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High Glucose Concentration Increases Macrophage Cholesterol Biosynthesis in Diabetes Through Activation of the Sterol Regulatory Element Binding Protein 1 (SREBP1): Inhibitory Effect of Insulin

Kaplan, Marielle PhD; Kerry, Rachel MSc*; Aviram, Michael DSc*; Hayek, Tony MD*

Journal of Cardiovascular Pharmacology: October 2008 - Volume 52 - Issue 4 - p 324-332
doi: 10.1097/FJC.0b013e3181879d98
Original Article
Free

Diabetes activates atherogenesis and macrophage foam cell formation. This study's goal was to determine whether insulin counteracts diabetes-induced macrophage foam cell formation, as well as to determine transcriptional mechanisms involved in this effect.

Insulin injection to diabetic mice reduced macrophage lipid peroxides levels, Ox-LDL uptake, and CD36 mRNA levels by 40%, 29%, and by 41% respectively, compared to age-matched untreated diabetic mice. These results were further assessed using an in vitro system. Addition of insulin to glucose-enriched cells led to a significant decrease in cellular lipid peroxidation by 43% compared to cells incubated with high concentrations of glucose with no insulin. This effect was correlated with a reduction in NADPH oxidase activity.

Macrophage cholesterol biosynthesis was then studied in cells from diabetic mice treated with insulin and in glucose-enriched macrophages incubated with insulin. Insulin treatment of diabetic mice significantly reduced macrophage cholesterol biosynthesis, HMG-CoA reductase mRNA expression, and protein expression by 81%, 54%, and 31% respectively, compared to macrophages isolated from nontreated diabetic mice. Similarly, insulin incubation with glucose-enriched macrophages significantly reduced macrophage cholesterol biosynthesis, HMG-CoA reductase mRNA expression, and protein expression by 84%, 42%, and 18%, respectively, compared to macrophages incubated with high glucose but without insulin. These effects were mediated by glucose and insulin ability to regulate the transcription factor SREBP-1. Whereas glucose upregulated SREBP-1 expression and maturation, insulin blocked SREBP1 cleavage, leading to reduced mature form of the transcription factor in the nucleus.

In conclusion, this study presents important novel insights on the events connecting diabetes and glucose stimulation of macrophage foam cell formation leading to atherosclerosis. Most important, the inhibitory effects of insulin on diabetes-mediated (and high glucose-induced) increased cholesterol synthesis were shown to involve modulation of SREBP-1 expression and its maturation.

From the *Lipid Research Laboratory, Faculty of Medicine, Technion, Rambam Medical Center, Haifa, Israel; and the †Clinical Biochemistry laboratory, Rambam Medical Center, Haifa, Israel.

Received for publication September 2, 2007; accepted July 1, 2008.

This work was supported by the Russell Berrie Foundation and D-Cure, “Diabetes Care in Israel.”

The authors state that they have no financial interest in the products mentioned within this article.

Reprints: Tony Hayek, MD, Internal Medicine Department E, Rambam Medical Center, 31096 Haifa, Israel (e-mail: t_hayek@rambam.health.gov.il).

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INTRODUCTION

Diabetes pathophysiology is associated with accelerated atherosclerosis.1 A critical event in the early stages of atherosclerosis is the accumulation of lipid-laden macrophage foam cells.2 Conversion of macrophages into foam cells involves several mechanisms, including increased cellular and lipoproteins lipid peroxidation, increased oxidized-LDL cellular uptake, decreased HDL-mediated cholesterol efflux, and increased cellular cholesterol biosynthesis.3 Studying the mechanisms involved in diabetes induction of foam cell formation is thus of major importance. The role of insulin, however, in atherosclerosis progression is uncertain. Hyperinsulinemia is considered to be an independent risk factor for atherosclerosis development, but there are some lines of evidence suggesting a protective role for insulin.4,5

