Stevioside is a non-caloric natural sweetener that does not induce a glycemic response, making it attractive as sweetener to diabetics and others on carbohydrate-controlled diets. Obesity is frequently associated with insulin resistance and increased inflammation and oxidative stress. Therefore, we investigated its effects on insulin resistance, inflammation and oxidative stress related to atherosclerosis in obese insulin-resistant mice.
Twelve-week-old mice were treated with stevioside (10 mg kg−1, n=14) or placebo (n=20) for 12 weeks.
Stevioside had no effect on weight and triglycerides, but lowered glucose and insulin. Stevioside treatment improved adipose tissue maturation, and increased glucose transport, insulin signaling and antioxidant defense in white visceral adipose tissues. Together, these increases were associated with a twofold increase of adiponectin. In addition, stevioside reduced plaque volume in the aortic arch by decreasing the macrophage, lipid and oxidized low-density lipoprotein (ox-LDL) content of the plaque. The higher smooth muscle cell-to-macrophage ratio was indicative for a more stable plaque phenotype. The decrease in ox-LDL in the plaque was likely due to an increase in the antioxidant defense in the vascular wall, as evidenced by increased Sod1, Sod2 and Sod3. Circulating adiponectin was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the aorta of stevioside-treated mice.
Stevioside treatment was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the vascular wall, leading to inhibition of atherosclerotic plaque development and inducing plaque stabilization.
The growing incidence of obesity and obesity-related cardiovascular risk factors have led to the quest for natural sweeteners that can substitute for sucrose, and do not provide calories. Much attention has been placed on glycosides that are extracted from Stevia rebaudiana Bertoni. The most abundant glycoside is stevioside, which is one of the major sweeteners in use in Japan and Korea.1
The increasing prevalence of obesity, a low-grade inflammatory and oxidative stress state, is closely associated with the rising incidence of type diabetes and cardiovascular diseases.2 Indeed, recent studies showed a positive relationship between obesity, inflammatory C-reactive protein,3 and oxidized low-density lipoprotein (ox-LDL).4, 5 Moreover, we showed that ox-LDL was associated with the incidence of the metabolic syndrome6 and with several metabolic syndrome components, especially visceral obesity.7, 8
Stevioside was found to exert antihyperglycemic and insulinotropic effects in a non-obese animal model of type 2 diabetes9 by acting directly on pancreatic cells.10 However, its effects on adipose tissue and obesity-associated insulin resistance, inflammation, oxidative stress and atherosclerosis have not been determined. Therefore, our aim was to measure these effects and identify underlying molecular pathways. We selected mice with combined leptin and LDL-receptor deficiency (double knockout (DKO) mice). These exhibit most of the metabolic syndrome components, which are associated with increased inflammation and oxidative stress, accelerated atherosclerosis and impaired cardiovascular function.11, 12, 13 Weight loss11 and treatment with statin12 were associated with improved insulin sensitivity and decreased macrophage and ox-LDL accumulation in their aortic arch. These improvements were associated with increased expression of the antioxidant superoxide dismutases (Sods) in the aorta. These observations prompted us to investigate the effect of stevioside on the expression of factors involved in the regulation of adipose tissue maturation, insulin signaling, inflammation and oxidative stress in the adipose tissue and aorta in relation to atherosclerosis in these mice. We found a relation between adipose tissue maturation, adiponectin, insulin signaling and the antioxidant defense in the adipose tissue and the aorta of stevioside-treated mice.
Research design and methods
Experimental protocol of animal studies
Experimental procedures in animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. DKO mice, on the C57BL6 background, were obtained as previously described.11, 13 All offspring were genotyped by PCR techniques.14 Mice were treated with stevioside (n=14) or placebo (n=20) for 12 weeks starting at the age of 12 weeks. Stevioside (molecular weight: 804) was dissolved in physiological saline solution (0.9% NaCl) (1 mg ml−1) and administered orally at a dose of 10 mg kg−1 day−1. Stevioside was purified as described previously with a 99.9% purity.15, 16 Placebo was the solvent without active compound. All mice were housed at 22 °C on a fixed 12/12-hour light–dark cycle and were fed standard chow containing 4% fat. Food and water were available ad libitum. Food intake was ≈5.7 g day−1 and was not affected by the treatment.
