Oral Insulin Supplementation Attenuates Atherosclerosis Progression in Apolipoprotein E-Deficient Mice
Abstractayn, http://www.100md.com
Objective— The role of insulin in atherosclerosis progression in diabetes is uncertain. We examined the effects of oral insulin supplementation on atherogenesis in apolipoprotein E-deficient (E0) mice.ayn, http://www.100md.com
Methods and Results— One-month-old male E0 mice were orally supplemented with human insulin (0.1, 0.5, and 1 U/mL) or placebo for 3 months. At the end of the study, serum and macrophage oxidative stress and atherosclerosis progression were studied. Insulin reduced lesion size by 22% to 37% (P<0.05) in all study groups. Lipid peroxides serum levels were 18% lower (P<0.01), and serum paraoxonase activity was 30% higher (P<0.01) in mice supplemented with 1.0 U/mL insulin compared with controls. Insulin reduced mouse peritoneal macrophage (MPM) lipid peroxides content and superoxide anion release by up to 44% and 62%, respectively (P<0.01). In addition, oral insulin reduced MPM cholesterol content and cholesterol biosynthesis by up to 36% and 53%, respectively (P<0.01). In vitro incubation of E0 mice MPM with increasing insulin concentrations (0 to 100 µU/mL) resulted in a dose-dependent reduction of cholesterol synthesis by up to 66% (P<0.05).
Conclusions— In E0 mice, oral insulin supplementation attenuates the atherosclerotic process. This may be attributable to insulin-mediated reduction of oxidative stress in serum and macrophages as well as reduction in macrophage cholesterol content.kk5/l, 百拇医药
Key Words: oral insulin atherosclerosis oxidative stress paraoxonase macrophageskk5/l, 百拇医药
Introductionkk5/l, 百拇医药
Atherosclerosis is the major cause of morbidity and mortality in the western world, and diabetes mellitus is one of the major risk factors for atherosclerosis development.1 The role of insulin, however, in atherosclerosis progression is uncertain. Hyperinsulinemia is considered to be an independent risk factor for atherosclerosis development,1,2 but there are some lines of evidence suggesting a protective role for insulin.3kk5/l, 百拇医药
Recently, we demonstrated that oral insulin supplementation increased insulin serum levels in Balb-C mice.4 In the present study, we evaluated the effect of oral insulin supplementation on atherosclerosis progression in apolipoprotein E-deficient (E0) mice. The E0 mice model is widely used to study atherogenesis, because these mice develop severe hypercholesterolemia and accelerated atherosclerosis and their atherosclerotic lesion composition is similar to human atherosclerotic lesion.5 In addition, the E0 mice are under oxidative stress, and their serum lipids, as well as their harvested macrophages, contain oxidized lipids.6,7 Similarly, diabetic patients are under oxidative stress and prone to develop atherosclerosis.8 Thus, we questioned whether insulin supplementation to these mice has an effect on the oxidative status of serum and macrophages. Furthermore, because macrophages play an essential role in the development of atherosclerosis9,10 and cholesterol accumulation in macrophages leads to foam cell formation,9,10 we evaluated the effects of insulin supplementation on cholesterol metabolism in macrophages.
In the present study, we have demonstrated for the first time that oral insulin supplementation to the atherosclerotic E0 mice resulted in a significant reduction in aortic atherosclerotic lesion size, and this may be mediated by reduction of serum and macrophage oxidative status as well as reduction in macrophage cholesterol biosynthesis.{g], http://www.100md.com
Methods{g], http://www.100md.com
Materials{g], http://www.100md.com
Cytochrome C from horse heart, SOD from bovine erythrocytes, and BSA were purchased from Sigma. DMEM, HBSS, RPMI, PBS, FCS, streptomycin, and penicillin were purchased from Biological Industries. [3H]-acetate sodium salt or [14C]-mevalonate was purchased from Amersham.{g], http://www.100md.com
Mice Studies{g], http://www.100md.com
The study conformed with the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health. E0 mice were generously provided by Dr Jan Breslow, Rockefeller University, New York.
Human insulin (Humulin R, Eli Lilly) was given to 4-week-old male E0 mice in their drinking water for 3 months. Mice were supplemented with 0.1 U/mL (n=6), 0.5 U/mL (n=6), or 1 U/mL (n=6) of insulin. The placebo group received only water (n=6). All animals were fed an identical chow diet.iaw, 百拇医药
Mice Weight and Blood Pressureiaw, 百拇医药
Mice body weights and blood pressure were recorded before and at the end of the treatment period in all animals, as described previously.11 All blood pressure measurements were repeated 5 times for each animal.iaw, 百拇医药
Blood Glucose and Insuliniaw, 百拇医药
At the end of the study period, blood was collected from the retroorbital plexus. Blood glucose was determined using a Glucometer (Elite, Kyoto Daiichi Kagaku Co) and by using an enzymatic kit (Roche Diagnostics). Insulin concentrations were determined by radioimmunoassay with a commercial kit (DiaSorin). The principle of the assay is based on the competition between labeled insulin and insulin contained in standards or specimens to be assayed for a fixed and limited number of antibody binding sites. The amount of labeled insulin bound to the monoclonal antibody (which is 100% specific for human insulin) is inversely related to the amount of unlabelled human insulin present in the sample. The assay sensitivity, defined as the apparent concentration of analyte that can be distinguished from the zero standard, is below 4 µU/mL at 95% confidence limit. The assay accuracy has been checked by the dilution and recovery tests.
