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Dietary Intake of Sugar Substitutes Aggravates Cerebral Ischemic Injury and Impairs Endothelial Progenitor Cells in Mice

Originally publishedhttps://doi.org/10.1161/STROKEAHA.114.007308Stroke. 2015;46:1714–1718

Abstract

Background and Purpose—

In our current food supply, sugar substitutes are widely used in beverages and other food products. However, there is limited information about the link between dietary consumption of sugar substitutes and stroke to date. This study sought to determine the effect of various sugar substitutes on the cerebral ischemic injury and endothelial progenitor cells, which have been implicated to play an important role in vascular repair and revascularization in ischemic brain tissues, in mice.

Methods—

After treatment with sucrose and various sugar substitutes (the doses are in the range of corresponding acceptable daily intake levels) and vehicle for 6 weeks, mice were subjected to permanent left middle cerebral artery occlusion, and the infarct volumes, angiogenesis, and neurobehavioral outcomes were determined. In addition, the number and function of endothelial progenitor cells were also examined.

Results—

After long-term treatment with fructose, erythritol (sugar alcohols), acesulfame K (artificial sweeteners), or rebaudioside A (rare sugars), the cerebral ischemic injury (both infarct volumes and neurobehavioral outcomes) was significantly aggravated, angiogenesis in ischemic brain was reduced, and endothelial progenitor cell function was impaired in mice compared with control. However, the similar impairments were not found in sucrose (with the same dose as fructose’s)-treated mice.

Conclusions—

Long-term consumption of sugar substitutes aggravated cerebral ischemic injury in mice, which might be partly attributed to the impairment of endothelial progenitor cells and the reduction of angiogenesis in ischemic brain. This result implies that dietary intake of sugar substitutes warrants further attention in daily life.

Introduction

As the negative impact of sugar consumption on weight and other health outcomes has been increasingly recognized, many persons have turned to fructose and sugar substitutes (like artificial sweeteners, sugar alcohols, and rare sugars) as a way to reduce the risk of these consequences.13 But recently, accumulating evidences suggest that dietary consumption of these compounds may also increase the risk of metabolic disorder, which is one of major risk factors for cerebrocardiovascular diseases, such as stroke.13 Stroke is a devastating disease and the major cause of mortality and morbidity worldwide.4 However, there is limited information about the link between dietary consumption of sugar substitutes and stroke to date.

The integrity and function of the endothelium plays an important role in the prevention of cerebrocardiovascular diseases, such as stroke.5 Endothelial progenitor cells (EPCs) are immature cells that can differentiate into mature endothelial cells and could be recruited from bone marrow to the injury site to promote endothelial regeneration and neovascularization.6 In addition, EPCs have been used to successfully improve function recovery in ischemic organs, including brain after ischemic injury.5,79 Recent reports showed that EPCs may serve as a new marker for stroke outcomes.5,79 Therefore, EPC dysfunction and the consequent abnormality of endothelial regeneration may influence the susceptibility to cerebral ischemic injury.

On the basis of these findings, this study sought to determine the effect of various sugar substitutes consumption on the cerebral ischemic injury and EPCs in mice. Among sugar substitutes widely used in beverages and other food products,13 fructose, erythritol (sugar alcohols), acesulfame K (artificial sweeteners), and rebaudioside A (rare sugars) were used in this study.

Methods

Male C57BL/6 mice (10–12 week, 20–25g) used in these experiments were obtained from Sino-British SIPPR/BK Laboratory Animal Ltd (Shanghai, China). Mice were given fructose (Fru, 1.5 g/kg body weight), erythritol (Ery, 15 mg/kg body weight), acesulfame K (Ace, 4 mg/kg body weight), rebaudioside A (Reb, 1 mg/kg body weight), sucrose (Suc, 1.5 g/kg body weight), and water (Control) via drinking water for 6 weeks. The doses of these sugar substitutes (Sigma) are in the range of acceptable daily intake or estimated daily intake levels for food additives approved by US Food and Drug Administration (FDA) or European Union.2,3 Sucrose (Sigma), with the same dose as fructose’s, was denoted as positive control. The body weight was monitored every week, and fasting blood glucose levels were measured before and after 6 weeks of treatment. After long-term treatment, mice were subjected to permanent focal cerebral ischemia, and the infarct volumes, angiogenesis, and neurobehavioral outcomes were determined according to published protocols and our previous work.10,11 According to our described technique,7,12 mouse circulating EPCs and bone marrow–derived EPCs were isolated and cultured, and the functions (migration, tube formation and adhesion assay) of bone marrow–derived EPCs were examined. All animals received humane care, and the experimental procedures were in compliance with the institutional animal care guidelines.

Detailed methods are described in the online-only Data Supplement.

Statistical Analysis

Data are expressed as mean±SEM. Statistical significance of difference among groups was performed using 1-way ANOVA followed by Tukey post hoc analysis. A value of P<0.05 was considered statistically significant.