Blood from awaken mice was collected by tail bleeding into EDTA tubes after an overnight fast. During killing, total blood was collected by puncturing the vena cava. Plasma was obtained by centrifugation. Total cholesterol and triglycerides were measured with standard enzymatic assays (Boehringer, Mannheim, Germany), glucose with a glucometer (Menarini Diagnostics, Zaventem, Belgium) and plasma insulin with a mouse ELISA (Mercodia, Oxon, UK). To determine glucose tolerance, glucose was measured in samples obtained by tail bleeding before and 15, 30, 60, 120 and 240 min after intraperitoneal glucose administration (20% glucose solution; 2 g kg−1).11, 12 Adiponectin, interleukin-6 and tumor necrosis factor-α were measured with specific mouse ELISAs (R&D Systems, Uppsala, Sweden). Because the assay for ox-LDL in blood is based on a mouse monoclonal antibody, ox-LDL cannot be measured directly in mouse blood. Therefore, we measured the titer of autoantibodies against malondialdehyde (MDA)-modified LDL as a proxy for ox-LDL in mice, as described before.13, 17, 18
Real-time reverse transcription-PCR analysis
The levels of RNA expression in extracts of white visceral (IP) adipose tissue and aorta were measured by quantitative real-time reverse transcription-PCR. Total RNA was extracted with Trizol reagent (Invitrogen, Merelbeke, Belgium) and purified on RNeasy Mini kit columns (Qiagen, Venlo, The Netherlands). First-strand cDNA was generated from total RNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative real-time reverse transcription-PCR was performed using Power SYBR Green Master mix according to the supplier protocols (Applied Biosystems, Lennik, Belgium). Oligonucleotides (Invitrogen) used as forward and reverse primers were designed using the ‘Primer Express’ software (Applied Biosystems) and are summarized in Table 1. Primer sequences were validated for specificity by Basic Local Alignment Search Tool (BLAST).19 PCR fragments were validated for GC/AT ratio, length and amplification specificity with dissociation curve analysis and agarose gel electrophoresis.20 The level of RNA expression was calculated using the threshold cycle (Ct) value, normalized with the housekeeping gene, β-actin, and related to an external calibrator consisting of extracts from intra-abdominal adipose tissue or aorta of C57BL6 control mice. Subsequently, ΔΔCt (ΔCt,sample–ΔCt,calibrator) was determined, and the relative expression levels were calculated from 2–ΔΔCt. RNA expression levels are thus indicated as arbitrary units ±s.d.11, 12, 13
The extent of atherosclerosis was determined by analysis of ten 7-μm cross-sections of aortic root of 24-week placebo- and stevioside-treated DKO mice. Lipids were stained with oil red O, ox-LDL with mAb4E6,21 smooth muscle cells (SMC) with an α-actin-specific antibody (Dako, Heverlee, Belgium), macrophages with an antibody against mouse Mac-3 antigen (Pharmingen, Erembodegem, Belgium) and paraoxonase 1 (Pon1) with polyclonal antibodies from Santa Cruz Biotechnology (Tebu-bio, Boechout, Belgium). A color intensity threshold mask for immunoassaying was defined to detect the red color by sampling, and the same threshold was applied to all specimens. Blinded analysis was performed with the Quantimet 600 image analyzer (Leica, Groot Bijgaarden, Belgium). The positively immunostained area was expressed as a percentage of the total plaque area.11, 12
Groups were compared by means of the unpaired t-test with Welch's Correction (Graph Pad Prism version 5; La Jolla, CA, USA). Correlations were calculated using the nonparametric Spearman's correlation coefficient (Rs). The area under the curve of the glucose tolerance test was calculated using Graph Pad Prism version 5. P<0.05 was considered to be statistically significant.
Weight and blood analysis
Stevioside treatment had no effect on weight. It lowered blood glucose, insulin and cholesterol, but had no effect on triglycerides or glucose tolerance. Treatment with stevioside nearly doubled plasma adiponectin concentrations, but did not change plasma interleukin-6 and tumor necrosis factor-α concentrations. The titer of autoantibodies against MDA-modified LDL was decreased in stevioside-treated mice (Table 2) and correlated inversely with the plasma adiponectin concentration (Rs=−0.66; P<0.001).