Serum Lipids|'\, http://www.100md.com
Serum total cholesterol and triglycerides were measured by enzymatic spectrophotometric determination.12,13 Direct measurement of high-density lipoprotein cholesterol was performed using polyethylene glycol-modified enzymes as previously described.14|'\, http://www.100md.com
Serum Lipid Peroxidation|'\, http://www.100md.com
Basal serum lipid peroxidation state was determined by measuring lipid peroxides level.15 In this assay, the capacity of peroxide to convert iodide to iodine is determined photometrically at 365 nm.15|'\, http://www.100md.com
Serum Paraoxonase Activity|'\, http://www.100md.com
Arylesterase and paraoxonase activities were measured in serum samples spectrophotometrically using phenylacetate or paraoxon as the substrate, respectively.17 One unit of arylesterase or paraoxonase activity is equal to 1 µmol or 1 nmol of substrate hydrolyzed/mL per min, respectively.16
Histomorphometry of Atherosclerotic Lesions/:2, http://www.100md.com
At euthanasia, the aortic arch was rapidly dissected out and fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) before postfixation in 1% aqueous osmium tetroxide and embedding in epoxy resin as described in detail previously.11 Histomorphometry of areas occupied by atherosclerotic lesions was performed in thin transverse sections of the aortic arch using an Olympus Cue-2 image analysis system.11/:2, http://www.100md.com
Isolation of Mouse Peritoneal Macrophages/:2, http://www.100md.com
Mouse peritoneal macrophages (MPMs) were harvested 4 days after intraperitoneal injection of thioglycolate (72 µg/mouse).7 The MPMs from 6 mice in each group were pooled into two pools. The cell suspension (106/mL) was dispensed into 35-mm dishes and incubated (5% CO2, 95% air) for 2 hours. Cells were washed and additionally incubated under similar conditions./:2, http://www.100md.com
Macrophage Lipid Peroxides
The cell monolayers (3x106) were washed with PBS and lipid extracted with hexane; isopropanol (3:2, vol/vol). The upper hexane phase was collected and dried under nitrogen. NaOH 0.1N was added to the cells for protein determination by the Lowry method.17 The amount of lipid peroxides was determined in the dried samples.15]m(60, 百拇医药
LDL Oxidation by Macrophages]m(60, 百拇医药
LDL was isolated from plasma of fasted normolipidemic human volunteers by discontinuous density gradient ultracentrifugation.18 LDL (100 µg of protein/mL) was incubated without or with MPM (2x106) for 6 hours at 37°C in the presence of 5 µmol/L CuSO4 in RPMI medium (without phenol red). The extent of LDL oxidation was measured as previously described.7]m(60, 百拇医药
Superoxide Anion Generation by Macrophages]m(60, 百拇医药
The production of the superoxide anion (O2-) by MPM (2.5x105/well) with no addition of any stimulator was measured as the SOD inhibitable reduction of acetyl ferricytochrome C.7
Macrophage Cholesterol Mass!y$me, 百拇医药
Macrophage total cholesterol was extracted from the cells (3x106), and its amount in the dried samples was determined using the CHO (CHOD-PAP) Kit (Roche Diagnosticum, Mannheim, Germany). A cholesterol standard (Sigma) was used for the calibration curve.!y$me, 百拇医药
Macrophage Cholesterol Biosynthesis!y$me, 百拇医药
Cells (3x106) were incubation for 18 hours at 37°C with [3H]-acetate (1 µCi/mL) or with [14C]-mevalonate (2.5 µCi/mL). Cellular lipids were extracted, separated by thin layer chromatography on silica gel plates, and developed in hexane:ether:acetic acid (130:30:1.5, vol/vol/vol). Unesterified cholesterol spots were visualized by iodine vapor, using standard for identification, scraped into scintillation vials, and counted in a ß-counter.!y$me, 百拇医药
Statistical Analysis!y$me, 百拇医药
Statistical analysis was performed with SPSS 9.0 software (SPSS Inc). Data are expressed as mean±SD for each group. Comparisons for significant differences between two groups were performed by Student’s t test. For comparison of 4 groups, ANOVA was used. For single parameters, the 2 test was used.
Resultscu, 百拇医药
In Vivo Studiescu, 百拇医药
Effect of Oral Insulin on Atherosclerotic Lesion Developmentcu, 百拇医药
The effect of oral insulin supplementation on atherosclerosis development was analyzed in E0 mice supplemented with 3 different dosages of insulin for 3 months in comparison with placebo-treated mice. Insulin supplementation significantly (P<0.05) reduced the mean atherosclerotic lesion area by 37%, 28%, and 22% in mice supplemented with 0.1, 0.5, and 1.0 U/mL of insulin, respectively, compared with placebo-treated mice . In addition, the mean number of lesions per mouse in the group supplemented with 1.0 U/mL of insulin was 48% lower than the placebo group and the other supplemented mice (P<0.01, ). No major differences were seen in the histopathology of the 4 groups.cu, 百拇医药
fig.ommittedcu, 百拇医药
The effects of insulin supplementation to E0 mice on aortic atherosclerotic mean lesion area and on the number of lesions per mouse. Four groups of 4-week-old E0 mice (6 mice in each group) were included in the study. The placebo group received regular water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. Values are given as mean±SD for each study group. A, Mean size of the atherosclerotic plaque (lesion area). *P<0.05 vs placebo. B, Number of atherosclerotic plaques. *P<0.01 between group supplemented with 1.0 U/mL of insulin vs all other groups.
Effects of Oral Insulin Supplementation on Weight, Blood Pressure, Serum Glucose, Insulin, and Lipid Levels{f5+, 百拇医药
Baseline weight and blood pressure were similar in all study groups, and oral insulin supplementation had no significant effect on these parametersAs shown in the insulin was found in the serum of all study groups, and in the group receiving 1.0 U/mL, it was significantly higher (P<0.05) than in the other study groups, indicating that the oral insulin was indeed absorbed. Serum glucose levels were significantly (P<0.01) lower in all insulin-supplemented groups compared with controls.{f5+, 百拇医药
fig.ommitted{f5+, 百拇医药
Descriptive Data of the Study Groups{f5+, 百拇医药
Effects of Oral Insulin Supplementation on Serum Lipid Peroxidation{f5+, 百拇医药
The effect of oral insulin supplementation on serum lipid peroxidation was studied in mice supplemented with 1.0 U/mL of insulin. Insulin induced significant reduction (P<0.001) of lipid peroxides by 18% from 572±31 nmol/mL in the placebo-treated mice to 471±47 nmol/mL in insulin-supplemented mice.
Serum paraoxonase (PON1) was shown to reduce serum lipid peroxidation.19,20 Therefore, PON1 arylesterase activity was next measured. Insulin supplementation induced significant increase (P<0.001) of arylesterase activity by 30% from 49±3 U/mL in the placebo-treated mice to 69.8±2.5 U/mL in the insulin-supplemented mice. Similar results were obtained on using paraoxon as the substrate (data not shown).qv+]n$o, http://www.100md.com
Effects of Oral Insulin Supplementation on Macrophage Oxidative Stressqv+]n$o, http://www.100md.com
Previous studies demonstrated that macrophages from E0 mice contain lipid peroxides.7,8 Consequently, we determined the amount of lipid peroxides in insulin-supplemented mice MPM. Oral insulin supplementation to E0 mice resulted in a reduction in MPM lipid peroxides by 14%, 44%, and 41% in mice supplemented with 0.1, 0.5, or 1 U/mL insulin, respectively, compared with the level of lipid peroxides in placebo-treated mice MPM ( P<0.01). Accumulation of oxidized lipids in the macrophages affects their capability to release superoxide anions and to oxidize LDL.7 Indeed, the reduced lipid peroxide content was accompanied by a dose-dependent reduction in superoxide anion release from MPM by 49%, 55%, and 62% in mice supplemented with 0.1, 0.5, and 1 U/mL insulin, respectively, compared with placebo-treated mice (P<0.01). LDL oxidation after 6-hour incubation in the presence of 5 µmol/L CuSO4 with MPM harvested from E0 mice supplemented with 1 U/mL was lower by 15% compared with LDL oxidation by placebo-treated mice MPM (18.6±15 versus 21.9±2.1 nmol MDA/mg LDL protein, respectively, not statistically significant).