The regulation of cellular cholesterol biosynthesis involved specific transcription factors: the Sterol regulator elements binding proteins (SREBPs). SREBPs are nuclear factors that bind to the sterol regulatory element (SRE), which is common to the LDL receptor and the HMG CoA synthase genes. SREBPs are produced as membrane-bound precursors that are cleaved by a 2-step proteolytic process and release the nuclear SREBP (the mature form) into the nucleus to activate their target genes. This nuclear SREBP then enters the nucleus and activates the transcription of genes involved in cholesterol and fatty acid synthesis by its binding to sterol regulatory elements (SREs) located in a sterol-sensing domain that is shared by HMG CoA reductase and some additional proteins.6,7 SREBP-1 mainly regulates fatty acid synthesis, and SREBP-2 regulates mainly cholesterol metabolism.6 However, several genes involved in cholesterol metabolism such as HMGCoA reductase, are responsive to both SREBP-1 and SREBP-2.8 It was previously shown that insulin affects SREBP-1 processing,9 but the mechanism(s) by which insulin/glucose affects SREBP-1 expression and maturation need to be clarified.

We have previously shown that diabetes induction in mice, as well as macrophages incubations with glucose, increased cellular lipids peroxidation, as well cholesterol uptake and macrophage cholesterol biosynthesis.10,11

The goal of the present study was to analyze whether insulin treatment can reverse the proatherosclerotic effects of diabetes mediated by glucose on macrophage foam cell formation. Furthermore, we analyzed transcriptional mechanisms involved in diabetes/glucose involvement in macrophages cholesterol biosynthesis and the protective role of insulin in this process.

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METHODS

Mice

Balb C mice (6 weeks old) were randomly divided into three groups. (1) Diabetic mice were injected intraperitoneally with 200 mg/kg streptozotocin (STZ) within 5 minutes of preparation. Serum glucose levels were determined within 2 weeks and mice with serum glucose levels in the range of 250 to 400 mg/dl were included in study group. Mice were sacrificed at 16 weeks old (after 2 months of diabetes). (3) Diabetic+Insulin mice were injected intraperitoneally with 200 mg/kg STZ within 5 minutes of preparation. Serum glucose levels were determined within 2 weeks, and mice with serum glucose levels in the range of 250 to 400 mg/dl were included in the study group. Insulin was injected to the mice daily (beginning at 8 weeks old) at a dose of 1U/mice. Mice were sacrificed at 16 weeks old. (3) Nondiabetic Balb C Control mice were sacrificed at the age of 16 weeks old.

The mice protocol was approved by the committee for supervision of animal experiments of the Technion Israel Institute of Technology (approval No. IL-05-001-2005) and was conducted in accordance to the Israeli law for animal care.

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Cells

Mouse peritoneal macrophages (MPM) were harvested from the peritoneal fluid of Balb-C mice 4 days after intraperitoneal injection of thioglycolate. The cell suspension is dispensed into petri dishes and incubated in an incubator (5% CO2, 95% air) for 2 hours. The dishes are washed once with Dulbecco modified Eagle medium (DMEM) to remove nonadherent cells. Nonspecific esterase staining-Adherent MPMs were washed in PBS, fixed for 1 minute using 0.2% glutaraldehyde in PBS, and rinsed with water. The cells were stained for 1 hour at pH 6.3 at room temperature with naphthyl butyrate as the substrate by using a commercial kit (Sigma, St. Louis, MO) according to the manufacturer's instructions.12 Images were obtained using an Olympus BHA Microscope (System Microscope, Steindamm, Germany). Nonspecific esterase cytochemical staining of MPMs from either untreated mice as well as from diabetic mice revealed that the cells were positive for nonspecific esterase, indicating their monocytic origin. Only adherent seeded cells were used for experiments ensuring that only macrophages (and not undifferentiated monocytes) were studied.

The J-774 A.1 murine macrophage-like cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD). J-774 A.1 cells were plated at 2.5 × 105 cells/16-mm dish in DMEM supplemented with 10% fetal calf serum, 100 U penicillin/ml, 100 μg streptomycin/ml, and 2 mM glutamine. The cells were fed every 3 days and used for experiments within 7 days of plating.13 For in vitro experiments, J774 A.1 macrophages were incubated with D-glucose-enriched media (5 or 30 mM) or with 30 mM D-glucose mixed with 200 mU/L insulin, in the presence of fetal calf serum for 18 hours before the experiments.

