This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sartippour, M. R. Articles by Renier, G. Search for Related Content PubMed PubMed Citation Articles by Sartippour, M. R. Articles by Renier, G. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB GLUCOSE Related Collections Pathophysiology Risk Factors Cell signalling/signal transduction Physiological and pathological control of gene expression Lipid and lipoprotein metabolism

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:104.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Differential Regulation of Macrophage Peroxisome Proliferator–Activated Receptor Expression by Glucose

Role of Peroxisome Proliferator–Activated Receptors in Lipoprotein Lipase Gene Expression

Maryam Radimeh Sartippour; Geneviève Renier

From Centre Hospitalier de l’Université de Montréal Research Center, Notre-Dame Hospital, Department of Nutrition, University of Montreal, Montreal, Quebec, Canada.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Peroxisome proliferator–activated receptors (PPARs) are implicated in several metabolic disorders with altered glucose and lipid metabolism, including atherosclerosis and diabetes. In the present study, we evaluated the in vitro and ex vivo effects of high glucose concentrations on macrophage PPAR mRNA expression. Exposition of monocyte-derived macrophages isolated from healthy donors to a high glucose environment led to an increase in PPAR and PPARß mRNA expression. In contrast, this treatment significantly decreased human macrophage PPAR mRNA expression. Overexpression of PPAR and PPARß mRNA and inhibition of PPAR mRNA expression were also observed in monocyte-derived macrophages isolated from patients with type 2 diabetes. Because high glucose and PPAR agonists increase lipoprotein lipase (LPL) gene expression, the role of PPAR in the glucose-mediated upregulation of macrophage LPL gene expression was next evaluated. Incubation of murine J774 macrophages with high glucose concentrations increased the expression of PPAR at the mRNA and protein levels and enhanced nuclear protein binding to the peroxisome proliferator responsive element of the LPL promoter. Incubation of nuclear extracts in the presence of anti-PPAR and anti-PPARß antibodies decreased glucose-stimulated nuclear protein binding to the peroxisome proliferator responsive element. These results demonstrate that glucose is an important regulator of macrophage PPAR expression and suggest a role of PPAR and PPARß in the upregulation of macrophage LPL by glucose. Dysregulation of macrophage PPAR expression in type 2 diabetes may contribute, by altering arterial lipid metabolism and inflammatory response, to the accelerated atherosclerosis associated with diabetes.

Key Words: peroxisome proliferator–activated receptors • macrophage • glucose • lipoprotein lipase

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Peroxisome proliferator–activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors.^{1 2 3} Members of this family include PPAR, PPARß, and PPAR. PPARs are characterized by distinct tissue distribution patterns and metabolic functions. PPAR is expressed preferentially in tissues exhibiting high catabolic rates of fatty acids, such as liver and brown adipose tissue, and plays a key role in lipid metabolism.^{4 5} PPARß (also called NUC-1 or PPAR) is ubiquitously expressed, and its specific function is still unknown.^{5 6} PPAR is adipose tissue selective and is implicated as a mediator of adipocyte differentiation and regulation of glucose homeostasis.^{7 8} The activity of PPARs is regulated by various agents, including insulin, fatty acids, fibrates, leukotriene B₄, prostaglandin J₂, and synthetic thiazolidinedione drugs.^{9 10 11 12 13} PPARs function as ligand-dependent transcription factors, which, on heterodimerization with the 9-cis-retinoic acid receptor, bind to a specific response element termed peroxisome proliferator responsive element (PPRE), which is present in the promoter of various genes implicated in lipid metabolism, such as lipoprotein lipase (LPL).^{14 15}

Recent findings demonstrate that PPARs are involved in several metabolic diseases, such as obesity, dyslipidemia, atherosclerosis, and diabetes. Diabetes is a major risk factor for atherosclerosis.¹⁶ Accumulating evidence indicates that immune mechanisms play a critical role in the pathogenesis of atherosclerosis. Arguments that point to the monocyte/macrophage as a principal participant in atherogenesis include its role in arterial lipid metabolism and as a precursor of foam cells.^{17 18 19 20} Recent studies have demonstrated that PPARs are expressed in cells of the monocyte/macrophage lineage²¹ and in macrophage-derived foam cells of atherosclerotic lesions.^{22 23 24} It has been proposed that PPARs may regulate macrophage activation and lipid metabolism in monocytic cells.^{22 24 25 26 27}

Although macrophage PPARs may play a key role in the accelerated atherosclerosis associated with diabetes, the modulatory effect of a high glucose environment on macrophage PPAR expression has not yet been investigated. In the present study, we determined the in vitro effect of high glucose concentration on macrophage PPAR expression and examined the regulation of macrophage PPAR expression in human type 2 diabetes. In addition, on the basis of previous results demonstrating a stimulatory effect of glucose on macrophage LPL expression²⁸ and a key role of PPARs in the control of LPL gene expression,^{14 15} we also investigated the role of PPARs in the regulation of macrophage LPL mRNA expression by glucose.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
FCS was purchased from Hyclone Laboratories. DMEM was obtained from ICN Biochemicals Inc. RPMI 1640 medium, Hanks’ balanced salt solution, and Trizol reagent were purchased from GIBCO-BRL. Lymphoprep and penicillin-streptomycin were obtained from Nycomed Pharma As and Flow, respectively. D-Glucose was purchased from Sigma Chemical Co.