Results

Effects of Sugar Substitutes on Fasting Blood Glucose and Body Weight in Mice

There was no significant difference in baseline fasting blood glucose levels among all the groups of mice (data not shown). After 6 weeks of treatment, compared with control mice, fasting blood glucose levels were slightly but significantly increased in acesulfame K–treated mice but not in mice treated with fructose, erythritol, rebaudioside A, or sucrose (Figure IA in the online-only Data Supplement). There was no significant difference in body weights between sugar substitutes–treated mice and control animals (Figure IB in the online-only Data Supplement).

Sugar Substitutes Aggravate Cerebral Ischemic Injury and Reduced Angiogenesis in Ischemic Brain in Mice

The infarct volumes were significantly increased and the corresponding neurobehavioral outcomes were markedly impaired in sugar substitutes–treated, but not sucrose (with the same dose as fructose’s)-treated, mice compared with control (Figure 1A–1C). Furthermore, angiogenesis in ischemic brain was assessed at 3 days after cerebral ischemia. It was found that capillary density was significantly lower in the sugar substitutes–treated, but not sucrose-treated, mice compared with control (Figure 1D). These results suggest that long-term consumption of these sugar substitutes was able to aggravate cerebral ischemic injury and reduce angiogenesis in ischemic brain in mice.

Figure 1.

Figure 1. Sugar substitutes aggravate cerebral ischemic injury and reduced angiogenesis in ischemic brain in mice. A, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections (up) and cerebral infarct volumes (below). **P<0.01 vs control (Con), n=10 to 18. B and C, Neurobehavioral outcomes (B, Beam Test; C, Body Asymmetry Test). *P<0.05, **P<0.01 vs Con; n=10 to 21. D, The local angiogenesis in the ischemic brain in mice. CD31 immunostaining shows microvessels in ischemic brain of mice treated with sugar substitutes. The bar graph shows that the number of microvessels in sugar substitutes–treated mice was significantly reduced compared with that in vehicle-treated mice. **P<0.01 vs Con, n=6. Scale bar= 50 μm (left); 25 μm (right). Ace indicates acesulfame K; Ery, erythritol; Fru, fructose; Reb, rebaudioside A, and Suc, sucrose.

Sugar Substitutes Impair EPCs in Mice

No significant difference in the number of circulating EPCs (Sca-1/Flk-1 double-positive cells) was found among all the groups of mice (Figure 2A). However, bone marrow–derived EPC functions (including migration, tube formation, and adhesion function) were significantly impaired in sugar substitutes–treated, but not sucrose-treated, mice compared with control (Figure 2B–2D).

Figure 2.

Figure 2. Effects of sucrose and sugar substitutes on the number of circulating endothelial progenitor cells (EPCs) and functions of bone marrow–derived EPCs (BM-EPCs) in mice. A, The number of circulating EPCs determined by Sca-1/Flk-1 double-staining flow cytometry. n=7 to 17. B, Migration assay of BM-EPCs. C, Tube formation assay of BM-EPCs. D, Adhesion assay of BM-EPCs. n=5 to 12, **P<0.01 vs control (Con). Ace indicates acesulfame K; Ery, erythritol; Fru, fructose; Reb, rebaudioside A, hpf, high-power fields (magnification ×100); and Suc, sucrose. Scale bar=100 μm.

Discussion

This study showed the first direct evidence that long-term intake of various sugar substitutes in the amounts currently consumed impaired EPC functions and aggravated cerebral ischemic injury in mice. However, the similar impairments were not found in sucrose (with the same dose as fructose’s)-treated mice.

Fructose in the amounts currently consumed has been proposed to be hazardous to the health of some people, but the other 3 sugar substitutes used in the present work are generally regarded as safe for consumption.13 It is found that neither plasma glucose nor insulin levels are affected by sugar alcohols (including erythritol) consumed.13 Five artificial sweeteners (including acesulfame K) have been approved for human use by FDA and have gained popularity as a solution in maintaining the palatability of foods while substantially reducing or eliminating caloric content for many years.13 Rebaudioside A, a representative of rare sugars, has been regarded as safe for consumption in Asia and North America for many years and was recently permitted for use in the European Union and prized for its sweet taste and bulking properties but apparent low energy status.13 However surprisingly, in this study, we found that, besides fructose intake, chronic consumption of the other 3 sugar substitutes could also led to a significant increase in cerebral ischemic injury and reduction in EPC functions.

As aforementioned, EPCs participate in both vasculogenesis and vascular homestasis and have been used to successfully restore endothelial function and enhance angiogenesis in ischemic brain tissue.5,79 Therefore, EPC dysfunction and subsequent reduction of local angiogenesis in ischemic brain, as observed in this study, may partly contribute to the aggravated cerebral ischemic injury produced by chronic sugar substitutes intake (Figure 3). However, the mechanisms underlying EPC dysfunction remain to be investigated in further studies

Figure 3.

Figure 3. Putative mechanisms underlying sugar substitutes aggravating cerebral ischemic injury in mice. Chronic sugar substitutes intake impaired endothelial progenitor cell (EPC) functions, reduced the angiogenesis in ischemic brain, increased cerebral infarct volumes, and decreased the corresponding neurobehavioral outcomes in mice.