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Macrophage Oxidative Stress

Flow cytometric assay was determined by incubation of the cells with DCFH-DA. Under oxidative stress, DCFH is oxidized to 2′,7′-dichlorofluorescein (DCF), which is fluorescent. Cellular fluorescence was determined with a flow cytometer (FACS-SCAN; Becton Dickinson, San Jose, CA) at 510 to 540 nm after excitation of cells at 488 nm with an argon ion laser.14

Macrophages PD were assayed in MPM sonicate.1 Peritoneal macrophages were suspended in PBS (6 × 106/ml) and sonicated at 80W for 3 × 20 seconds. Cellular PD was determined in the sonicate by the PD assay using a spectrophotometer (Ultrospec 3000; Pharmacia Biotech, Cambridge, England).16

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Macrophages Cholesterol Metabolism

Macrophage Cholesterol Content

MPMs (3 × 106/well) sonicated in PBS containing 10 μmol/L butylated hydroxytoluene were extracted with 3 volumes of diethyl ether containing 5 α-cholestane (10 μL of 50 μg/mL stock) as internal standard. The upper phase was dried, and cholesterol content in the samples was measured after sonification by gas chromatography-mass spectrometry (GC-MS) analysis.17

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Macrophage Uptake of Ox-LDL

Macrophages were incubated at 37°C for 3 hours with FITC-conjugated Ox-LDL at a concentration of 10 mg of protein/l. The uptake of the lipoproteins was determined by flow cytometry. Measurements of cellular fluorescence determined by FACS were done at 510 nm to 540 nm after excitation of the cells at 488 nm with an argon ion laser. A total of 10,000 events were registered for each experiment. Cellular fluorescence was quantitated by mean fluorescence intensity (MFI).18

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Cellular Cholesterol Biosynthesis

Cellular cholesterol biosynthesis was assayed after incubation of macrophages (3 × 106/well) with DMEM containing 2% BSA followed by additional 3 hours of incubation at 37°C with [3H]-acetate (3.3 μCi/mL). Cellular lipids were extracted with hexane:isopropanol (3:2, v:v), and the upper phase was dried under nitrogen. The lipids were then separated by TLC and developed in hexane:ether:acetic acid (130:30:1.5, v:v:v). Unesterified cholesterol spots were visualized by iodine vapor (by using standard for identification) and counted with ß-counter.19

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Cellular Cholesterol Efflux

Mouse peritoneal macrophages were incubated with [3H]-labeled cholesterol for 18 hours at 37°C followed by cell wash in ice-cold PBS (×3) and further incubation in the absence or presence of 100 mg of HDL protein/ml for 3 hours at 37°C. Cellular and medium [3H]-labels were quantitated, and HDL-mediated cholesterol efflux was calculated as the ratio of [3H]-label in the medium/[3H]-label in the medium+[3H]-label in cells.

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mRNA Expression of p47phox and of HMGCoA Reductase

mRNA expression of p47phox and of HMGCoA reductase was analyzed by RT-PCR. Total RNA was extracted from cells with Tri-reagent (Molecular Research Center, Cincinnati, OH). cDNA were generated from 2 μg of total RNA using reverse transcriptase (RT; Boehringer-Mannheim, Mannheim, Germany) and oligo (dT) primers (Boehringer-Mannheim). The RT reaction was carried out at 37°C for 1 hour and at 95°C for 5 minutes. Products of the RT reaction were diluted 1:10 or 1:2.5 and subjected to PCR amplifications using specific primers as listed bellow:

Mouse p47phox: the forward primer 5′-ACATCACAGGCCCCATCATCCTTC-3′, the reverse primer 5′-ATGGATTGTCCTTTGTGCC-3′.20 Mouse HMG-CoA reductase: the forward primer 5′-GGGACGGTGACACTTACCATCTGTATGATG-3′, the reverse primer 5′-ATCATCTTGGAGAGATAAAACTGCCA-3′.21 The amplification conditions were: denaturation at 94°C for 30 seconds, annealing at 56°C for 20 seconds, and extension at 72°C for 60 seconds. The β-actin gene was used as housekeeping gene using the forward primer 5′-CTGCCATTTGCAGTGGCAAAGTGG-3′ and the reverse primer 5′-TTGTCATGGATGACCTTGGCCAGG-3′. Specific PCR products obtained for mouse p47phox, HMG CoA reductase and β-actin (615, 882, and 439 bp, respectively) were analyzed on a 1% agarose gel in Tris borate/EDTA buffer, visualized by staining with 1 mg/mL ethidium bromide, and documented on Polaroid film.