Human and Murine Macrophages
Human monocytes were isolated as previously described.^{29 30} Peripheral blood mononuclear cells were isolated by density centrifugation with Ficoll, allowed to aggregate in the presence of FCS, and then further purified by the rosetting technique. After density centrifugation, highly purified monocytes (85% to 90%) were recovered, as assessed by flow cytometry (FACScan, Becton Dickinson). Differentiation of monocytes into macrophages was achieved by culturing the freshly isolated monocytes in RPMI 1640 medium (2 000 000/mL) containing 20% (vol/vol) autologous serum for 4 days.

The J774 murine macrophage cell line was obtained from American Type Culture Collection (ATCC). Murine macrophages were cultured in DMEM containing 10% FCS and 100 µg/mL penicillin-streptomycin (FCS-DMEM). For experiments assessing the effect of increasing concentrations of glucose, a customized preparation of FCS-DMEM was used; this preparation contained 5.6 mmol/L of glucose to which varying amounts of glucose were added to make up the desired final glucose concentrations.

Patients
The study group consisted of 7 patients with type 2 diabetes and 7 healthy control subjects. They gave written consent to participate in this study, which was approved by the Center Hospitalier de l’Université de Montréal Research and Ethics committees. All patients recruited from our outpatient clinic were normotriglyceridemic and treated with glyburide and metformin. None of the patients was primarily insulin dependent. Characteristics of the study population are presented in Table 1. Healthy controls, matched with patients for sex, age, and body mass index, were recruited from the hospital staff and relatives. Subjects with infectious or inflammatory conditions or with cardiac, renal, or pulmonary decompensated diseases or who were treated with anti-inflammatory or antioxidant drugs were excluded from the study.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Study Population

Analysis of PPAR mRNA Expression
The levels of PPAR mRNAs in human and murine macrophages were assessed by semiquantitative polymerase chain reaction (PCR) and Northern blot analysis, respectively.

PCR Technique
Cytoplasmic RNA for use in the PCR reaction was extracted from human macrophages by an improvement of the acid-phenol technique of Chomczynski and Sacchi,³¹ precipitated, and resuspended in diethyl pyrocarbonate water. cDNA was synthesized from RNA by incubating total cellular RNA isolated from human macrophages with 0.1 µg random primers (Pharmacia) for 5 minutes at 98°C and then by incubating the mixture with reverse transcription buffer for 60 minutes at 37°C. The cDNA obtained was amplified by using 0.8 µmol/L of 2 synthetic primers for PPAR, PPARß, PPAR, and GAPDH. Primers (sense and antisense) used in the PCR reaction are shown in Table 2. A 510-bp human PPAR cDNA fragment, a 406-bp murine PPARß cDNA fragment, a 421-bp murine PPAR cDNA fragment, and a 456-bp human GAPDH cDNA fragment were amplified enzymatically by repeated cycles. An aliquot of each reaction mixture was then subjected to electrophoresis on 1% agarose gel. The intensity of the bands was measured by an image analysis scanning system (Alpha Imager 2000, Packard Instrument Co).

View this table: [in this window] [in a new window]   Table 2. Sequences of Oligonucleotides

Northern Blot Analysis
Ten million J774 macrophages were plated in plastic Petri dishes (100x20 mm, Falcon). After treatment with appropriate agents, macrophages were lysed with guanidine isothiocyanate. Total RNA was purified by centrifugation through a cesium chloride gradient.³² Total RNA (20 µg) was separated on a 1.2% agarose gel containing 2.2 mol/L formaldehyde.³³ The blots were prehybridized for 8 hours. The mRNA expression was analyzed by hybridization with [³²P]dCTP (specific activity 3000 Ci/mmol, Amersham)–labeled murine PPAR and S28 cDNA probes. Hybridization was detected by autoradiography. RNA expression was quantified by high-resolution optical densitometry.

Analysis of PPAR Protein Expression
After appropriate treatments, murine macrophages were pelleted and lysed in 50 mmol/L Tris HCl (pH 7.4), 150 mmol/L NaCl, and 1% Nonidet P-40. Samples were centrifuged at 13 000 rpm for 15 minutes at 4°C, and supernatants were collected. Protein concentrations were determined with a colorimetric assay (Bio-Rad) by use of BSA as a standard. Samples (50 µg proteins) were applied to 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane by using a Bio-Rad transfer blotting system at 100 V for 60 minutes. Membranes were blocked overnight at 4°C with a solution of PBS–0.3% Tween 20 containing 1% BSA and 5% FCS. After a wash with PBS–0.05% Tween 20, membranes were incubated for 3 hours at room temperature with anti-PPAR antibody (1/1000; kindly provided by Dr W. Wahli, Université de Lausanne, Lausanne, Switzerland) in PBS–0.05% Tween 20 and FCS 1%. After a further wash, membranes were incubated with IgG antibodies linked to the horseradish peroxidase in PBS–0.05% Tween 20 and FCS 5% for 1 hour at room temperature. Membranes were washed with PBS containing 0.05% Tween 20. Antigen detection was performed with an enhanced chemiluminescence detection system (Amersham).

