (Table 4). None of the 3 agents significantly affected the mRNA levels of GSK3B gene (data not shown).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
TTP has antiinflammatory properties with therapeutic potential for the prevention and treatment of inflammation-related diseases. Previous studies have shown that TTP gene expression is induced in mammalian cells by a wide range of agents (1). However, most of the same agents also increase the expression levels of proinflammatory cytokines in the same cells and tissues (8). We have shown that TTP gene expression is induced by CPE in mouse 3T3-L1 adipocytes (17). In this study, we determined that CPE also exhibited the ability to rapidly increase TTP mRNA and protein in mouse RAW264.7 macrophages. However, CPE also increased the levels of mRNAs coding for proinflammatory cytokines, including TNF, COX2, and IL6 in the same cells, but to much less extent than LPS and with a slower response than TTP.

One major finding of this study is that CPE increased TTP mRNA and protein levels in mouse RAW macrophages. TTP is a highly phosphorylated protein (32,33) with antiinflammatory functions through down-regulation of proinflammatory cytokines (1). TTP binds to AU-rich elements in some mRNA sequences and destabilizes those transcripts encoding proteins such as TNF{alpha} (6–9), GM-CSF (10), and COX2 (12). The size of TTP was similar in CPE- and LPS-stimulated TTP in RAW cells (Fig. 1) (19), suggesting that TTP might be phosphorylated in CPE-treated RAW cells. CPE increased TTP mRNA levels after 30 min treatment and TTP protein was clearly detected after 90 min treatment in RAW cells. The current finding that CPE increased TTP gene expression in macrophages is consistent with previous findings that TTP gene expression is increased by CPE in adipocytes (17). These results suggest that nutritional supplements with cinnamon may therefore affect modulating immunological responses in mammalian species.

The relative ratios of anti- and proinflammatory proteins are proposed to be important in the understanding of modulation of inflammatory responses (34). The 2-fold increases in TTP expression by CPE in RAW macrophages were sustained during the 4 h of treatment. However, the expression of a number of proinflammatory cytokine genes was gradually increased in RAW cells by CPE after treatment for longer times. Because TTP mRNA levels in unstimulated RAW cells were 6- to 3000-fold of TNF, COX2, GM-CSF, and IL6 mRNA levels (Table 1), the net increases of TTP mRNA molecules were still larger than those of the proinflammatory cytokine mRNA molecules in the same cells (Supplemental Table 2). Therefore, CPE may have benefits in improving inflammation-related conditions. However, additional studies are required to draw a firm conclusion, because the relationships between TTP mRNA levels, protein levels, and the interactions between TTP protein and its proinflammatory cytokine mRNA target molecules are not completely understood.

Another major finding in this study is that CPE and LPS, but not insulin, increased the levels of GLUT1 mRNA, the major form of GLUT family mRNAs in RAW macrophages. These results suggest that CPE and LPS may have profound effects on the regulation of energy metabolism in immunity-related macrophages, because glucose is the major metabolic substrate critically important for the host response to injury and infection (25,26). Our PCR results showed that GLUT1 mRNA levels were ~8-fold of GLUT3 and 1300-fold of GLUT4 mRNA levels and GLUT2 mRNA was undetectable in RAW cells. Previous studies reported that GLUT1 was the major if not the only GLUT in murine peritoneal macrophages that is induced by LPS, TNF{alpha}, burn injury, and Pseudomonas aeruginosa infection (25). Furthermore, increased GLUT1 gene expression results in enhanced glucose uptake in these macrophages (25,27). Our ability to detect GLUT3 and GLUT4 mRNAs in RAW cells is probably due to the more sensitive real-time PCR method we used. This pattern of GLUT family gene expression in RAW macrophages is different from those in rat liver and skeletal muscle, in which GLUT2 and GLUT4 are the major GLUT mRNAs in respective tissues (35). The ineffectiveness of insulin on GLUT1 gene expression in RAW cells is in agreement with a previous report that showed by flow cytometry that GLUT1 protein does not respond to insulin in either resting or phorbol 12-myristate 13-acetate/LPS-activated white blood cells from healthy people (36).