PCR was conducted using increasing concentrations of cDNA (2.5 to 10 ng) for the different genes studied (p47, HMGCoA-reductase). The range of linearity of the amplification was determined for each gene, and results of the different experiments were analyzed according to the linear range. In this instance, changes in gene expression could be detected and interpreted.

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SREBP Determination by Immunoblotting

Cell Lysate Preparation

J.774 A.1 macrophages were stimulated with glucose under the specific experimental conditions. Cells were then washed, scraped in 2 ml of PBS, and pelleted (1000 × g for 10 minutes at 4°C). Pellets were suspended in 0.2 to 0.5 mL of lysis buffer [50 mM Tris Base pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, protease cocktail inhibitor (Sigma)].22

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Nuclear Extract Preparation

2 × 107 cells were harvested and washed twice with ice-cold PBS and resuspended in 0.5 ml of buffer A (10 mM Hepes, 10 mM KCl, 1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF, 1.5% NP-40). The cells were kept on ice for 15 minutes, vortexed for 10 seconds, and centrifuged at 16,000 × g at 4°C for 1 minute. The pellet was resuspended in 0.2 ml of Buffer C (20 mM Hepes, 0.4 M NaCl, 1 mM EDTA, 0.5 mM PMSF), placed on ice for 15 minutes, and centrifuged at 16,000 × g at 4°C for 5 minutes. The supernatant was saved as nuclear extract and stored at -80°C.22

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Immunoblotting

Cell lysates and nuclear fractions were separated electrophoretically by SDS-PAGE (7.5% acrylamide and 10% acrylamide, respectively) and transferred to nitrocellulose membranes. Membranes were incubated with antibodies for HMGCR (goat polyclonal antihuman, C-18, sc-27578; Santa Cruz Biotechnology, Santa Cruz, CA) and SREBP-1 [mouse monoclonal antibody (2A4), sc- 13551; Santa Cruz Biotechnology, Santa Cruz, CA].22 Donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP secondary antibodies were obtained from Santa Cruz Biotechnology. Detection was performed with ECL (Amersham Biosciences, Sweden).

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Statistics

Student t test (two-tailed) was performed when comparing 2 arrays of data, and analysis of variance was used when more than 2 groups were compared. Results are expressed as mean ± SD.

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RESULTS

In the present study, we investigated the effect of insulin on diabetes induction of foam cell formation by analyses of macrophage lipids peroxidation, macrophage uptake of oxidized LDL by the cells, and macrophages cholesterol biosynthesis.

Diabetes was induced in Balb-C mice using STZ injection. Glucose blood levels of control nondiabetic mice averaged 98 ± 13 mg/dL. STZ injection increased serum glucose concentration by 3.5 fold up to 340 ± 70 mg/dL. Insulin treatment reduced glucose concentration compared to diabetic mice by 59% up to 140 ± 10 mg/dL.

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Foam Cell Formation in Diabetic Mice Treated With Insulin