DNA Binding Assay
The isolation of nuclei was performed as previously described.³⁴ Briefly, 5x10⁷ J774 cells were collected, washed with cold PBS, and lysed in 1 mL of ice-cold buffer A (15 mmol/L KCl, 2 mmol/L MgCl₂, 10 mmol/L HEPES, 0.1% phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40). After a 10-minute incubation on ice, lysed cells were centrifuged, and the nuclei were washed with buffer A without Nonidet P-40. The nuclei were then lysed in a buffer containing 2 mol/L KCl, 25 mmol/L HEPES, 0.1 mmol/L EDTA, and 1 mmol/L dithiothreitol. After a 15-minute incubation period, a dialysis buffer (25 mmol/L HEPES, 1 mmol/L dithiothreitol, 0.1% phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 0.1 mmol/L EDTA, and 11% glycerol) was added to the nuclei preparation. Nuclei were collected by centrifugation for 20 minutes at 13 000 rpm. Aliquots (50 µL) of the supernatants were frozen at -70°C, and protein concentration was determined. DNA retardation (mobility shift) electrophoresis assays were performed as previously described by Fried and Crothers.³⁵ Briefly, 5 µg nuclear extracts were incubated for 15 minutes in the presence of 5x binding buffer (125 mmol/L HEPES, pH 7.5, 50% glycerol, 250 mmol/L NaCl, 0.25% Nonidet P-40, and 5 mmol/L dithiothreitol) in the presence or absence of 200 ng anti-PPAR, anti-PPARß (kindly provided by Dr W. Wahli, Université de Lausanne, Lausanne, Switzerland), or anti-PPAR (Calbiochem) antibodies. End-labeled double-stranded consensus sequences of the LPL promoter PPAR-enhancing element (20 000 cpm per sample) were then added to the samples for 30 minutes. Samples were analyzed on a 4% nondenaturing polyacrylamide gel containing 0.01% Nonidet P-40. The specificity of the nuclear protein binding was assessed by incubating the nuclear proteins isolated from murine macrophages with labeled DNA probe in the presence of a 1000-molar excess of unlabeled DNA probe.

DNA Probes
The cDNA probe for detection of murine PPAR was prepared by PCR. cDNA was obtained from total RNA by using a reverse transcription reaction. Two synthetic primers (Table 2) spanning bases 1175 to 1759 were used to enzymatically amplify a 584-bp region of the PPAR probe. The PPAR probe was purified by Sephaglas BandPrep Kit (Pharmacia). The cDNA probe for murine S28 was purchased from ATCC. A 20-mer double-stranded oligonucleotide (Table 2) containing the consensus sequence for the PPRE of the human LPL gene promoter¹⁴ was synthesized with the aid of an automated DNA synthesizer. After annealing, the double-stranded oligonucleotide was labeled with [-³²P]ATP by using the Boehringer-Mannheim 5' end-labeling kit.

Statistical Analysis
All values were expressed as the mean±SEM. For single comparisons, data were analyzed by the Student t test or Mann-Whitney rank sum test. For multiple comparisons, data were analyzed by ANOVA, followed by the Tukey test or the Dunn test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of High Glucose Concentrations on Human Monocyte–Derived Macrophage PPAR, PPARß, and PPAR mRNA Levels
To evaluate the effects of high glucose concentrations on macrophage PPAR mRNA expression, monocyte-derived macrophages obtained from healthy control subjects were cultured for 48 hours in the presence of 5.6 or 30 mmol/L glucose. High glucose concentration significantly enhanced PPAR and PPARß mRNA expression (PPAR mRNA levels [fold increase over control values] for 30 mmol/L glucose, 2.44±0.73, P=0.036; PPARß mRNA levels [fold increase over control values] for 30 mmol/L glucose, 1.47±0.66, P=0.016; Figure 1A). In contrast, high glucose concentration significantly decreased PPAR mRNA expression (PPAR mRNA levels [fold decrease under control values] for 30 mmol/L glucose, 1.64±0.08, P<0.001; Figure 1A). PPAR, PPARß, and PPAR mRNA levels normalized to the levels of GAPDH mRNA (Figure 1A) are illustrated in Figure 1B.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of high glucose concentrations on PPAR, PPARß, and PPAR mRNA expression by human monocyte–derived macrophages. Monocyte-derived macrophages obtained from healthy control subjects were cultured in the presence of 5.6 or 30 mmol/L glucose. After 2 days, cells were lysed. A, PPAR, PPARß, PPAR, and GAPDH mRNA expression was analyzed by PCR. B, PPAR , PPARß, and PPAR mRNA levels were normalized to the levels of GAPDH mRNA. Data represent the mean±SEM of 4 different experiments. *P<0.05, **P<0.02, and ***P<0.001 vs control.