Insulin signaling and oxidative stress in visceral adipose tissue
The RNA expressions of Insr, Irs1, Irs2, Glut4, Fabp4 and Lxrα in the visceral adipose tissue of placebo-treated DKO mice were lower than those in adipose tissue of lean control C57BL6 mice. Stevioside treatment increased their expressions. However, all expressions were still lower in stevioside-treated mice than in lean controls (Figure 1). Stevioside treatment also increased plasma adiponectin that correlated Insr, Irs1 and Irs2 (Figure 1).
Previously, obesity in DKO mice was shown to be associated with increased oxidative stress due to a lack of antioxidant enzymes.13 Therefore, we measured Sod3, because it was shown to have a protective role against hyperglycemia-induced reactive oxygen species (ROS).22 Sod3 was decreased in placebo-treated DKO mice compared with lean control mice; stevioside normalized its expression from 0.64±0.28 to 1.06±0.47 (P<0.05). In addition, stevioside increased the expression of Cat in DKO mice from 0.30±0.08 to 0.51±0.12 (P<0.001), important for further conversion of H2O2, generated by SOD, to water. The expressions of antioxidant enzymes correlated negatively with the titer of autoantibodies against MDA-modified LDL (Rs=−0.41, P<0.05 for Sod3 and Rs=−0.58, P<0.001 for Cat). Furthermore, both Sod3 and Cat correlated strongly with plasma adiponectin (Rs=0.778 and 0.741, respectively; both P<0.001) and Irs2 (Rs=0.407, P<0.05 and Rs=0.530, P<0.01, respectively).
Figure 2 shows representative sections of the aortic arch of placebo- and stevioside-treated DKO mice with lipids stained with oil red O, macrophages with an antibody against Mac-3 and ox-LDL with 4E6. Stevioside inhibited atherosclerosis by reducing macrophage, ox-LDL and lipids. Furthermore, stevioside treatment increased the SMC area of the plaque. This increase together with the reduction of macrophages resulted in an increase of the SMC-to-macrophage ratio. Plaque macrophages correlated with ox-LDL (Rs=0.33, P<0.01) that correlated with lipids (Rs=0.48, P<0.01). Plaque ox-LDL correlated with the titer of autoantibodies against MDA-modified LDL (Rs=0.45, P<0.001). There was no difference in PON1 staining in control and stevioside-treated mice (9.0±4.4 vs 6.5±4.1%).
Insulin signaling, oxidative stress and inflammation in the aorta
To get a better insight in the pathways that are involved in decreasing the oxidative stress in the aorta of stevioside-treated DKO mice, we measured the RNA expression of Sods. Figure 3 shows that all Sods were lower in the aorta of DKO mice than in lean control mice and that stevioside increased their expressions. All Sods were inversely related with the ox-LDL content of the plaque (Figure 3). In addition, the titer of autoantibodies against MDA-LDL, used as a proxy for ox-LDL in the circulation, correlated negatively with Sod1 (Rs=−0.49, P<0.05), Sod2 (Rs=−0.51, P<0.01) and Sod3 (Rs=−0.58, P<0.001). Previously, we found that overexpression of Sod depends on induction of Ppars, especially Pparγ.23 Pparγ was lower in the aorta of DKO mice, and stevioside restored its expression (Figure 3). Pparγ expression correlated with Sod1 (Rs=0.49, P<0.05), Sod2 (Rs=0.57, P<0.01) and Sod3 (Rs=0.67, P<0.001). Adiponectin correlated with Pparγ, Sod1 and Sod3 (Figure 3). Stevioside treatment had no effect on Ppara (data not shown).
As in adipose tissues, the expressions of Insr, Irs1, Irs2, Glut4, Fabp4 and Lxrα in the aorta of placebo-treated DKO mice were lower than those in the aorta of lean control C57BL6 mice (data not shown). Stevioside treatment only increased the expression of Irs2. The expression of Irs2 correlated with Sod1 (Figure 3).