We investigated whether insulin administration to diabetic mice affects their macrophages conversion into foam cell by determination of cellular lipids peroxidation and cholesterol cellular metabolism. Macrophage total peroxides and lipid peroxides levels in diabetic mice treated with insulin were decreased by 41% and by 40%, respectively, compared to age-matched untreated diabetic mice. Macrophage total peroxides, and lipid peroxides levels in diabetic mice were increased by 286% and by 61%, respectively, compared to age-matched control mice (Figure 1, A and B). Macrophage uptake of Ox-LDL and cellular cholesterol efflux were studied in diabetic mice treated with insulin compared to diabetic mice. Similarly, macrophage Ox-LDL uptake and CD36 mRNA levels in diabetic mice treated with insulin were also decreased by 29% and by 41%, respectively, compared to age-matched diabetic mice. Macrophage Ox-LDL uptake and CD36 mRNA levels in diabetic mice were increased by 20% and by 98%, respectively, compared with control, age-matched mice (Figure 1, C and D). HDL-mediated cholesterol efflux from macrophages was significantly increased by 163% on using macrophages from nontreated diabetic mice treated with insulin, compared to MPM from diabetic mice (21 ± 3% HDL-mediated cholesterol efflux from macrophages in insulin-treated diabetic mice and 8 ± 2% in nontreated diabetic mice, P < 0.01, n = 4). HDL-mediated cholesterol efflux from macrophages was significantly decreased by 71% in macrophages from diabetic mice, compared to MPM from control mice (HDL-mediated cholesterol efflux from macrophages was 27 ± 4% in control mice and 8 ± 2% in diabetic mice, P < 0.01, n = 4). P47 mRNA expression was determined in diabetic mice administrated with insulin. Macrophages isolated from diabetic mice, diabetic mice treated with insulin, and control mice exhibited p47 mRNA expression levels (normalized to b-actin levels) of 0.81, 0.41, and 0.42 Arbitrary units, respectively, therefore showing that insulin significantly abolished diabetes effect on p47 expression in macrophages.

FIGURE 1

FIGURE 1

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The Effect of Insulin on Glucose-Induced Foam Cell Formation

To determine whether insulin effects on mouse peritoneal macrophage lipid peroxidation and cholesterol accumulation resulted from a direct effect on the cells and to verify whether these effects were common to different sources of macrophages, we turned to an in vitro system, using J-774 A.1 macrophages that were incubated with high levels of glucose (30 mM) in the presence or absence of insulin. Macrophages incubated with low concentration of glucose (5 mM) were used as control. Addition of insulin to glucose-enriched cells led to a significant decrease in cellular lipid peroxidation by 43% as measured by DCFH flow cytometry analysis, compared to cells incubated with the high concentrations of glucose, but with no insulin (Figure 2, A and B). Figure 2B illustrated the graphical expression of flow cytometry analysis of cellular lipid peroxidation. Shift of the peak to the right indicates an increase in lipids peroxidation. Treatment of macrophages with 30 mM glucose indeed resulted in a clear shift to the right of the cells' fluorescence, compared to cells treated with the low levels of glucose (5 mM). Treatment of the cells with insulin (in addition to the high glucose concentrations) reversed the high glucose effect on cellular peroxidation, and the cells' fluorescence was shifted toward the left part, indicating a reduction in fluorescence and hence in cellular peroxidation (Figure 2B). We have previously shown that NADPH oxidase was involved in glucose-induced macrophage oxidative stress; therefore, we next analyzed the effect of insulin on NADPH oxidase, by determination of mRNA expression of the cytosolic component of NADPH oxidase, p47phox. p47 mRNA expression was significantly down-egulated by 26% in glucose-enriched macrophages after their incubation with insulin, compared to macrophages incubated with high glucose but without insulin. An important observation is that p47 mRNA expression was significantly upregulated by 60% after macrophage incubations with high glucose concentration compared to macrophages that were incubated with low glucose concentration (Figure 2C). Finally, we have investigated the effect of insulin on macrophage CD36 mRNA expression. Incubations of glucose-enriched macrophages with insulin led to a significant downregulation of the Ox-LDL receptor, CD36, mRNA expression by 37% compared to cells incubated only with glucose (Figure 2D, P < 0.01, n = 4). Again, on using glucose-enriched cells compared to macrophages that were treated with low glucose, a 73% increment in CD-36 mRNA expression was noted (Figure 2D).

