Acta Diabetologica

, Volume 47, Supplement 1, pp 97–103

Effects of intermittent high glucose on oxidative stress in endothelial cells

Authors

  • Qin-Min Ge
    • Department of Endocrinology, Xinhua HospitalShanghai Jiaotong University School of Medicine
  • Yan Dong
    • Department of Endocrinology, Xinhua HospitalShanghai Jiaotong University School of Medicine
  • Hong-Mei Zhang
    • Department of Endocrinology, Xinhua HospitalShanghai Jiaotong University School of Medicine
    • Department of Endocrinology, Xinhua HospitalShanghai Jiaotong University School of Medicine
Original Article

DOI: 10.1007/s00592-009-0140-5

Cite this article as:
Ge, Q., Dong, Y., Zhang, H. et al. Acta Diabetol (2010) 47: 97. doi:10.1007/s00592-009-0140-5

Abstract

The objective of this study is to explore the mechanism of oxidative stress induced by intermittent high glucose in porcine iliac endothelial cells (PIECs). The PIECs were exposed to intermittent or constant high glucose for 3 or 6 days, and the mean fluorescent intensity (MFI) was measured via intracellular reactive oxygen species (ROS) captured by flow cytometry. The NADPH oxidase activity was measured by chemiluminescence with lucigenin. Intermittent high glucose induced a greater over-production of ROS than constant high glucose in PIECs; the NADPH oxidase activity was increased under both constant and intermittent high glucose conditions, being more marked in the latter (P < 0.05). In conclusion, intermittent high glucose induced more ROS in PIECs than constant high glucose, this effect seemed to be, at least in part related to the enhanced activation of NADPH oxidase. Glucose fluctuation may be involved in the development of vascular complications.

Keywords

Intermittent high glucoseEndotheliumOxidative stressReactive oxygen speciesNADPH oxidaseDiabetes

Introduction

The morbidity of diabetes mellitus has increased these years. It is of great significance to explore the patho-physiology and prevention of chronic diabetic complications. Recent studies have confirmed that hyperglycemia is the central initiating factor for all types of diabetic complications. The golden standard HbA1c used to reflect the severity of hyperglycemia can only represent the long-term blood glucose control but not the fluctuation of plasma glucose. Studies have shown that the risks of development or progression of complications are different even though the patients have the same HbA1c level. In fact, the level of glucose in vivo is always fluctuating. Postprandial hyperglycemia is an independent risk factor for cardiovascular complications in diabetic patients [1, 2], so there may be relationship between intermittent high glucose and diabetic complications. Vascular endothelial cells are important targets of hyperglycemic insult. There is growing evidence that an acute surge of plasma glucose may contribute to the endothelial dysfunction, but the mechanisms underlying this event are not fully understood. Studies have shown that temporary hyperglycemia induces adhesion of monocyte to endothelial cells compared with consistent hyperglycemia in SD rats [3, 4]. A previous research [5] using Kakizaki rat thoracic aorta also indicated that repetitive fluctuations in blood glucose enhance monocyte adhesion to the endothelium.

It has been suggested that oxidative stress plays a key role in the pathogenesis of endothelial dysfunction. Oxidative stress is a state of reactive oxygen species (ROS) overproduction. Here, we compare the differences of ROS production and NADPH oxidase activity between the effects of intermittent high glucose and constant high glucose on porcine iliac endothelial cells (PIECs).

Materials and methods

Reagents

DMEM was purchased from Hyclone company (Hyclone, USA). Newborn bovine serum was purchased from PAA company (PAA, USA). Dihydroethidium (DHE), dihydrorhodamine123 (DHR123), lucigenin, d-glucose and NADPH were purchased from Sigma Company (Sigma, USA).

Cell culture

Porcine iliac endothelial cells were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Science. Cells were cultured on 25 cm dishes and propagated in DMEM medium supplemented with 10% heat-inactivated newborn bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air −5% carbon dioxide. Medium was changed every day.