Furthermore, stevioside treatment decreased the expressions of the chemotactic receptor Ccr2 and its ligand Ccl2, important for the recruitment of monocytes/macrophages to the vascular wall, supporting decreased inflammation. In addition, it decreased the expression of the inflammatory Nfκb1 and increased the expression of its inhibitor Nfκbiα, leading to a decreased Nfκb1/Nfκbiα ratio. This ratio was inversely related to Sod1 and Irs2, and positively to the ox-LDL content of the plaque (Figure 4). In contrast, stevioside treatment did not lower Icam1, Cd44, Cd68 and Cd36 and did not increase Abca1 (data not shown).
In normal physiology, adipose tissue stores energy in the form of triglycerides, which can be broken down to release free fatty acids. In obese individuals, impaired adipogenesis is characterized by hypertrophic adipocytes, which are less responsive to insulin and have a higher basal rate of fatty acids release. This leads to increased fatty acids in the serum and ectopic fat accumulation resulting in impaired insulin signaling in non-adipose tissues.24 We found that stevioside treatment improved adipogenesis and glucose uptake in visceral adipose tissue, evidenced by higher expressions of Lxrα, Fabp4 and Glut4.25, 26 The Glut427 and Fabp428 are direct transcriptional targets for the LXR/retinoid X-receptor heterodimer. Thus, the induction of Lxrα in adipose tissue of stevioside-treated mice supports the increased expression of Glut4, which favors glucose uptake, and of Fabp4, which improves fatty acid metabolism. The decrease in circulating insulin could be explained by a lower need for insulin due to improved insulin signaling, supported by the increased expression of Irs1 and Irs2 in the adipose tissue of stevioside-treated mice. Irs1 is involved in the differentiation of preadipocytes into adipocytes,29 whereas Irs2 regulates the insulin-induced glucose uptake.30 The improved adipocyte differentiation was associated with an increase in circulating adiponectin, which correlated with Irs1 and Irs2.
We then investigated whether decreased hyperglycemia and improved insulin sensitivity were associated with a reduction of the obesity-induced oxidative stress. Previously, it was demonstrated that the decreased expression of antioxidant enzymes in adipose tissues of obese mice was associated with increased ROS production.31 Stevioside treatment partially restored the expressions of Sod3 and Cat. Both genes correlated strongly with adiponectin and Irs2.
In aggregate, we present a novel action of stevioside on gene expression in adipose tissue associated with improved adipogenesis, glucose uptake, insulin signaling and antioxidant defense. These observations prompted us to investigate its effect on oxidative stress and insulin signaling in the aorta in relation to atherosclerosis.
In the aortic arch, a reduction in macrophage, ox-LDL and lipids were associated with the inhibition of atherosclerosis in stevioside-treated mice. The decrease in macrophages was likely due to reduced expressions of Ccl2 and its chemotactic receptor Ccr2. In contrast, the expressions of Icam1 and Cd44, which also mediate the interactions of monocytes with other arterial cells, were not affected by stevioside treatment.
The correlation between plaque macrophages and ox-LDL is in agreement with macrophages being the principal cellular source of ox-LDL.17 Thus, the decrease in ox-LDL could in part be due to a decrease in macrophages. In addition, it can be explained by increased expression of ROS-scavenging enzymes. As in the adipose tissue, we found increased expression of the antioxidant Sods. Previously, we showed that their induction, and the associated decrease in ox-LDL, depends on the increase in Pparα activity.23 Their increase in the vessel wall can also be due to the increase in plasma adiponectin. Indeed, adiponectin correlated with Sod,32 and was inversely related to ox-LDL in diabetics.33 The subsequent decrease in ROS resulted in higher Irs2 expression, and thus improved insulin signaling.34 In addition, overexpression of adiponectin increased the Sods in mice.35 Where there was ample evidence of diminished ROS and ox-LDL generation by induction of anti-oxidant enzymes, there was no evidence of enhanced ox-LDL clearance in stevioside-treated mice. Indeed, the expressions of the scavenger receptors CD36 and CD68 remained low.