The results reported here demonstrate that CPE, unlike insulin, is able to promote TTP, GLUT1, and proinflammatory gene expression in RAW macrophages. Our results suggest that RAW macrophages are probably insensitive to insulin, because none of the mRNA levels evaluated were significantly affected by insulin treatment, except that insulin slightly decreased TTP and VEGFB mRNA levels in cells treated for 120 min and increased INSR mRNA levels in cells treated for 240 min. These results are in agreement with the noninsulin-dependent glucose uptake in macrophage-rich tissues, such as liver, spleen, and lung (37), which is opposite to the effect of insulin on GLUT4 gene expression in 3T3-L1 adipocytes (38). Previous studies suggest that cinnamon exhibits insulin-like activity in cells, animals, and people with type 2 diabetes. Both cinnamon and insulin increase antiinflammatory TTP gene expression in mouse adipocytes (17,28), increase the activity of autophosphorylation of the INSRβ and decrease the activity of tyrosine phosphatase in vitro (39), and stimulate glucose uptake and glycogen biosynthesis, activate glycogen synthase, and inhibit glycogen synthase kinase-3β (40). CPE potentiates in vivo insulin-regulated glucose utilization via increasing glucose uptake (41). It also decreases glucose and increases insulin in blood of rats (42) and decreases blood pressure (43). Cinnamon powder also decreased the levels of glucose, triglycerides, and LDL cholesterol in people with type 2 diabetes (44) and delayed gastric emptying without affecting satiety (45). Cinnamon and its polyphenol extract exhibit both insulin-dependent effects in insulin-sensitive cells like adipocytes and insulin-independent effects in insulin-insensitive cells like macrophages. Therefore, the benefit of cinnamon is likely due to its multiple effects, including insulin potentiation and antiinflammatory effects.

The major differences in gene expression affected by CPE and LPS were that: 1) CPE had a greater effect on GLUT1 gene expression than LPS, but LPS had much greater effects than CPE in TTP and proinflammatory cytokine gene expression; 2) CPE increased only TTP gene expression, but LPS also induced TTP, TNF, and COX2 gene expression in RAW cells treated for 30 min; 3) GM-CSF mRNA levels were induced only by LPS by not by CPE in RAW cells; and 4) CPE did not affect GLUT3 gene expression and increased GLUT4 gene expression, but LPS decreased both in RAW cells. These differences may explain the observed beneficial effects of CPE as nutrient supplement and the detrimental effects of LPS. The sustained TTP gene expression patterns stimulated by CPE and LPS in RAW macrophages are similar to those by CPE in 3T3-L1 adipocytes (17), by LPS in RAW264.7 cells (19), and by glucocorticoids in A549 lung epithelial cells and rat tissues (46) but are different from the transient induction by insulin in HIR3.5 preadipocytes (3) and 3T3-L1 adipocytes (28).

In summary, this study reports several novel findings: 1) CPE rapidly induces TTP mRNA and protein and induces TTP gene expression more rapidly than the proinflammatory cytokine mRNAs encoding TNF{alpha}, COX2, and IL6 in mouse macrophages; 2) the net increase in TTP mRNA levels was greater than the proinflammatory cytokines measured; 3) CPE increases more GLUT1 gene expression than LPS; 4) CPE effects on gene expression patterns are different from those of LPS in RAW macrophages during the initial treatment; and 5) insulin does not exhibit major effects on the expression of these genes. Our results indicate that CPE is capable of affecting immune responses by regulating anti- and proinflammatory as well as the gene expression of GLUT in macrophages.


   ACKNOWLEDGMENTS
 
We thank Marilyn M. Polansky and Noella A. Bryden for technical assistance.


   FOOTNOTES
 
1 Supported by the USDA-Agricultural Research Service Human Nutrition Research Program 107 through Cris 235-51520-037-00D. Back

2 Author disclosures: H. Cao, J. F. Urban Jr, and R. A. Anderson, no conflicts of interest. Back

3 Supplemental Tables 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

4 Abbreviations used: CPE, cinnamon polyphenol extract; COX2 (PTGS2), cyclooxgenase-2 (prostaglandin-endoperoxide synthase 2); CT, cycle of threshold; DMSO, dimethylsulfoxide; GLUT, glucose transporter; GM-CSF (CSF2), granulocyte-macrophage colony-stimulating factor (colony-stimulating factor 2); GSK3B, glycogen synthase kinase-3 β; HuR (ELAVL1), Hu antigen R (embryonic lethal, abnormal vision-like 1); IFN{gamma}, interferon-{gamma}; IL, interleukin; INSR, insulin receptor; LPS, lipopolysaccharide; RPL32, ribosomal protein L32; TNF{alpha}, tumor necrosis factor-{alpha}; TTP, tristetraprolin; VEGF, vascular endothelial growth factor; ZFP36, zinc finger protein 36; ZFP36L, ZFP36-like. Back

Manuscript received 14 November 2007. Initial review completed 10 January 2008. Revision accepted 28 February 2008.


   LITERATURE CITED
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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