FIGURE 2

FIGURE 2

HDL-mediated cholesterol efflux from macrophages was significantly increased by 350% on using macrophages incubated with high glucose in the presence of insulin, compared to macrophages incubated only with glucose (18 ± 2% HDL-mediated cholesterol efflux from macrophages incubated with high glucose in the presence of insulin and 4 ± 2% in nontreated diabetic mice, P < 0.01, n = 4). HDL-mediated cholesterol efflux from macrophages was significantly decreased by 300% in macrophages incubated with high concentrations of glucose, compared to macrophages incubated with low concentrations of glucose (HDL-mediated cholesterol efflux from macrophages was 12 ± 3% in macrophages incubated with low concentrations of glucose and 4 ± 2% in macrophages incubated with high concentrations of glucose, P < 0.01, n = 4).

Most important, macrophage cholesterol accumulation was significantly affected because total cholesterol content of macrophages incubated with insulin with high concentration of glucose was lower by 41% compared to macrophages incubated only with glucose. Macrophage cholesterol content in macrophages incubated with insulin and high glucose was 33.8 ± 3.4 nmol/mg cell protein compared to 57 ± 5 nmol/mg cell protein in macrophages incubated with glucose only. Incubations of macrophage with high glucose concentrations led to an increase in macrophage cholesterol content by 418% compared to macrophages incubated with low concentrations of glucose. Macrophage cholesterol content in macrophages incubated with high glucose was 57 ± 5 nmol/mg cell protein compared to 11 ± 0.9 nmol/mg cell protein in macrophages incubated with low glucose concentrations.

To verify whether insulin incubation with the cells affected glucose uptake by the cells, we have determined glucose levels in media of cells incubated with glucose in the presence of insulin. Media from J-774 A.1 macrophages incubated with 5 mM glucose, 5 mM glucose with 200 mU insulin, 30 mM glucose, or 30 mM glucose with 200 mU insulin exhibited glucose levels of 121 ± 29, 136 ± 48, 1765 ± 48, 1962 ± 108 mg/mg cell protein, therefore showing no effect of insulin on glucose uptake in vitro.

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Effect of Insulin on Macrophage Cholesterol Biosynthesis

We next determined the regulation of macrophage cholesterol biosynthesis by insulin. We used 2 different models of macrophages, ie, the ex vivo model (MPMs isolated from diabetic mice that were treated with insulin), and an in vitro model of macrophages (glucose-enriched J-7774 A.1 macrophages that were incubated with insulin). As cholesterol biosynthesis rate-limiting step involves activation of the enzyme HMG-CoA reductase, we determined macrophage cholesterol biosynthesis as well as HMG-CoA reductase mRNA expression and HMG-CoA reductase protein expression. Insulin treatment of diabetic mice significantly reduced macrophage cholesterol biosynthesis, HMG-CoA Reductase mRNA expression, and HMGCOA reductase protein expression by 81%, 54%, and 31%, respectively, compared to macrophages isolated from nontreated diabetic mice (Figure 3, A, B, and C). Macrophages from diabetic mice exhibited a significant increase in their cholesterol biosynthesis, HMG-CoA Reductase mRNA expression, and HMGCOA reductase protein expression by 7-fold, 39%, and 83%, respectively, compared to macrophages isolated from control nondiabetic mice (Figure 3, A, B, and C).

FIGURE 3

FIGURE 3

Similarly, insulin incubation with glucose-enriched macrophages significantly reduced macrophage cholesterol biosynthesis, HMG-CoA Reductase mRNA expression, and HMGCoA reductase protein expression by 84%, 42%, and 18%, respectively, compared to macrophages incubated with high glucose but without insulin (Figure 3, D, E, and F). Glucose-enriched macrophages exhibited a significant increase in their cholesterol biosynthesis, HMG-CoA reductase mRNA expression, and HMGCoA reductase protein expression by 6.3-fold, 110%, and 151%, respectively, compared to macrophages incubated with low concentrations of glucose (Figure 3, D, E, and F).