fig.ommitted4, 百拇医药
Effects of oral insulin supplementation to E0 mice on macrophage oxidative stress. Four groups of 4-week-old E0 mice were included in the study. The placebo group received water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. MPMs were then harvested from the mice. The amount of cellular lipid peroxides (A) and the amount of superoxide anions released from the cells after 1 hour of incubation at 37°C (B) were measured. Results are given as mean±SD. *P<0.01 vs placebo.4, 百拇医药
Effects of Oral Insulin Supplementation on Macrophage Cholesterol Metabolism4, 百拇医药
Accumulation of cholesterol in macrophages converts macrophages into foam cells and is the hallmark of atherosclerotic lesion formation.9,10 Thus, we next measured the amount of total cholesterol in insulin-treated mice MPM. Oral insulin supplementation to E0 mice resulted in a dose-dependent reduction in MPM cellular cholesterol content Whereas in mice supplemented with 0.1 U/mL insulin no significant effect on MPM cholesterol levels was observed, there was a significant reduction (P<0.01) in MPM cholesterol content by 31% and 36% in mice supplemented with 0.5 or 1 U/mL of insulin, respectively, compared with the amount of cholesterol in placebo-mice MPM
fig.ommittedjo?o@, 百拇医药
Effects of oral insulin supplementation to E0 mice on macrophage cholesterol metabolism. Four groups of 4-week-old E0 mice were included in the study. The placebo group received water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. MPMs were then harvested from the mice. A, Cells (3x106) were washed and their lipids were extracted with hexane:isopropanol (3:2, vol/vol) and cellular cholesterol mass was determined. B, Cells were washed and incubated with [3H]-acetate for 18 hours at 37°C for cholesterol biosynthesis determination. Results represent mean±SD of 3 different experiments, each one performed in triplicate. *P<0.01 vs placebo.jo?o@, 百拇医药
To examine if decreased cholesterol biosynthesis may be the cause for reduced cholesterol mass, the ability of macrophages harvested from the insulin-supplemented E0 mice to synthesize cholesterol from [3H]-acetate was next determined. As seen in there was a significant reduction (P<0.01) in MPM cholesterol biosynthesis (by 42% to 53%) in mice supplemented with 0.1 to 1 U/mL of insulin compared with placebo-treated mice MPM
In Vitro Studiese$7[?7, 百拇医药
Effects of Insulin on Macrophage Oxidative Stress In Vitro: Concentration Studye$7[?7, 百拇医药
To assess the direct effects of insulin on macrophage oxidative stress, MPMs from 4-month-old E0 mice were incubated for 18 hours with increasing concentrations of insulin (0 to 100 µU/mL). As can be seen in there was a dose-dependent decrease in MPM lipid peroxide content (up to 46%) after adding 1 µU/mL insulin compared with lipid peroxides content in nontreated MPM. However, the decrease was not statistically significant, and higher insulin concentrations were less effective (adding 10 or 100 µU/mL of insulin reduced MPM lipid peroxides content only by 12% and 20%, respectively,. The amount of superoxide anions released from MPM after their incubation with increasing insulin concentrations (0 to 100 µU/mL) was then measured . Maximal reduction in superoxide anion release was observed with 1 µU/mL of insulin (26%), but the reduction was not statistically significant.
fig.ommitted.5, 百拇医药
Effects of increasing concentrations of insulin on macrophage oxidative stress in vitro. MPM were harvested from 4-month-old E0 mice. The cells were incubated for 18 hours at 37°C with increasing insulin concentrations (0, 0.01, 0.1, 1, 10, and 100 µU/mL) in DMED+5%FCS after cell wash. A, Cellular lipids were extracted using hexane:isopropanol (3:2, vol/vol), for lipid peroxides determination. B, Amount of superoxide anion released from the cells was determined after 1 hour of incubation at 37°C. C, Cells were additionally incubated for 6 hours with LDL (100 µg of protein/mL) in the presence of 5 µmol/L CuSO4. The extent of LDL oxidation was determined by the TBARS assay. Results are given as mean±SD of 3 different experiments each performed in triplicate..5, 百拇医药
We determined next the capability of insulin-treated MPM to oxidize LDL. The addition of insulin (0 to 0.1 µU/mL) to the cells caused a reduction in their capability to oxidize LDL by up to 32%, and this effect was insulin dose-dependentnot statistically significant). However, higher concentrations were less effective, and incubation with 100 µU/mL of insulin resulted in oxidation values similar to those obtained in nontreated cells
Effects of Insulin on Macrophage Cholesterol Metabolism In Vitro: Concentration Study2, 百拇医药
To assess the direct effects of insulin on cholesterol biosynthesis, MPMs from 4-month-old E0 mice were incubated for 18 hours with increasing concentrations of insulin (0 to 100 µU/mL). Adding insulin to the cells resulted in a dose-dependent reduction by up to 66% in cholesterol biosynthesis from [3H]-acetate, compared with the values obtained in nontreated MPM ( P<0.05). The concentration of 0.1 µU/mL was significant (P<0.05) to 0.01 µU/mL, and the concentration of 1 µU/mL was significant (P<0.01) to 0.1 µU/mL. However, a significant reduction in cholesterol biosynthesis from [14C]-mevalonate was seen only at concentrations of 1.0 to 100 µU/mL of insulin ( P<0.01 between each concentration).2, 百拇医药
fig.ommitted2, 百拇医药
Effects of increasing concentrations of insulin on macrophage cholesterol metabolism in vitro. MPMs were harvested from 4-month-old E0 mice. The cells were incubated with increasing insulin concentrations (0, 0.01, 0.1, 1, 10, and 100 µU/mL) for 18 hours at 37°C in DMED+5%FCS. The cells were then washed and additionally incubated for 18 hours at 37°C with [3H]-acetate (A) or with [14C]-mevalonate (B) for cholesterol biosynthesis determination. Results are given as mean±SD of 3 different experiments, each one performed in triplicate. *P<0.01 (vs 0 concentration), &P<0.05 (vs 0.01 µU/mL), #P<0.01 (vs 0.1 µU/mL), @P<0.01 (vs 1 µU/mL), +P<0.01 (vs 10 µU/mL).