Endothelial cells in growth medium were equally seeded into 6-well culture plates, 5 × 105 per well. The growth medium was changed to 0.1% serum medium when cells had grown to approximately 90% confluence. 24 h later, 25 mM mannitol and glucose medium of different concentrations were added into the wells, respectively. The PIECs were grouped as follows: (1) constant normal glucose medium (5.5 mM), (2) constant high glucose medium (25 mM), (3) normal and high glucose medium alternating every 12 h, and (4) osmotic control was assured by incubating cells with 25 mM mannitol. Cells were incubated at 37°C in a humidified atmosphere of 95% air −5% carbon dioxide for 3 or 6 days.

Analysis of cellular ROS levels

Fluorescent microscopy

Cells were treated with 10 μmol/L dihydroethidium (DHE), and incubated for 1 h at 37°C, and then were washed three times with PBS. The fluorescence of PIECs was observed by fluorescent microscopy (excitation 520 nm, emission 610 nm).

Flow cytometry

DHR123 was used as ROS capture, and the cells at a concentration of 1 μmol/L were incubated together with DHR123 in culture medium for 1 h. Blank controls were set, in which DHR123 incubation was omitted. DHR123 was oxidized intracellularly to form the fluorescent compound, rhodamine 123 (Rh123) by ROS, and was maintained there. After DHR123 incubation, PIECs were trypsinized, harvested, washed twice with PBS and directly collected before an immediate detection of mean fluorescence intensity (MFI) of Rh123 for 10 × 103 cells each sample by flow cytometry was made to measure cellular ROS levels.

Measurement of NADPH oxidase activity

NADPH oxidase activity was measured using lucigenin chemiluminescence. Cells were trypsinized, pelleted by centrifugation, and resuspended at 1 × 10cells/mL with cold Krebs-Hepes buffer containing NaCl 119 mmol/L, Hepes 20 mmol/L, KCl 4.6 mmol/L, CaCl2 1.2 mmol/L, Na2HPO4 0.15 mmol/L, KH2PO4 0.4 mmol/L, MgSO4 1.0 mmol/L, NaHCO3 25.0 mmol/L and glucose 5.5 mmol/L (pH 7.4). 300 μl cellular suspensions were put into a 96-well white plate in a luminescence reader and dark-adapted lucigenin (10 μmol/L) was added to start the reaction. Chemiluminescence was recorded every 15 s for 10 min. The lucigenin chemiluminescence was expressed as counts per minute per 10cells. NADPH (final concentration 100 μmol/L) was added after measurement of background lucigenin chemiluminescence and measurements were performed for another 10 min. The difference between the values obtained before and after adding NADPH was calculated and it represented the activity of NADPH oxidase.

Statistical analysis

Each experiment was carried out in duplicate or triplicate and three independent experiments were performed. Results were expressed as mean ± standard deviation (SD) and analyzed with SPSS 11.5 software. Groups were compared using analysis of variance (ANOVA). Statistical significance was set at P < 0.05.

Results

The cellular ROS levels measured by fluorescent microscopy

Cultured with constant high glucose or intermittent high glucose for 3 days, the intracellular fluorescence was intensified as compared with normal glucose concentration group or mannitol control group (Fig. 1). The intermittent high glucose group showed more marked fluorescence than the constant high glucose group (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs00592-009-0140-5/MediaObjects/592_2009_140_Fig1_HTML.jpg
Fig. 1

ROS of PIEC detected with probe HE by fluorescent microscopy ×200. a PIECs with 5.5 mM glucose concentration, b PIECs with 25 mM mannitol, c PIECs with 25 mM glucose, d PIECs with intermittent high glucose (25–5.5 mM) for 3 days

The cellular ROS levels measured by flow cytometry

There was no statistically significant increase in MFI of Rh123 in mannitol- versus normal glucose-treated cells at 3 or 6 days (P > 0.05) (Figs. 2, 3). MFI of PIECs under constant high glucose concentrations (25 mM) or intermittent high glucose (25–12.5 or 25–5.5 mM) was higher than that in normal glucose concentration group and mannitol control group at 3 or 6 days (P < 0.05) (Figs. 2, 3), and the change was more marked at 6 days (P < 0.05). As compared with constant high glucose group, intermittent high glucose group showed more evident change (Figs. 2, 3).
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Fig. 2