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Regulation of SREBP Upon Glucose and Insulin Treatment of Macrophages

To gain an insight into the mechanism by which glucose and insulin regulate the expression of HMG-CoA reductase, we analyzed their effects on posttranslational activation of the transcription factor SREBP-1 because it is known to regulate the expression of HMGCoA Reductase in a feedback manner. To test whether glucose and insulin treatment affect the amount of the mature form of SREBP-1, we performed Western blots with a specific antibody against SREBP-1. To determine whether glucose and insulin affect SREBP-1 regulation before or after SREBP-1 activation, we determined both the precursor form (in the cytosol) and the mature form (in the cytosol and the nucleus) of SREBP-1. The levels of cytosolic precursor SREBP-1, nuclear precursor SREBP-1, and nuclear mature SREBP-1 were significantly increased by 104%, 178%, and 122%, respectively, after cell treatment with high glucose concentrations compared to cells treated with low levels of glucose (Figure 4, A, B, and C). To further evidence that glucose activated HMGCoA Reductase gene expression in an SREBP-dependent pathway, we used 25-hydroxycholesterol (25-OH). Addition of 25-OH to glucose-enriched macrophages completely blocked the effect of glucose on cholesterol biosynthesis. Cholesterol biosynthesis increases from 4071 cpm/mg cell protein in control cells to 24946 cpm/mg cell protein in glucose-treated cells, whereas cells treated with glucose together with 25-OH show a cholesterol biosynthesis of 5611 cpm/mg cell protein. Furthermore, addition of 25-OH to glucose-enriched macrophages abrogated the formation of nuclear mature SREBP-1. Densitometric analysis of the nuclear SREBP-1 fragment was 0.49 ± 0.03 in control cells and 0.15 ± 0.01 AU in control cells incubated with 25-OH, whereas it was 1.47 ± 0.09 in glucose-treated cells and of 0.338 ± 0.02 in glucose-treated cells incubated with 25-OH. These data further support the involvement of SREBP-1 in glucose effect on cellular cholesterol biosynthesis.

FIGURE 4

FIGURE 4

The levels of nuclear precursor SREBP-1 and nuclear mature SREBP-1 were significantly decreased by 88% and 100%, respectively, after cells treatment with insulin in the presence of high glucose concentrations compared to cells treated only with glucose, whereas insulin treatment did not affect the level of cytosolic precursor SREBP-1 (Figure 4, A, B, and C).

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DISCUSSION

This study presents novel findings on the cellular mechanisms involved in diabetes-induced foam cell formation as well as on insulin effects and mechanisms of action. We have shown that high glucose-induced increased cholesterol biosynthesis involve activation of the transcription of the cytosolic precursors SREBP-1 and the formation of mature SREBP-1. Insulin effects reside in its ability to inhibit the formation of mature SREBP-1. We have also shown that the glucose atherogenic effects on foam cell formation, including increased such as cellular lipid peroxidation and oxidized lipoprotein uptake, were abolished by treatment with insulin in vitro and in vivo.

Cholesterol accumulation in macrophages can result from increased lipoproteins uptake, increased cholesterol biosynthesis, decreased cholesterol efflux, and increased cellular peroxidation. This study clearly shows that all of these processes were adversely affected by diabetes induction and by macrophage incubation with high glucose concentration. These changes were able to induce foam cell formation acceleration as total cholesterol content in macrophages incubated with high glucose, whereas insulin treatment reduced it. Moreover, insulin was able to inhibit diabetes-induced lipid peroxidation through the inhibition of NADPH oxidase expression, cholesterol accumulation (inhibition of CD36, the Ox-LDL receptor), and inhibition of HMGCoA reductase expression. The present study demonstrated a significant increase in cellular cholesterol biosynthesis (both in vitro and in vivo) in glucose-enriched cells and in macrophages from diabetic mice, paralleled by an upregulation of the cholesterol biosynthesis rate-limiting enzyme, HMG-CoA reductase.

Mouse peritoneal macrophages are widely used as a tool for understanding macrophage cholesterol and lipoproteins metabolism in vitro. Even though peritoneal macrophages are not arterial macrophages and obviously they are not participating in the formation of atheroma, this model is accepted for studies of macrophage cholesterol metabolism and foam cell formation, as these cells mimic arterial macrophages present in atheroma areas.23,24

Macrophages are insulin-sensitive cells. However, they are different from other insulin-responsive cells, such as muscle and adipocytes, because they lack the insulin-sensitive glucose-transporter GLUT4.25 Thus, the effects of insulin observed in the present study do not reflect the classical effect of insulin on glucose cellular transport, which allows the uptake of glucose by the cells through GLUT4. In this case we are evaluating a specific effect of insulin on macrophage foam cell formation that could be distinct from its effects on glucose.