Discussionj6, 百拇医药
Supplementation of oral insulin to E0 mice resulted in decreased aortic atherosclerotic mean plaque area and reduced the number of atherosclerotic lesions. These effects of oral insulin were associated with reduced oxidative stress both in serum and in MPM as well as reduced cholesterol biosynthesis in macrophages.j6, 百拇医药
Hyperinsulinemia is an independent cardiovascular risk factor.1,2 However, the contribution of insulin itself to the atherosclerotic process is unknown, because insulin resistance could be responsible for the increased risk rather than the compensatory hyperinsulinemia observed with insulin resistance.3 As part of insulin resistance, there is increased lipid peroxidation attributable to increased production of free radicals,8 hyperglycemia increases intracellular reactive oxygen species,21 and postprandial hyperglycemia may be a risk factor for cardiovascular disease in diabetic patients.22
There are some lines of evidence suggesting that insulin may have beneficial effects on the cardiovascular system. Insulin causes vasodilation in healthy humans3,23 and improves endothelial relaxation through nitric oxide production.3 Insulin attenuates the vascular smooth muscle cell response to vasoactive substances24 and may decrease large arteries’ stiffness.3 Insulin has been shown to lower blood pressure in some studies,24 whereas in another study it had no effect.25;[px^hv, 百拇医药
In contrast, insulin may be related to increased risk for atherosclerosis either secondary to obesity, hyperlipidemia, hypertension, hyperuricemia, and the use of oral contraceptives2 or through inhibition of lipolysis and by induction of vascular smooth muscle cell proliferation.3;[px^hv, 百拇医药
Findings in animal models are few and inconsistent. The administration of subcutaneous insulin to rats caused an increase in triglyceride content in the aorta and in intimal thickening.26 In rabbits fed with an atherogenic diet, the administration of subcutaneous insulin significantly reduced plasma lipid levels, but continued administration of insulin during atherosclerosis development resulted in reinforcement of the lipemia with aggravation of atherosclerosis.27 Interestingly, in that study,27 after switching to a normal diet at the age of 2 months, insulin accelerated the reduction in hypercholesterolemia. In a similar study in rabbits, no effects on atherosclerosis or on lipid serum levels were demonstrated.28 In contrast, we were able to demonstrate in the present study that adding insulin at 1 U/mL to the drinking water of the atherosclerotic E0 mice for 3 months significantly reduced the number of atherosclerotic lesions per mouse, and all insulin concentrations resulted in significant reduction in mean atherosclerotic lesion area. Oral insulin supplementation resulted in a dose-dependent increase in serum insulin concentrations. However, portal insulin levels may be several times higher than those found in the systemic circulation.29
LDL oxidation is important in atherosclerosis progression,30 and in E0 mice, increased plasma lipoprotein lipid peroxidation was documented.7 In our study, insulin supplementation to E0 mice caused a significant reduction in serum lipid peroxides and a significant increase in serum paraoxonase (PON1) activities. The increase in PON1 activity may explain the reduced serum oxidative stress, because it was shown that paraoxonase reduces lipid peroxides levels in oxidized lipoproteins.20 Reduced serum PON1 activity is seen in atherosclerotic patients, including diabetic patients,31 in insulin-deficient (streptozotocin-induced) diabetic rats,32 in E0 mice,22 and in rabbits fed an atherogenic diet.33 Thus, similar to its effect on E0 mice, insulin supplementation may increase PON1 activity in diabetes.{+gxp6), 百拇医药
It was recently shown that E0 mice macrophages, which contain oxidized lipids, release increased amounts of superoxide anions and oxidize LDL at an enhanced rate.7 In that study,7 the reduction of oxidative stress by antioxidants supplementation reduced the amount of lipid peroxides in macrophages and their ability to oxidize LDL. In the present study, insulin supplementation to the E0 mice resulted in the reduction in macrophage lipid peroxides content in association with reduction in the cell’s ability to release superoxide anions and to oxidize LDL. The reduction in macrophage oxidative stress may again be explained by increased paraoxonase activity, because paraoxonase was shown to reduce specific oxidized lipids in macrophages and in atherosclerotic lesion.34,35 In addition, the decreased oxidation observed in our study could be the result of increased reduced glutathione (GSH) concentration. GSH is the major antioxidant in cells, and it has been shown that on incubation of cells with insulin, the amount of GSH was significantly increased.36 In addition, it was shown that increasing GSH cellular content in the cells reduced their capability to release superoxide anions and to oxidized LDL.37 Furthermore, in diabetic rat ventricular myocytes, GSH homeostasis is functionally coupled to insulin signaling.38
To assess the direct role of insulin on macrophages, we performed in vitro experiments. By incubating E0 mice macrophages with increasing concentrations of insulin (0 to 100 µU/mL), we observed no significant trend toward reduced oxidative stress in macrophages. First, this could be the result of differences between the time period macrophages acutely were exposed to insulin in the in vitro studies compared with the chronic exposure in the ex vivo studies. Second, the intracellular concentrations of insulin may have been different in the ex vivo versus the in vitro studies. Third, oral insulin supplementation may have triggered other, not yet identified, antioxidative pathways that could explain the lack of a significant effect in the in vitro studies.\62&}r, 百拇医药
Accumulation of cholesterol in macrophages is an essential step in atherosclerosis progression,9,10 and insulin was found to regulate cholesterol metabolism in macrophages.39,40 In our study, we demonstrated that insulin inhibited cholesterol synthesis in peritoneal macrophages in a dose-dependent manner, both in the ex vivo and in the in vitro studies. Thus, the reduced macrophage cholesterol content in insulin supplemented mice may be attributable to the inhibition of cholesterol synthesis induced by insulin. This is in agreement with other observations that insulin therapy depresses the rate of cholesterologenesis in patients with non-insulin-dependent diabetes mellitus.41 Also, increased cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase activity42 were previously demonstrated in MPM from insulin-deficient mice. In that study, insulin addition resulted in reduced HMGCoA-reductase activity and reduced cholesterol biosynthesis.42 However, in the present study, mevalonate blocked the inhibitory effect of insulin on cholesterol biosynthesis only at low insulin concentrations, suggesting that the inhibition of cholesterol biosynthesis by insulin may involve other, still undetermined, pathways.
In summary, in E0 mice, oral insulin supplementation attenuates the atherosclerotic process. It is conceivable that the effects of oral insulin supplementation on the atherosclerotic process are mediated via effects on oxidative stress in serum and in macrophages as well as effects on macrophage cholesterol content and biosynthesis.n, 百拇医药
Acknowledgmentsn, 百拇医药
This study was supported by a research grant from the Danone Institute, Israel.n, 百拇医药
Received July 17, 2002; accepted September 26, 2002.n, 百拇医药
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Watson PA, Nesterova A, Burant CF, Klemm DJ, Reuch JE. Diabetes-related changes in camp response element-binding protein content enhance smooth muscle cell proliferation and migration. J Biol Chem. 2001; 276: 46142–46150.
Ceriello A. Mechanisms of tissue damage in the postprandial state. Int J Clin Pract Suppl. 2001; 123: 7–12.}'1(;0k, 百拇医药
Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL. Hyperinsulinemia produces both sympathetic neural activation and vasodilatation in normal humans. J Clin Invest. 1991; 87: 2446–2252.}'1(;0k, 百拇医药
Saito F, Hori MT, Fittingoff M, Tuck ML. Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells. J Clin Invest. 1993; 92: 1161–1168.}'1(;0k, 百拇医药
Rowe JW, Young JB, Minaker KL, Stevens AL, Pallotta J, Landsberg L. Effect of insulin and glucose infusions on sympathetic nervous system activity in normal men. Diabetes. 1981; 30: 219–225.}'1(;0k, 百拇医药
Sato Y, Shiraishi S, Oshida Y, Ishiguro T, Sakamoto N. Experimental atherosclerosis-like lesions induced by hyperinsulinism in Wistar rats. Diabetes. 1989; 38: 91–96.}'1(;0k, 百拇医药
Marquie G. Effect of insulin in the induction and regression of experimental cholesterol atherosclerosis in the rabbit. Postgrad Med J. 1978; 54: 80–85.}'1(;0k, 百拇医药
Nordestgaard BG, Agerholm-Larsen B, Stender S. Effect of exogenous hyperinsulinaemia on atherogenesis in cholesterol-fed rabbits. Diabetologia. 1997; 40: 512–520.