Regulation of Monocyte-Derived Macrophage PPAR mRNA Expression in Patients With Type 2 Diabetes
To investigate the regulation of macrophage PPARs in diabetes, PPAR mRNA expression was determined in monocyte-derived macrophages isolated from patients with type 2 diabetes. Macrophages of diabetic patients expressed higher PPAR and PPARß mRNA levels than did macrophages of control subjects (PPAR mRNA levels [fold increase over control values],1.93±0.16, P=0.003; PPARß mRNA levels [fold increase over control values], 1.92±0.35, P=0.048; Figure 2A and 2B). Macrophages of diabetic patients also expressed significantly lower PPAR mRNA levels compared with levels in macrophages of control subjects (PPAR mRNA levels [fold decrease under control values], 1.31±0.11, P=0.026; Figure 2C).

View larger version (11K): [in this window] [in a new window]   Figure 2. PPAR mRNA expression in monocyte-derived macrophages isolated from patients with type 2 diabetes. Monocytes were isolated from 7 control subjects and 7 diabetic patients and cultured for 4 days in RPMI medium supplemented with 20% autologous serum. At the end of the incubation period, monocyte-derived macrophages were lysed, and PPAR, PPARß, and PPAR mRNA expression was analyzed by PCR. PPAR (A), PPARß (B), and PPAR (C) mRNA levels were normalized to the levels of GAPDH mRNA. *P<0.05 and **P<0.005 vs control.

Role of PPARs in Upregulation of Macrophage LPL Gene Expression in Response to Glucose
On the basis of our previous observations that LPL, a key target gene for PPARs, is upregulated by glucose in J774 murine macrophages,²⁸ we next determined whether incubation of J774 cells in the presence of high glucose concentrations might induce changes at the level of the LPL gene promoter binding PPAR protein.

As part of a setup for subsequent gel-shift data, PPAR mRNA and protein levels in J774 cells exposed to high glucose concentrations were first determined. Incubation of J774 cells with increasing glucose concentrations (5.6, 10, 20, and 30 mmol/L) for 48 hours increased, in a dose-dependent manner, PPAR mRNA expression by these cells (Figure 3A). Under these experimental conditions, no modulation of the mRNA expression of S28, used as an internal control, was observed (Figure 3B). PPAR mRNA levels normalized to the levels of S28 mRNA are illustrated in Figure 3C.

View larger version (38K): [in this window] [in a new window]   Figure 3. Effect of high glucose concentration on murine macrophage PPAR mRNA expression. J774 cells were cultured for 48 hours in the presence of increasing glucose concentrations. At the end of the incubation period, cells were lysed, and total RNA was extracted and analyzed by Northern blot analysis for PPAR (A) and S28 mRNA (B) expression. PPAR mRNA levels were normalized to the levels of S28 mRNA (C). Data shown in panel C represent the results of 4 independent experiments.

Determination of PPAR protein levels in J774 cells exposed for 48 hours in the presence of 30 mmol/L glucose demonstrated a significant increase in the expression levels of this receptor over control values (PPAR protein levels [fold increase over control values] for 30 mmol/L glucose, 2.95±0.43; Figure 4).

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of high glucose on murine macrophage PPAR protein expression. Murine macrophages were incubated for 2 days with 5.6 or 30 mmol/L glucose. At the end of the incubation period, cells were lysed, and Western blot analysis was carried out as described in Methods. PPAR was detected by using the polyclonal anti-rat PPAR antibody. Lanes are as follows: lane 1, molecular weight markers; lane 2, 5.6 mmol/L glucose; and lane 3, 30 mmol/L glucose.

Exposure of J774 cells to a high glucose environment for 60 hours resulted in a significant increase in the binding of nuclear proteins to the PPRE consensus sequence of the LPL promoter (Figure 5). The specificity of these proteins is demonstrated by the fact that they were effectively competed by excess unlabeled PPRE oligonucleotide. In the glucose-treated nuclear extracts, antibodies against PPAR and PPARß decreased the binding activity to the PPRE sequence, whereas anti-PPAR antibody was ineffective (Figure 5).

View larger version (45K): [in this window] [in a new window]   Figure 5. Effect of high glucose concentration on the binding activity of nuclear proteins extracted from murine macrophages to the regulatory PPRE sequence of the human LPL gene promoter. Murine macrophages were exposed for 60 hours to 5.6 or 30 mmol/L glucose. The nuclear proteins isolated from these cells were incubated with double-stranded PPAR regulatory element of the LPL gene. Retardation was assessed on a 4% nondenaturating polyacrylamide gel. Data represent the results of one of 3 representative experiments. Lanes are as follows: lane 1, 5.6 mmol/L glucose; lane 2, 30 mmol/L glucose; lane 3, 30 mmol/L glucose+competitor; lane 4, 30 mmol/L glucose+anti-PPAR antibody; lane 5, 30 mmol/L glucose+ anti-PPARß antibody; and lane 6, 30 mmol/L glucose+anti-PPAR antibody.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Glucose is the primary metabolic substrate of macrophages.³⁶ Enhanced glucose metabolism occurs in these cells in response to mitogenic or immunologic stimulation. Evidence has been provided that high glucose regulates in vitro macrophage lipid metabolism and function. Indeed, it has been shown that macrophages cultured in a high glucose environment overexpress LPL, a key enzyme in the catabolism of triglyceride-rich lipoproteins,²⁸ and secrete large amounts of various proinflammatory cytokines, including tumor necrosis factor- and interleukin-6.^{37 38}