In addition, because EPCs has been implicated in playing an important role in vascular repair and revascularization in various ischemic organs besides brain tissue, and EPC is inversely correlated with several cardiovascular risk factors,5,79 EPC dysfunction produced by sugar substitutes might also aggravate other ischemic diseases except stroke, such as acute myocardial infarction, peripheral vascular ischemic diseases, and so on, which remains to be test by further studies.

In summary, long-term consumption of fructose, acesulfame K, rebaudioside, and erythritol might aggravate the cerebral ischemic injury, which might partly result from the impairment of EPCs and the reduction of angiogenesis in ischemic brain. This result implies that dietary intake of sugar substitutes warrants further attention in daily life. Further studies are required to better understand the mechanisms of this response.

Footnotes

X.-H. Dong, X. Sun, and Dr Jiang contributed equally.

The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.007308/-/DC1.

Correspondence to He-Hui Xie, PhD, or Alex F. Chen, PhD, Department of Science for Identifying Chinese Materia Medica, The Second Military Medical University, Shanghai 200433, China. E-mail or

References

  • 1. Swithers SEArtificial sweeteners produce the counterintuitive effect of inducing metabolic derangements. Trends Endocrinol Metab. 2013; 24:431–441. doi: 10.1016/j.tem.2013.05.005.CrossrefMedlineGoogle Scholar
  • 2. Payne AN, Chassard C, Lacroix CGut microbial adaptation to dietary consumption of fructose, artificial sweeteners and sugar alcohols: implications for host-microbe interactions contributing to obesity. Obes Rev. 2012; 13:799–809. doi: 10.1111/j.1467-789X.2012.01009.x.CrossrefMedlineGoogle Scholar
  • 3. Gardner C, Wylie-Rosett J, Gidding SS, Steffen LM, Johnson RK, Reader D, et al; American Heart Association Nutrition Committee of the Council on Nutrition, Physical Activity and Metabolism, Council on Arteriosclerosis, Thrombosis and Vascular Biology, Council on Cardiovascular Disease in the Young, and the American D. Nonnutritive sweeteners: current use and health perspectives: a scientific statement from the American Heart Association and the American Diabetes Association. Circulation. 2012; 126:509–519. doi: 10.1161/CIR.0b013e31825c42ee.LinkGoogle Scholar
  • 4. Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJGlobal and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet. 2006; 367:1747–1757. doi: 10.1016/S0140-6736(06)68770-9.CrossrefMedlineGoogle Scholar
  • 5. Zhao YH, Yuan B, Chen J, Feng DH, Zhao B, Qin C, et al. Endothelial progenitor cells: therapeutic perspective for ischemic stroke. CNS Neurosci Ther. 2013; 19:67–75. doi: 10.1111/cns.12040.CrossrefMedlineGoogle Scholar
  • 6. Fadini GP, Losordo D, Dimmeler SCritical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use. Circ Res. 2012; 110:624–637. doi: 10.1161/CIRCRESAHA.111.243386.LinkGoogle Scholar
  • 7. Marrotte E, Chen DD, Hakim JS, Chen AFRestoration of endothelial progenitor cell function with manganese superoxide dismutase accelerates wound healing in diabetic mice. J Clin Invest. 2010; 120:4207–4219.CrossrefMedlineGoogle Scholar
  • 8. Khakoo AY, Finkel TEndothelial progenitor cells. Annu Rev Med. 2005; 56:79–101. doi: 10.1146/annurev.med.56.090203.104149.CrossrefMedlineGoogle Scholar
  • 9. Fan Y, Shen F, Frenzel T, Zhu W, Ye J, Liu J, et al. Endothelial progenitor cell transplantation improves long-term stroke outcome in mice. Ann Neurol. 2010; 67:488–497. doi: 10.1002/ana.21919.CrossrefMedlineGoogle Scholar
  • 10. Zhu W, Fan Y, Frenzel T, Gasmi M, Bartus RT, Young WL, et al. Insulin growth factor-1 gene transfer enhances neurovascular remodeling and improves long-term stroke outcome in mice. Stroke. 2008; 39:1254–1261. doi: 10.1161/STROKEAHA.107.500801.LinkGoogle Scholar
  • 11. Ma J, Peng C, Guo W, Dong YF, Dong XH, Sun X, et al. A modified model of middle cerebral artery electrocoagulation in mice. CNS Neurosci Ther. 2012; 18:796–798. doi: 10.1111/j.1755-5949.2012.00370.x.CrossrefMedlineGoogle Scholar
  • 12. Xie HH, Zhou S, Chen DD, Channon KM, Su DF, Chen AFGTP cyclohydrolase I/BH4 pathway protects EPCs via suppressing oxidative stress and thrombospondin-1 in salt-sensitive hypertension. Hypertension. 2010; 56:1137–1144. doi: 10.1161/HYPERTENSIONAHA.110.160622.LinkGoogle Scholar

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