Field JB. Extraction of insulin by the liver. Annu Rev Med. 1973; 24: 309–314.{, 百拇医药
Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997; 272: 20963–20966.{, 百拇医药
Aviram M. Does paraoxonase play a role in susceptibility to cardiovascular disease? Mol Med Today. 1999; 5: 356–381.{, 百拇医药
Patel BN, Mackness MI, Hart DW, Arrol S, Boot-Handford RP, Durrington PN. Serum esterase activities and hyperlipidemia in the streptozocin-diabetic rats. Biochim Biophys Acta. 1990; 1035: 113–116.{, 百拇医药
Mackness M, Boullier A, Hennuyer N, Mackness B, Hall M, Tailleux A, Duriez p, Delfly B, Durrington P, Fruchart JC, Duverger N, Caillaud JM, Castro G, Bouiller A. paraoxonase activity is reduced by a pro-atherogenic diet in rabbits. Biochem Biophys Res Commun. 2000; 269: 232–236.{, 百拇医药
Aviram M, Hardak E, Vaya J, Mahmood S, Milo S, Hoffman A, Billecke S, Dragonov D, Rosenblat M. Human serum paraoxonases (PON 1), Q and R selectively decrease lipid peroxides in coronary and carotid atherosclerotic lesions: PON 1 esterase and peroxidase-like activities. Circulation. 2000; 101: 2510–2517.
Fuhrman B, Volkova N, Aviram M. Oxidative stress increases the expression of the CD36 scavenger receptor and cellular uptake of oxidized low-density lipoprotein in macrophages from atherosclerotic mice: protective role of antioxidants and paraoxonase. Atherosclerosis. 2002; 161: 307–316.;sp, http://www.100md.com
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Rosenblat M, Aviram M. Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDL. Free Radic Biol Med. 1998; 24: 305–317.;sp, http://www.100md.com
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Andersen E, Hellstrom P, Hellstrom K. Cholesterol biosynthesis in nonketotic diabetics before and during insulin therapy. Diabetes Res Clin Pract. 1987; 3: 207–214.;sp, http://www.100md.com
Kraemer FB. Insulin deficiency alters cellular cholesterol metabolism in murine macrophages. Diabetes. 1986; 35: 764–770.(Raanan Shamir Naim Shehadeh Mira Rosenblat Orly Eshach-Adiv Raymond Coleman Marielle Kaplan Shadi Ha)
Objective— The role of insulin in atherosclerosis progression in diabetes is uncertain. We examined the effects of oral insulin supplementation on atherogenesis in apolipoprotein E-deficient (E0) mice.ayn, http://www.100md.com
Methods and Results— One-month-old male E0 mice were orally supplemented with human insulin (0.1, 0.5, and 1 U/mL) or placebo for 3 months. At the end of the study, serum and macrophage oxidative stress and atherosclerosis progression were studied. Insulin reduced lesion size by 22% to 37% (P<0.05) in all study groups. Lipid peroxides serum levels were 18% lower (P<0.01), and serum paraoxonase activity was 30% higher (P<0.01) in mice supplemented with 1.0 U/mL insulin compared with controls. Insulin reduced mouse peritoneal macrophage (MPM) lipid peroxides content and superoxide anion release by up to 44% and 62%, respectively (P<0.01). In addition, oral insulin reduced MPM cholesterol content and cholesterol biosynthesis by up to 36% and 53%, respectively (P<0.01). In vitro incubation of E0 mice MPM with increasing insulin concentrations (0 to 100 µU/mL) resulted in a dose-dependent reduction of cholesterol synthesis by up to 66% (P<0.05).
Conclusions— In E0 mice, oral insulin supplementation attenuates the atherosclerotic process. This may be attributable to insulin-mediated reduction of oxidative stress in serum and macrophages as well as reduction in macrophage cholesterol content.kk5/l, 百拇医药
Key Words: oral insulin atherosclerosis oxidative stress paraoxonase macrophageskk5/l, 百拇医药
Introductionkk5/l, 百拇医药
Atherosclerosis is the major cause of morbidity and mortality in the western world, and diabetes mellitus is one of the major risk factors for atherosclerosis development.1 The role of insulin, however, in atherosclerosis progression is uncertain. Hyperinsulinemia is considered to be an independent risk factor for atherosclerosis development,1,2 but there are some lines of evidence suggesting a protective role for insulin.3kk5/l, 百拇医药
Recently, we demonstrated that oral insulin supplementation increased insulin serum levels in Balb-C mice.4 In the present study, we evaluated the effect of oral insulin supplementation on atherosclerosis progression in apolipoprotein E-deficient (E0) mice. The E0 mice model is widely used to study atherogenesis, because these mice develop severe hypercholesterolemia and accelerated atherosclerosis and their atherosclerotic lesion composition is similar to human atherosclerotic lesion.5 In addition, the E0 mice are under oxidative stress, and their serum lipids, as well as their harvested macrophages, contain oxidized lipids.6,7 Similarly, diabetic patients are under oxidative stress and prone to develop atherosclerosis.8 Thus, we questioned whether insulin supplementation to these mice has an effect on the oxidative status of serum and macrophages. Furthermore, because macrophages play an essential role in the development of atherosclerosis9,10 and cholesterol accumulation in macrophages leads to foam cell formation,9,10 we evaluated the effects of insulin supplementation on cholesterol metabolism in macrophages.
In the present study, we have demonstrated for the first time that oral insulin supplementation to the atherosclerotic E0 mice resulted in a significant reduction in aortic atherosclerotic lesion size, and this may be mediated by reduction of serum and macrophage oxidative status as well as reduction in macrophage cholesterol biosynthesis.{g], http://www.100md.com
Methods{g], http://www.100md.com
Materials{g], http://www.100md.com
Cytochrome C from horse heart, SOD from bovine erythrocytes, and BSA were purchased from Sigma. DMEM, HBSS, RPMI, PBS, FCS, streptomycin, and penicillin were purchased from Biological Industries. [3H]-acetate sodium salt or [14C]-mevalonate was purchased from Amersham.{g], http://www.100md.com
Mice Studies{g], http://www.100md.com
The study conformed with the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health. E0 mice were generously provided by Dr Jan Breslow, Rockefeller University, New York.
Human insulin (Humulin R, Eli Lilly) was given to 4-week-old male E0 mice in their drinking water for 3 months. Mice were supplemented with 0.1 U/mL (n=6), 0.5 U/mL (n=6), or 1 U/mL (n=6) of insulin. The placebo group received only water (n=6). All animals were fed an identical chow diet.iaw, 百拇医药
Mice Weight and Blood Pressureiaw, 百拇医药
Mice body weights and blood pressure were recorded before and at the end of the treatment period in all animals, as described previously.11 All blood pressure measurements were repeated 5 times for each animal.iaw, 百拇医药
Blood Glucose and Insuliniaw, 百拇医药
At the end of the study period, blood was collected from the retroorbital plexus. Blood glucose was determined using a Glucometer (Elite, Kyoto Daiichi Kagaku Co) and by using an enzymatic kit (Roche Diagnostics). Insulin concentrations were determined by radioimmunoassay with a commercial kit (DiaSorin). The principle of the assay is based on the competition between labeled insulin and insulin contained in standards or specimens to be assayed for a fixed and limited number of antibody binding sites. The amount of labeled insulin bound to the monoclonal antibody (which is 100% specific for human insulin) is inversely related to the amount of unlabelled human insulin present in the sample. The assay sensitivity, defined as the apparent concentration of analyte that can be distinguished from the zero standard, is below 4 µU/mL at 95% confidence limit. The assay accuracy has been checked by the dilution and recovery tests.