PPARs are key nuclear factors in nutrient gene interaction that translate nutritional signals into changes in the expression of genes implicated in lipid and glucose metabolism.^{14 15} In adipose tissue, skeletal muscle, and hepatic cells, PPAR mRNA expression has been shown to be under hormonal control, as reflected by the induction at the transcriptional level of PPAR and PPAR gene expression by insulin and glucocorticoids.^{39 40 41 42} The observation that PPARs are transcriptionally active in human macrophages has raised the question of the nature of the regulators of PPAR mRNA expression and the target genes for PPARs in these cells. Insight into the regulation of PPAR expression in activated macrophages has recently been provided by Ricote et al,²⁴ who demonstrated an induction of PPAR expression in these cells by colony-stimulating factor, granulocyte/macrophage colony–stimulating factor, and oxidized LDL.

The present study demonstrates for the first time that high glucose levels regulate PPAR expression in human macrophages; PPAR and PPARß are increased, whereas PPAR is decreased. These results identify the macrophage PPAR genes as response genes for glucose action. The molecular mechanism(s) by which glucose upregulates macrophage PPAR and PPARß mRNA levels is presently unknown. One may hypothesize that such an effect may result from the direct interaction of glucose with a putative glucose responsive element present in the promoter of these genes. DNA motifs that could mediate glucose responsiveness of genes include the cis-acting carbohydrate responsive element (5'-CACGTGNNNGCG-3'), the CACGTG motif related to the consensus sequence binding site for the c-myc family of transcription factors, and other glucose responsive elements, such as the stimulatory protein 1 sites.^{43 44 45 46} The presence of a stimulatory protein 1 site (-130 to -125) and of sequences similar to the cis-acting carbohydrate responsive element (-313 to -302) and the CACGTG motif (-869 to -862 and -140 to -135) in the promoter of the PPAR gene⁴⁷ suggests that glucose may induce PPAR gene expression through this site(s). Our preliminary results (data not shown) showing that high glucose increases the level of binding of nuclear proteins to stimulatory protein 1 and carbohydrate responsive elements present in the PPAR promoter seem to support this possibility. Alternatively, intracellular accumulation of fatty acids resulting from glucose interaction with lipid metabolism may be responsible for PPAR gene induction. This possibility is supported by one recent study demonstrating, in hepatic cells, an upregulatory effect of fatty acids on the steady-state PPAR mRNA levels.⁴¹

Negative regulation of PPAR gene expression in glucose-treated macrophages could theoretically involve tumor necrosis factor-. Indeed, it has been shown that high glucose stimulates macrophage tumor necrosis factor- secretion³⁸ and that this cytokine, in turn, exerts a suppressive effect on PPAR mRNA expression.^{48 49}

Because PPARs regulate lipid metabolism, genes involved in macrophage lipid metabolism, such as LPL, are likely candidates as target genes for PPARs in these cells. This view is supported by the observation that PPAR agonists increase LPL gene expression^{14 15 50 51} and by the demonstration of a PPRE site in the regulatory sequence of the human LPL gene.¹⁴ The parallel induction of PPAR and LPL gene expression in glucose-treated macrophages is consistent with the view that the transcriptional effect of glucose on macrophage LPL that we previously reported²⁸ may involve this PPAR isoform. This possibility is further supported by the fact that anti-PPAR decreases the enhanced binding of nuclear proteins isolated from glucose-treated macrophages to the PPRE regulatory domain of the LPL gene. The upregulation of PPARß by high glucose also suggests a potential role of this isoform in the stimulation of LPL gene expression by glucose. Our finding that anti-PPARß antibody decreases the glucose-induced binding activity to the PPRE is consistent with this view. Finally, despite the reduction of PPAR expression by glucose, it cannot be excluded that induction of a potent PPAR ligand by glucose may be sufficient to activate this isoform and thereby stimulate the LPL target gene. However, this possibility is not supported by our results, which show that anti-PPAR antibody does not decrease the glucose-induced binding activity to the PPRE regulatory domain of the LPL gene.

Genes involved in the control of inflammation may represent additional targets for PPARs in macrophages. Indeed, it has been previously shown that mice rendered deficient for PPAR display a prolonged response to inflammatory stimuli¹¹ and that PPAR negatively regulates genes implicated in macrophage activation.^{25 26} On the basis of these results and observations that glucose-treated macrophages exhibit an increase in cytokine production^{37 38} and an inhibition of macrophage PPAR expression, it is tempting to speculate that the stimulatory effect of glucose on macrophage function may involve, at least partly, a suppression of PPAR activation in these cells. Whether PPAR and PPAR exert differential effects on macrophage function and whether the different subcellular localizations of PPAR and PPAR proteins in macrophages, ie, PPAR in the nucleus and PPAR in the cytoplasmic compartment,²⁷ are responsible for these effects remain to be investigated.