Stevioside treatment also reduced the lipid content of the plaques. This could be due to decreased accumulation of lipids due to lowering of blood cholesterol and decreasing accumulation of ox-LDL in the plaque. Most likely the lowering of plaque lipids is not due to higher lipid efflux. Indeed, the expression of Abca1 that is important for inducing the reverse cholesterol transport, and thereby reducing plaque lipids,12 was not increased in stevioside-treated mice. The latter could be due to the lack of effect on LXRα. Indeed, in rosuvastatin-treated DKO mice an increase in LXRα was associated with higher Abca1.12
The lower macrophage, ox-LDL and lipid contents of the atherosclerotic plaques in the aortic arch of DKO mice are characteristic for a more stable plaque phenotype, which is also supported by the higher SMC-to-macrophage ratio. Interestingly, this higher ratio was not only due to a decrease in macrophages, but also due to an increase in SMC. The latter could be due to the decrease in ox-LDL. Indeed, ox-LDL was found to induce metalloproteases, which degrade matrix proteins, important for SMC migration, and, in addition, ox-LDL induces SMC apoptosis.36
It has been shown that Pparγ exerts anti-inflammatory actions by antagonizing the transcriptional activity of Nfκb1.37 Accordingly, stevioside-treated mice showed a lower Nfκb1/Nfκbiα ratio. Interestingly, the lower Nfκb1/Nfκbiα ratio in aortic extracts of stevioside-treated mice correlated inversely with Sod1. The suppression of Nfκb1 could partly be due to the suppression of O2− radicals by Sod1. O2− radicals cause the phosphorylation and ubiquitination of Nfκbiα with the subsequent translocation of Nfκb1 into the nucleus and the transcription of proinflammatory genes.38 Thus, the reduction in oxidative stress may have led to the reduction in inflammation, associated with decreased monocyte infiltration, and thus a further decrease in ROS and ox-LDL.
In conclusion, this is the first report showing an association between stevioside treatment and increased adiponectin and insulin sensitivity, improved antioxidant defense and reduced atherosclerosis. The improved antioxidant defense can be attributed mainly to increased expressions of Sods. The latter correlated with decreased accumulation of ox-LDL in circulation and the vessel wall. The decrease of ox-LDL by stevioside is particularly important in view of our recent observation that ox-LDL is associated with the metabolic syndrome components obesity, hyperglycemia and insulin resistance, and hyperlipidemia in the general population.7, 8 The decrease in ox-LDL was not due to an increase in PON1 in stevioside-treated mice.
A limitation of our study is that it is performed in mice. However, all our data were obtained at a daily dosage that is considered to be safe. Indeed, JECFA (Joint FAO/WHO Expert Committee on Food Additives), an international food safety organization that provides guidance on the safety of food additives, has set a permanent Acceptable Daily Intake (ADI) at 4 mg kg−1 body weight expressed as steviol, corresponding to 11 mg kg−1 of stevioside (http://www.fao.org/ag/agn/agns/jecfa_new_en.asp). The selected mice are hyperlipidemic and hyperglycemic. Therefore, we do not know the effect of stevioside in mice that are only hyperglycemic.
In conclusion, stevioside treatment was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the vascular wall of obese insulin-resistant mice. The improved metabolism led to the inhibition of atherosclerotic plaque development and inducing plaque stabilization. As yet, conflicting data about stevioside's effects on insulin resistance and diabetes in humans have been published.39, 40 However, the study groups were very small. Moreover, patients were already treated with PPAR agonists and/or statins, which are known to increase insulin sensitivity. Thus, large-scale studies in humans are warranted. Particularly, the action of stevioside for preventing insulin resistance in obese persons requires further attention.
Conflict of interest
The authors declare no conflict of interest.
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This study was supported in part by the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen (Program G.0548.08), the OT/06/56 program and Interuniversity Attraction Poles Program – Belgian Science Policy (P6/30). We thank Hilde Bernar, Michèle Landeloos and Roxane Menten for excellent technical assistance.
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Geeraert, B., Crombé, F., Hulsmans, M. et al. Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant mice. Int J Obes 34, 569–577 (2010). https://doi.org/10.1038/ijo.2009.261
- oxidative stress
- insulin signaling
- natural sweetener
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