Serum Lipids|'\, http://www.100md.com
Serum total cholesterol and triglycerides were measured by enzymatic spectrophotometric determination.12,13 Direct measurement of high-density lipoprotein cholesterol was performed using polyethylene glycol-modified enzymes as previously described.14|'\, http://www.100md.com
Serum Lipid Peroxidation|'\, http://www.100md.com
Basal serum lipid peroxidation state was determined by measuring lipid peroxides level.15 In this assay, the capacity of peroxide to convert iodide to iodine is determined photometrically at 365 nm.15|'\, http://www.100md.com
Serum Paraoxonase Activity|'\, http://www.100md.com
Arylesterase and paraoxonase activities were measured in serum samples spectrophotometrically using phenylacetate or paraoxon as the substrate, respectively.17 One unit of arylesterase or paraoxonase activity is equal to 1 µmol or 1 nmol of substrate hydrolyzed/mL per min, respectively.16
Histomorphometry of Atherosclerotic Lesions/:2, http://www.100md.com
At euthanasia, the aortic arch was rapidly dissected out and fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) before postfixation in 1% aqueous osmium tetroxide and embedding in epoxy resin as described in detail previously.11 Histomorphometry of areas occupied by atherosclerotic lesions was performed in thin transverse sections of the aortic arch using an Olympus Cue-2 image analysis system.11/:2, http://www.100md.com
Isolation of Mouse Peritoneal Macrophages/:2, http://www.100md.com
Mouse peritoneal macrophages (MPMs) were harvested 4 days after intraperitoneal injection of thioglycolate (72 µg/mouse).7 The MPMs from 6 mice in each group were pooled into two pools. The cell suspension (106/mL) was dispensed into 35-mm dishes and incubated (5% CO2, 95% air) for 2 hours. Cells were washed and additionally incubated under similar conditions./:2, http://www.100md.com
Macrophage Lipid Peroxides
The cell monolayers (3x106) were washed with PBS and lipid extracted with hexane; isopropanol (3:2, vol/vol). The upper hexane phase was collected and dried under nitrogen. NaOH 0.1N was added to the cells for protein determination by the Lowry method.17 The amount of lipid peroxides was determined in the dried samples.15]m(60, 百拇医药
LDL Oxidation by Macrophages]m(60, 百拇医药
LDL was isolated from plasma of fasted normolipidemic human volunteers by discontinuous density gradient ultracentrifugation.18 LDL (100 µg of protein/mL) was incubated without or with MPM (2x106) for 6 hours at 37°C in the presence of 5 µmol/L CuSO4 in RPMI medium (without phenol red). The extent of LDL oxidation was measured as previously described.7]m(60, 百拇医药
Superoxide Anion Generation by Macrophages]m(60, 百拇医药
The production of the superoxide anion (O2-) by MPM (2.5x105/well) with no addition of any stimulator was measured as the SOD inhibitable reduction of acetyl ferricytochrome C.7
Macrophage Cholesterol Mass!y$me, 百拇医药
Macrophage total cholesterol was extracted from the cells (3x106), and its amount in the dried samples was determined using the CHO (CHOD-PAP) Kit (Roche Diagnosticum, Mannheim, Germany). A cholesterol standard (Sigma) was used for the calibration curve.!y$me, 百拇医药
Macrophage Cholesterol Biosynthesis!y$me, 百拇医药
Cells (3x106) were incubation for 18 hours at 37°C with [3H]-acetate (1 µCi/mL) or with [14C]-mevalonate (2.5 µCi/mL). Cellular lipids were extracted, separated by thin layer chromatography on silica gel plates, and developed in hexane:ether:acetic acid (130:30:1.5, vol/vol/vol). Unesterified cholesterol spots were visualized by iodine vapor, using standard for identification, scraped into scintillation vials, and counted in a ß-counter.!y$me, 百拇医药
Statistical Analysis!y$me, 百拇医药
Statistical analysis was performed with SPSS 9.0 software (SPSS Inc). Data are expressed as mean±SD for each group. Comparisons for significant differences between two groups were performed by Student’s t test. For comparison of 4 groups, ANOVA was used. For single parameters, the 2 test was used.
Resultscu, 百拇医药
In Vivo Studiescu, 百拇医药
Effect of Oral Insulin on Atherosclerotic Lesion Developmentcu, 百拇医药
The effect of oral insulin supplementation on atherosclerosis development was analyzed in E0 mice supplemented with 3 different dosages of insulin for 3 months in comparison with placebo-treated mice. Insulin supplementation significantly (P<0.05) reduced the mean atherosclerotic lesion area by 37%, 28%, and 22% in mice supplemented with 0.1, 0.5, and 1.0 U/mL of insulin, respectively, compared with placebo-treated mice . In addition, the mean number of lesions per mouse in the group supplemented with 1.0 U/mL of insulin was 48% lower than the placebo group and the other supplemented mice (P<0.01, ). No major differences were seen in the histopathology of the 4 groups.cu, 百拇医药
fig.ommittedcu, 百拇医药
The effects of insulin supplementation to E0 mice on aortic atherosclerotic mean lesion area and on the number of lesions per mouse. Four groups of 4-week-old E0 mice (6 mice in each group) were included in the study. The placebo group received regular water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. Values are given as mean±SD for each study group. A, Mean size of the atherosclerotic plaque (lesion area). *P<0.05 vs placebo. B, Number of atherosclerotic plaques. *P<0.01 between group supplemented with 1.0 U/mL of insulin vs all other groups.
Effects of Oral Insulin Supplementation on Weight, Blood Pressure, Serum Glucose, Insulin, and Lipid Levels{f5+, 百拇医药
Baseline weight and blood pressure were similar in all study groups, and oral insulin supplementation had no significant effect on these parametersAs shown in the insulin was found in the serum of all study groups, and in the group receiving 1.0 U/mL, it was significantly higher (P<0.05) than in the other study groups, indicating that the oral insulin was indeed absorbed. Serum glucose levels were significantly (P<0.01) lower in all insulin-supplemented groups compared with controls.{f5+, 百拇医药
fig.ommitted{f5+, 百拇医药
Descriptive Data of the Study Groups{f5+, 百拇医药
Effects of Oral Insulin Supplementation on Serum Lipid Peroxidation{f5+, 百拇医药
The effect of oral insulin supplementation on serum lipid peroxidation was studied in mice supplemented with 1.0 U/mL of insulin. Insulin induced significant reduction (P<0.001) of lipid peroxides by 18% from 572±31 nmol/mL in the placebo-treated mice to 471±47 nmol/mL in insulin-supplemented mice.