Human diabetes is associated with a high incidence of atherosclerosis.¹⁶ Recent evidence demonstrates that PPAR is expressed in macrophage foam cells of human atherosclerotic lesions.^{22 23 24} Furthermore, expression of PPAR, PPARß, and PPAR has been documented in vascular cells, including macrophages, smooth muscle cells,^{52 53} and endothelial cells.⁵⁴ PPARs may interfere with atherogenesis by regulating arterial lipid metabolism and/or inflammation. Our findings that human type 2 diabetes is associated with altered macrophage PPAR gene expression further suggest that changes in macrophage PPAR activation may occur in the vascular wall in the hyperglycemic state and may influence atherogenesis. Dysregulation of macrophage PPARs in the arterial wall may contribute, by increasing the in vivo macrophage production of LPL and of proinflammatory cytokines, to the accelerated atherosclerosis associated with diabetes. Given the potential proatherogenic effect of macrophage LPL in the arterial wall, study of the biological role of these transcription factors in the regulation of macrophage LPL expression seems especially relevant to the development of new strategies in the prevention and treatment of atherosclerosis.

	Acknowledgments

 
This study was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Canada, and the Association Diabète Québec. The authors thank Dr W. Wahli (Université de Lausanne, Lausanne, Switzerland) for providing the anti-PPAR and anti-PPARß antibodies and Dr O. Serri (University of Montreal, Metabolic Unit of Notre-Dame Hospital, Montreal, Quebec, Canada) for the referral of the diabetic patients and for his helpful comments in the preparation of the manuscript.

	Footnotes

 
Reprint requests to Dr Geneviève Renier, Notre-Dame Hospital, Research Center, 3rd Floor, Door Y-3622, 1560 Sherbrooke St East, Montreal, Quebec H2L 4 M1, Canada.