There are some lines of evidence suggesting that insulin may have beneficial effects on the cardiovascular system. Insulin causes vasodilation in healthy humans and improves endothelial relaxation through nitric oxide production.26 Insulin is a key hormone regulating glucose and lipid metabolism. Besides its ability to regulate glucose transport, it seems that insulin possesses intrinsic effects that allow it to directly inhibit macrophage foam cell formation. Previous reports have shown that insulin directly affects the activity of genes that have insulin-responsive regions in their promoters.27 According to our results, insulin regulates the expression of genes involved in cholesterol accumulation and foam cell formation such as CD-36 (scavenger receptor for oxidized LDL) and p-47 (a subunit of the NADPH complex). Moreover insulin resistance could be responsible for the increased risk rather than the compensatory hyperinsulinemia observed with insulin resistance.26 Indeed, a recent study showed an association between insulin resistance and reduced CD36 expression.28 Insulin resistance was shown to be associated with increased lipid peroxidation attributable to increased production of free radicals,29 along with hyperglycemia leading to an increase on intracellular reactive oxygen species.30

The sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) family that have been shown to regulate gene expression of several enzymes implicated in cholesterol, lipids, and glucose metabolism.6 To date, 3 members of the SREBP family, SREBP-1a, SREBP-1c, and SREBP-2, have been characterized. All SREBPs are synthesized as transcriptionally inactive precursors that are bound to the endoplasmic reticulum (ER) and nuclear envelope.7 They are activated by proteolytic cleavage in the Golgi apparatus to produce the N-terminal mature transcription factors that migrate into the nucleus, where they can bind to sterol response elements (SRE) in the promoter region of target genes and modulate the gene transcription.6,7 The anti-SREBP antibody used in this study is common to antigens against SREBP1a and SREBP1c. SREBPs represent a family of membrane-bound transcription factors, which directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines. SREBP-2 favors cholesterologenesis, whereas SREBP-1 is a potent activator of all SREBP-responsive genes, including these that mediate the synthesis of cholesterol. A large number of studies have demonstrated that SREBP-1c is tightly regulated by nutritional and hormonal status, especially at the transcriptional level, in various tissues.31 For instance, fasting decreases SREBP-1c mRNA and protein levels, whereas they are markedly increased upon feeding with a high carbohydrate diet.31 Insulin itself was shown to be a potent inducer of SREBP-1c transcription in various cell models and in rodent tissues, including liver, adipose tissue, and skeletal muscle.9 To date, the mechanism by which insulin triggers the transcription of SREBP-1c is not fully defined.

The present study demonstrated that the SREBP-1 precursor protein was increased after cell treatment with glucose, and this effect was reversed by the addition of insulin. These results suggest that SREBP-1 synthesis and its processing are both required for the transcriptional effects of glucose on the HMGCoA Reductase gene and hence on cholesterol biosynthesis in macrophages. Moreover, insulin inhibitory effects on high glucose-mediated changes in SREBP-1 expression were shown to reside in its ability to inhibit SREBP-1 processing rather than an effect on SREBP1 synthesis. In contrast to our study, previous studies have shown that insulin activates SREBP-1 expression. These studies however concentrate on the intrinsic effects of insulin, whereas our study analyzed insulin effects on glucose-mediated induction of cholesterol biosynthesis leading to cholesterol accumulation and foam cell formation.

In conclusion, the present study presents important novel insights on the events connecting diabetes and glucose stimulation of macrophages foam cell formation leading to atherosclerosis. Most important, the inhibitory effects of insulin on diabetes-mediated (and high glucose-induced) increased cholesterol synthesis were shown to involve modulation of SREBP-1 expression and its maturation.

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Keywords:

diabetes; macrophages; atherosclerosis; oxidative stress; cholesterol biosynthesis

© 2008 Lippincott Williams & Wilkins, Inc.