Serum paraoxonase (PON1) was shown to reduce serum lipid peroxidation.19,20 Therefore, PON1 arylesterase activity was next measured. Insulin supplementation induced significant increase (P<0.001) of arylesterase activity by 30% from 49±3 U/mL in the placebo-treated mice to 69.8±2.5 U/mL in the insulin-supplemented mice. Similar results were obtained on using paraoxon as the substrate (data not shown).qv+]n$o, http://www.100md.com
Effects of Oral Insulin Supplementation on Macrophage Oxidative Stressqv+]n$o, http://www.100md.com
Previous studies demonstrated that macrophages from E0 mice contain lipid peroxides.7,8 Consequently, we determined the amount of lipid peroxides in insulin-supplemented mice MPM. Oral insulin supplementation to E0 mice resulted in a reduction in MPM lipid peroxides by 14%, 44%, and 41% in mice supplemented with 0.1, 0.5, or 1 U/mL insulin, respectively, compared with the level of lipid peroxides in placebo-treated mice MPM ( P<0.01). Accumulation of oxidized lipids in the macrophages affects their capability to release superoxide anions and to oxidize LDL.7 Indeed, the reduced lipid peroxide content was accompanied by a dose-dependent reduction in superoxide anion release from MPM by 49%, 55%, and 62% in mice supplemented with 0.1, 0.5, and 1 U/mL insulin, respectively, compared with placebo-treated mice (P<0.01). LDL oxidation after 6-hour incubation in the presence of 5 µmol/L CuSO4 with MPM harvested from E0 mice supplemented with 1 U/mL was lower by 15% compared with LDL oxidation by placebo-treated mice MPM (18.6±15 versus 21.9±2.1 nmol MDA/mg LDL protein, respectively, not statistically significant).
fig.ommitted4, 百拇医药
Effects of oral insulin supplementation to E0 mice on macrophage oxidative stress. Four groups of 4-week-old E0 mice were included in the study. The placebo group received water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. MPMs were then harvested from the mice. The amount of cellular lipid peroxides (A) and the amount of superoxide anions released from the cells after 1 hour of incubation at 37°C (B) were measured. Results are given as mean±SD. *P<0.01 vs placebo.4, 百拇医药
Effects of Oral Insulin Supplementation on Macrophage Cholesterol Metabolism4, 百拇医药
Accumulation of cholesterol in macrophages converts macrophages into foam cells and is the hallmark of atherosclerotic lesion formation.9,10 Thus, we next measured the amount of total cholesterol in insulin-treated mice MPM. Oral insulin supplementation to E0 mice resulted in a dose-dependent reduction in MPM cellular cholesterol content Whereas in mice supplemented with 0.1 U/mL insulin no significant effect on MPM cholesterol levels was observed, there was a significant reduction (P<0.01) in MPM cholesterol content by 31% and 36% in mice supplemented with 0.5 or 1 U/mL of insulin, respectively, compared with the amount of cholesterol in placebo-mice MPM
fig.ommittedjo?o@, 百拇医药
Effects of oral insulin supplementation to E0 mice on macrophage cholesterol metabolism. Four groups of 4-week-old E0 mice were included in the study. The placebo group received water, whereas the other 3 groups received increasing insulin concentrations (0.1, 0.5, and 1 U/mL) in their drinking water for 3 months. MPMs were then harvested from the mice. A, Cells (3x106) were washed and their lipids were extracted with hexane:isopropanol (3:2, vol/vol) and cellular cholesterol mass was determined. B, Cells were washed and incubated with [3H]-acetate for 18 hours at 37°C for cholesterol biosynthesis determination. Results represent mean±SD of 3 different experiments, each one performed in triplicate. *P<0.01 vs placebo.jo?o@, 百拇医药
To examine if decreased cholesterol biosynthesis may be the cause for reduced cholesterol mass, the ability of macrophages harvested from the insulin-supplemented E0 mice to synthesize cholesterol from [3H]-acetate was next determined. As seen in there was a significant reduction (P<0.01) in MPM cholesterol biosynthesis (by 42% to 53%) in mice supplemented with 0.1 to 1 U/mL of insulin compared with placebo-treated mice MPM
In Vitro Studiese$7[?7, 百拇医药
Effects of Insulin on Macrophage Oxidative Stress In Vitro: Concentration Studye$7[?7, 百拇医药
To assess the direct effects of insulin on macrophage oxidative stress, MPMs from 4-month-old E0 mice were incubated for 18 hours with increasing concentrations of insulin (0 to 100 µU/mL). As can be seen in there was a dose-dependent decrease in MPM lipid peroxide content (up to 46%) after adding 1 µU/mL insulin compared with lipid peroxides content in nontreated MPM. However, the decrease was not statistically significant, and higher insulin concentrations were less effective (adding 10 or 100 µU/mL of insulin reduced MPM lipid peroxides content only by 12% and 20%, respectively,. The amount of superoxide anions released from MPM after their incubation with increasing insulin concentrations (0 to 100 µU/mL) was then measured . Maximal reduction in superoxide anion release was observed with 1 µU/mL of insulin (26%), but the reduction was not statistically significant.
fig.ommitted.5, 百拇医药
Effects of increasing concentrations of insulin on macrophage oxidative stress in vitro. MPM were harvested from 4-month-old E0 mice. The cells were incubated for 18 hours at 37°C with increasing insulin concentrations (0, 0.01, 0.1, 1, 10, and 100 µU/mL) in DMED+5%FCS after cell wash. A, Cellular lipids were extracted using hexane:isopropanol (3:2, vol/vol), for lipid peroxides determination. B, Amount of superoxide anion released from the cells was determined after 1 hour of incubation at 37°C. C, Cells were additionally incubated for 6 hours with LDL (100 µg of protein/mL) in the presence of 5 µmol/L CuSO4. The extent of LDL oxidation was determined by the TBARS assay. Results are given as mean±SD of 3 different experiments each performed in triplicate..5, 百拇医药
We determined next the capability of insulin-treated MPM to oxidize LDL. The addition of insulin (0 to 0.1 µU/mL) to the cells caused a reduction in their capability to oxidize LDL by up to 32%, and this effect was insulin dose-dependentnot statistically significant). However, higher concentrations were less effective, and incubation with 100 µU/mL of insulin resulted in oxidation values similar to those obtained in nontreated cells
Effects of Insulin on Macrophage Cholesterol Metabolism In Vitro: Concentration Study2, 百拇医药
To assess the direct effects of insulin on cholesterol biosynthesis, MPMs from 4-month-old E0 mice were incubated for 18 hours with increasing concentrations of insulin (0 to 100 µU/mL). Adding insulin to the cells resulted in a dose-dependent reduction by up to 66% in cholesterol biosynthesis from [3H]-acetate, compared with the values obtained in nontreated MPM ( P<0.05). The concentration of 0.1 µU/mL was significant (P<0.05) to 0.01 µU/mL, and the concentration of 1 µU/mL was significant (P<0.01) to 0.1 µU/mL. However, a significant reduction in cholesterol biosynthesis from [14C]-mevalonate was seen only at concentrations of 1.0 to 100 µU/mL of insulin ( P<0.01 between each concentration).2, 百拇医药
fig.ommitted2, 百拇医药
Effects of increasing concentrations of insulin on macrophage cholesterol metabolism in vitro. MPMs were harvested from 4-month-old E0 mice. The cells were incubated with increasing insulin concentrations (0, 0.01, 0.1, 1, 10, and 100 µU/mL) for 18 hours at 37°C in DMED+5%FCS. The cells were then washed and additionally incubated for 18 hours at 37°C with [3H]-acetate (A) or with [14C]-mevalonate (B) for cholesterol biosynthesis determination. Results are given as mean±SD of 3 different experiments, each one performed in triplicate. *P<0.01 (vs 0 concentration), &P<0.05 (vs 0.01 µU/mL), #P<0.01 (vs 0.1 µU/mL), @P<0.01 (vs 1 µU/mL), +P<0.01 (vs 10 µU/mL).