Received May 19, 1999; accepted August 9, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Schoonjans K, Martin G, Staels B, Auwerx J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol. 1997;8:159–166.[Medline] [Order article via Infotrieve]
2. Desvergne B, Wahli W. PPAR: a key nuclear factor in nutrient/gene interactions? In: Baeuerle PA, ed. Inducible Gene Expression. Vol 1. Boston, Mass: Birkhäuser; 1995.
3. Desvergne B, IJpenberg A, Devchand PR, Wahli W. The peroxisome proliferator-activated receptors at the cross-road of diet and hormonal signalling. J Steroid Biochem Mol Biol. 1998;65:65–74.[Medline] [Order article via Infotrieve]
4. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci U S A. 1994;91:7355–7359.[Abstract/Free Full Text]
5. Braissant O, Foufelle F, Scotto C, Dauca M, Whali W. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-, -ß, and - in the adult rat. Endocrinology. 1996;137:354–366.[Abstract]
6. Schmidt A, Endo N, Rutledge SJ, Vogel R, Shinar D, Rodan GA. Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. Mol Endocrinol. 1992;6:1634–1641.[Abstract]
7. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA. Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology. 1994;135:798–800.[Abstract]
8. Tontonoz P, Hu E, Devine J, Beale EG, Spiegelman BM. PPAR2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1995;15:351–357.[Abstract]
9. Rieusset J, Andreelli F, Auboeuf D, Roques M, Vallier P, Riou JP, Auwerx J, Laville M, Vidal H. Insulin acutely regulates the expression of the peroxisome proliferator-activated receptor- in human adipocytes. Diabetes. 1999;48:699–705.[Abstract]
10. Keller H, Dreyer C, Medin J, Mahfoudi A, Ozato K, Wahli W. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. Proc Natl Acad Sci U S A. 1993;90:2160–2164.[Abstract/Free Full Text]
11. Devchand PR, Keller H, Peters JM, Vasquez M, Gonzalez FJ, Whali W. The PPARalpha-leukotriene B4 pathway to inflammation control. Nature. 1996;384:39–43.[Medline] [Order article via Infotrieve]
12. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy-delta 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell. 1995;83:803–812.[Medline] [Order article via Infotrieve]
13. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor . J Biol Chem. 1995;270:12953–12956.[Abstract/Free Full Text]
14. Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, Staels B, Auwerx J. PPAR and PPAR activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J. 1996;15:5336–5348.[Medline] [Order article via Infotrieve]
15. Auwerx J, Schoonjans K, Fruchart JC, Staels B. Transcriptional control of triglyceride metabolism: fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR. Atherosclerosis. 1996;124(suppl):S29–S37.
16. Nathan DM. Long-term complications of diabetes mellitus. N Engl J Med. 1993;328:1676–1685.[Free Full Text]
17. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131–138.[Abstract/Free Full Text]
18. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid protein adducts. J Clin Invest. 1991;87:90–99.
19. Gerrity RG. The role of the monocyte in atherogenesis, I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181–190.[Abstract]
20. Joris I, Zand T, Nunnari JJ, Krolikowski FJ, Majno G. Studies on the pathogenesis of atherosclerosis, I: adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats. Am J Pathol. 1983;113:341–358.[Abstract]
21. Greene ME, Blumberg B, McBride OW, Yi HF, Kronquist K, Kwan K, Hsieh L, Greene G, Nimer SD. Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Expr. 1995;4:281–299.[Medline] [Order article via Infotrieve]
22. Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM. PPAR promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell. 1998;93:241–252.[Medline] [Order article via Infotrieve]
23. Marx N, Sukhova G, Murphy C, Libby P, Plutzky J. Macrophages in human atheroma contain PPAR gamma: differentiation-dependent peroxisomal proliferator-activated receptor gamma (PPAR gamma) expression and reduction of MMP-9 activity through PPAR gamma activation in mononuclear phagocytes in vitro. Am J Pathol. 1998;153:17–23.[Abstract/Free Full Text]
24. Ricote M, Huang J, Fajas L, Li A, Welch J, Najib J, Witztum JL, Auwerx J, Palinski W, Glass CK. Expression of the peroxisome proliferator-activated receptor (PPAR) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1998;95:7614–7619.[Abstract/Free Full Text]
25. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature. 1998;391:79–82.[Medline] [Order article via Infotrieve]
26. Jiang C, Ting AT, Seed B. PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998;391:82–86.[Medline] [Order article via Infotrieve]
27. Chinetti G, Griglio S, Antonucci M, Torra IP, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of proliferator-activated receptors and induces apoptosis of human monocyte-derived macrophages. J Biol Chem. 1998;273:25573–25580.[Abstract/Free Full Text]
28. Sartippour MR, Lambert A, Laframboise M, St.-Jacques P, Renier G. Stimulatory effect of glucose on macrophage lipoprotein lipase expression and production. Diabetes. 1998;47:431–438.[Abstract]
29. Mentzer SJ, Guyre PM, Burakoff SJ, Faller DV. Spontaneous aggregation as a mechanism for human monocyte purification. Cell Immunol. 1986;101:312–319.[Medline] [Order article via Infotrieve]
30. Hoover ML, Chapman SW, Cuchens MA. A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. J Immunol Methods. 1985;78:71–85.[Medline] [Order article via Infotrieve]
31. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
32. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294–5299.[Medline] [Order article via Infotrieve]
33. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980;77:5201–5205.[Abstract/Free Full Text]
34. Han JH, Beutler B, Huez G. Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages. Biochim Biophys Acta. 1991;1090:22–28.[Medline] [Order article via Infotrieve]
35. Fried M, Crothers DM. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981;9:6505–6525.[Abstract/Free Full Text]
36. Calder PC. Fuel utilization by cells of the immune system. Proc Nutr Soc. 1995;54:65–82.[Medline] [Order article via Infotrieve]
37. Morohoshi M, Fujisawa K, Uchimura I, Numano F. The effect of glucose and advanced glycosylation end products on IL-6 production by human monocytes. Ann N Y Acad Sci. 1995;748:562–570.[Abstract]
38. Morohoshi M, Fujisawa K, Uchimura I, Numano F. Glucose-dependent interleukin-6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes. 1996;45:954–959.[Abstract]
39. Park KS, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Nikoulina SE, Henry RR. PPAR-gamma gene expression is elevated in skeletal muscle of obese and type II diabetic subjects. Diabetes. 1997;46:1230–1234.[Abstract]
40. Vidal-Puig AJ, Considine RV, Jimenez-Linan M, Werman A, Pories WJ, Caro JF, Flier JS. Peroxisome proliferator-activated receptor gene expression in human tissues: effects of obesity, weight loss, and regulation by insulin and glucocorticoids. J Clin Invest. 1997;99:2416–2422.[Medline] [Order article via Infotrieve]
41. Steineger HH, Sorensen HN, Tugwood JD, Skrede S, Spydevold O, Gautvik KM. Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferator-activated receptor (PPAR) in hepatic cells: hormonal modulation of fatty-acid-induced transcription. Eur J Biochem. 1994;225:967–974.[Medline] [Order article via Infotrieve]
42. Lemberger T, Staels B, Saladin R, Desvergne B, Auwerx J, Wahli W. Regulation of the peroxisome proliferator-activated receptor gene by glucocorticoids. J Biol Chem. 1994;269:24527–24530.[Abstract/Free Full Text]
43. Shih HM, Towle HC. Definition of the carbohydrate response element of the rat S14 gene: evidence for a common factor required for carbohydrate regulation of hepatic genes. J Biol Chem. 1992;267:13222–13228.[Abstract/Free Full Text]
44. Shih H, Towle HC. Definition of the carbohydrate response element of the rat S14 gene: context of the CAGTG motif determines the specificity of carbohydrate regulation. J Biol Chem. 1994;269:9380–9387.[Abstract/Free Full Text]
45. Daniel S, Kim KH. Sp1 mediates glucose activation of the acetyl-CoA carboxylase promoter. J Biol Chem. 1996;271:1385–1392.[Abstract/Free Full Text]
46. Chen YQ, Su M, Walia RR, Hao Q, Covington JW, Vaughan DE. Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells. J Biol Chem. 1998;273:8225–8231.[Abstract/Free Full Text]
47. Gearing KL, Crickmore A, Gustafsson JA. Structure of the mouse peroxisome proliferator activated receptor alpha gene. Biochem Biophys Res Commun. 1994;199:255–263.[Medline] [Order article via Infotrieve]
48. Xing H, Northrop JP, Grove JR, Kilpatrick KE, Su JL, Ringold GM. TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPAR gamma without effects on Pref-1 expression. Endocrinology. 1997;138:2776–2783.[Abstract/Free Full Text]
49. Zhang B, Berger J, Hu E, Szalkowski D, White-Carrington S, Spiegelman BM, Moller DE. Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha. Mol Endocrinol. 1996;10:1457–1466.[Abstract]
50. Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res. 1996;37:907–925.[Abstract]
51. Staels B, Schoonjans K, Fruchart JC, Auwerx J. The effects of fibrates and thiazolidinediones on plasma triglyceride metabolism are mediated by distinct peroxisome proliferator-activated receptors (PPARs). Biochimie. 1997;79:95–99.[Medline] [Order article via Infotrieve]
52. Iijima K, Yoshizumi M, Ako J, Eto M, Kim S, Hashimoto M, Sugimoto N, Liang Y-Q, Sudoh N, Toba K, Ouchi Y. Expression of peroxisome proliferator-activated receptor (PPAR) in rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1998;247:353–356.[Medline] [Order article via Infotrieve]
53. Marx N, Schönbeck U, Lazar MA, Libby P, Plutzky J. Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998;83:1097–1103.[Abstract/Free Full Text]
54. Inoue I, Shino K, Noji S, Awata T, Katayama S. Expression of peroxisome proliferator-activated receptor (PPAR) in primary cultures of human vascular endothelial cells. Biochem Biophys Res Commun. 1998;246:370–374.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				R. Nielsen, L. Grontved, H. G. Stunnenberg, and S. Mandrup Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery Mol. Cell. Biol., August 1, 2006; 26(15): 5698 - 5714. [Abstract] [Full Text] [PDF]

				H. W. Cohen, S. M. Hailpern, and M. H. Alderman Glucose-Cholesterol Interaction Magnifies Coronary Heart Disease Risk for Hypertensive Patients Hypertension, May 1, 2004; 43(5): 983 - 987. [Abstract] [Full Text] [PDF]

				I. Pineda Torra, Y. Jamshidi, D. M. Flavell, J.-C. Fruchart, and B. Staels Characterization of the Human PPAR{alpha} Promoter: Identification of a Functional Nuclear Receptor Response Element Mol. Endocrinol., May 1, 2002; 16(5): 1013 - 1028. [Abstract] [Full Text] [PDF]

				M.-C. Beauchamp and G. Renier Homocysteine Induces Protein Kinase C Activation and Stimulates c-Fos and Lipoprotein Lipase Expression in Macrophages Diabetes, April 1, 2002; 51(4): 1180 - 1187. [Abstract] [Full Text] [PDF]

				T. R. Hughes, T. S. Tengku-Muhammad, S. A. Irvine, and D. P. Ramji A Novel Role of Sp1 and Sp3 in the Interferon-gamma -mediated Suppression of Macrophage Lipoprotein Lipase Gene Transcription J. Biol. Chem., March 22, 2002; 277(13): 11097 - 11106. [Abstract] [Full Text] [PDF]

				Y. Zhang, J. J. Repa, K. Gauthier, and D. J. Mangelsdorf Regulation of Lipoprotein Lipase by the Oxysterol Receptors, LXRalpha and LXRbeta J. Biol. Chem., November 9, 2001; 276(46): 43018 - 43024. [Abstract] [Full Text] [PDF]

				A.-Y. Tu and J. J. Albers Glucose Regulates the Transcription of Human Genes Relevant to HDL Metabolism: Responsive Elements for Peroxisome Proliferator-Activated Receptor Are Involved in the Regulation of Phospholipid Transfer Protein Diabetes, August 1, 2001; 50(8): 1851 - 1856. [Abstract] [Full Text] [PDF]

				E. Chevillotte, J. Rieusset, M. Roques, M. Desage, and H. Vidal The Regulation of Uncoupling Protein-2 Gene Expression by omega -6 Polyunsaturated Fatty Acids in Human Skeletal Muscle Cells Involves Multiple Pathways, Including the Nuclear Receptor Peroxisome Proliferator-activated Receptor beta J. Biol. Chem., March 30, 2001; 276(14): 10853 - 10860. [Abstract] [Full Text] [PDF]

				F. Zheng, A. Fornoni, S. J. Elliot, Y. Guan, M. D. Breyer, L. J. Striker, and G. E. Striker Upregulation of type I collagen by TGF-beta in mesangial cells is blocked by PPARgamma activation Am J Physiol Renal Physiol, April 1, 2002; 282(4): F639 - F648. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services