Discussionj6, 百拇医药
Supplementation of oral insulin to E0 mice resulted in decreased aortic atherosclerotic mean plaque area and reduced the number of atherosclerotic lesions. These effects of oral insulin were associated with reduced oxidative stress both in serum and in MPM as well as reduced cholesterol biosynthesis in macrophages.j6, 百拇医药
Hyperinsulinemia is an independent cardiovascular risk factor.1,2 However, the contribution of insulin itself to the atherosclerotic process is unknown, because insulin resistance could be responsible for the increased risk rather than the compensatory hyperinsulinemia observed with insulin resistance.3 As part of insulin resistance, there is increased lipid peroxidation attributable to increased production of free radicals,8 hyperglycemia increases intracellular reactive oxygen species,21 and postprandial hyperglycemia may be a risk factor for cardiovascular disease in diabetic patients.22
There are some lines of evidence suggesting that insulin may have beneficial effects on the cardiovascular system. Insulin causes vasodilation in healthy humans3,23 and improves endothelial relaxation through nitric oxide production.3 Insulin attenuates the vascular smooth muscle cell response to vasoactive substances24 and may decrease large arteries’ stiffness.3 Insulin has been shown to lower blood pressure in some studies,24 whereas in another study it had no effect.25;[px^hv, 百拇医药
In contrast, insulin may be related to increased risk for atherosclerosis either secondary to obesity, hyperlipidemia, hypertension, hyperuricemia, and the use of oral contraceptives2 or through inhibition of lipolysis and by induction of vascular smooth muscle cell proliferation.3;[px^hv, 百拇医药
Findings in animal models are few and inconsistent. The administration of subcutaneous insulin to rats caused an increase in triglyceride content in the aorta and in intimal thickening.26 In rabbits fed with an atherogenic diet, the administration of subcutaneous insulin significantly reduced plasma lipid levels, but continued administration of insulin during atherosclerosis development resulted in reinforcement of the lipemia with aggravation of atherosclerosis.27 Interestingly, in that study,27 after switching to a normal diet at the age of 2 months, insulin accelerated the reduction in hypercholesterolemia. In a similar study in rabbits, no effects on atherosclerosis or on lipid serum levels were demonstrated.28 In contrast, we were able to demonstrate in the present study that adding insulin at 1 U/mL to the drinking water of the atherosclerotic E0 mice for 3 months significantly reduced the number of atherosclerotic lesions per mouse, and all insulin concentrations resulted in significant reduction in mean atherosclerotic lesion area. Oral insulin supplementation resulted in a dose-dependent increase in serum insulin concentrations. However, portal insulin levels may be several times higher than those found in the systemic circulation.29
LDL oxidation is important in atherosclerosis progression,30 and in E0 mice, increased plasma lipoprotein lipid peroxidation was documented.7 In our study, insulin supplementation to E0 mice caused a significant reduction in serum lipid peroxides and a significant increase in serum paraoxonase (PON1) activities. The increase in PON1 activity may explain the reduced serum oxidative stress, because it was shown that paraoxonase reduces lipid peroxides levels in oxidized lipoproteins.20 Reduced serum PON1 activity is seen in atherosclerotic patients, including diabetic patients,31 in insulin-deficient (streptozotocin-induced) diabetic rats,32 in E0 mice,22 and in rabbits fed an atherogenic diet.33 Thus, similar to its effect on E0 mice, insulin supplementation may increase PON1 activity in diabetes.{+gxp6), 百拇医药
It was recently shown that E0 mice macrophages, which contain oxidized lipids, release increased amounts of superoxide anions and oxidize LDL at an enhanced rate.7 In that study,7 the reduction of oxidative stress by antioxidants supplementation reduced the amount of lipid peroxides in macrophages and their ability to oxidize LDL. In the present study, insulin supplementation to the E0 mice resulted in the reduction in macrophage lipid peroxides content in association with reduction in the cell’s ability to release superoxide anions and to oxidize LDL. The reduction in macrophage oxidative stress may again be explained by increased paraoxonase activity, because paraoxonase was shown to reduce specific oxidized lipids in macrophages and in atherosclerotic lesion.34,35 In addition, the decreased oxidation observed in our study could be the result of increased reduced glutathione (GSH) concentration. GSH is the major antioxidant in cells, and it has been shown that on incubation of cells with insulin, the amount of GSH was significantly increased.36 In addition, it was shown that increasing GSH cellular content in the cells reduced their capability to release superoxide anions and to oxidized LDL.37 Furthermore, in diabetic rat ventricular myocytes, GSH homeostasis is functionally coupled to insulin signaling.38
To assess the direct role of insulin on macrophages, we performed in vitro experiments. By incubating E0 mice macrophages with increasing concentrations of insulin (0 to 100 µU/mL), we observed no significant trend toward reduced oxidative stress in macrophages. First, this could be the result of differences between the time period macrophages acutely were exposed to insulin in the in vitro studies compared with the chronic exposure in the ex vivo studies. Second, the intracellular concentrations of insulin may have been different in the ex vivo versus the in vitro studies. Third, oral insulin supplementation may have triggered other, not yet identified, antioxidative pathways that could explain the lack of a significant effect in the in vitro studies.\62&}r, 百拇医药
Accumulation of cholesterol in macrophages is an essential step in atherosclerosis progression,9,10 and insulin was found to regulate cholesterol metabolism in macrophages.39,40 In our study, we demonstrated that insulin inhibited cholesterol synthesis in peritoneal macrophages in a dose-dependent manner, both in the ex vivo and in the in vitro studies. Thus, the reduced macrophage cholesterol content in insulin supplemented mice may be attributable to the inhibition of cholesterol synthesis induced by insulin. This is in agreement with other observations that insulin therapy depresses the rate of cholesterologenesis in patients with non-insulin-dependent diabetes mellitus.41 Also, increased cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase activity42 were previously demonstrated in MPM from insulin-deficient mice. In that study, insulin addition resulted in reduced HMGCoA-reductase activity and reduced cholesterol biosynthesis.42 However, in the present study, mevalonate blocked the inhibitory effect of insulin on cholesterol biosynthesis only at low insulin concentrations, suggesting that the inhibition of cholesterol biosynthesis by insulin may involve other, still undetermined, pathways.
In summary, in E0 mice, oral insulin supplementation attenuates the atherosclerotic process. It is conceivable that the effects of oral insulin supplementation on the atherosclerotic process are mediated via effects on oxidative stress in serum and in macrophages as well as effects on macrophage cholesterol content and biosynthesis.n, 百拇医药
Acknowledgmentsn, 百拇医药
This study was supported by a research grant from the Danone Institute, Israel.n, 百拇医药
Received July 17, 2002; accepted September 26, 2002.n, 百拇医药
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