ROS of PIECs treated with intermittent high glucose detected by flow cytometry https://static-content.springer.com/image/art%3A10.1007%2Fs00592-009-0140-5/MediaObjects/592_2009_140_Figa_HTML.gif 5.5 mmol/L glucose, without DHR123 probe. https://static-content.springer.com/image/art%3A10.1007%2Fs00592-009-0140-5/MediaObjects/592_2009_140_Figb_HTML.gif 5.5 mmol/L glucose, with DHR123 probe. https://static-content.springer.com/image/art%3A10.1007%2Fs00592-009-0140-5/MediaObjects/592_2009_140_Figc_HTML.gif mannitol or different high glucose. a 25 mM mannitol, b 25 mM glucose for 3 days, c intermittent high glucose (25–5.5 mM) for 3 d, d intermittent high glucose (25–12.5 mM) for 3 days, e intermittent high glucose (25–5.5 mM) for 6 days, f intermittent high glucose (25–12.5 mM) for 6 days, g 25 mM glucose for 6 days

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Fig. 3

ROS of PIECs treated with intermittent high glucose detected by flow cytometry N normal glucose (5.5 mM), M mannitol (25 mM), H constant high glucose (25 mM), H-L1 intermittent glucose (25–5.5 mM), H-L2 intermittent high glucose (25–12.5 mM). *P < 0.01 versus N, P < 0.05 versus H, ∆∆P < 0.01 versus H

The NADPH oxidase activity

The NADPH oxidase activity was increased under both constant 25 mM glucose and intermittent high glucose compared with normal glucose concentration and mannitol control group for 3 or 6 days (P < 0.01) (Fig. 4; Table 1). After 3 days, the NADPH oxidase activity was higher under fluctuating glucose concentration than that in constant high glucose (P < 0.05). After 6 days, the difference of changes in chemiluminescence between intermittent high glucose and constant high glucose-treated PIECs was even more marked (P < 0.01). As compared with normal glucose concentration group, the NADPH oxidase activity in mannitol group was slightly increased, but the difference was not statistically significant (P > 0.05) (Fig. 4; Table 1).
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Fig. 4

Changes in chemiluminescence for NADPH oxidase activity detection after 3 or 6 days of experiment. N normal glucose (5.5 mM), H high glucose (25 mM), H-L1 intermittent glucose (25–5.5 mM), H-L2 intermittent high glucose (25–12.5 mM), M mannitol

Table 1

Activity of NADPH oxidase (n = 3, \( \bar{\chi } \pm s \))

Group (days)

N

H

H-L1

H-L2

M

3

39.58 ± 10.21

275.53 ± 34.63**

396.65 ± 24.03**∆∆

348.15 ± 41.68**

46.03 ± 8.96

6

56.03 ± 15.73

449.45 ± 43.54**

845.05 ± 78.80**∆∆

596.60 ± 35.35**∆∆

62.64 ± 14.48

Compared with N: *P < 0.05; **P < 0.01. Compared with H: P < 0.05; ∆∆P < 0.01

Discussion

The risks of cardiac, cerebral and peripheral vascular diseases are increased two- to sevenfold in diabetes mellitus [6]. Increased risks are related to fasting plasma glucose (FPG), postprandial plasma glucose, and average levels of plasma glucose as measured by HbA1c. Plasma glucose concentration in normal subjects is strictly controlled within a narrow range, but excessive glucose excursion after ingestion of a meal seems to be a common phenomenon even in treated diabetic individuals, whose plasma glucose concentration often changes markedly within a single day. It is now recognized that both hyperglycemia at 2 h during an oral glucose challenge and glucose fluctuations per se are strong predictors of both macrovascular disease and microvascular complications, and it has been suggested [7] that these hyperglycemic “spikes” may play a direct and significant role in the pathogenesis of diabetic vascular complications. Recently, the possibility that acute increases of glucose levels, particularly in the postprandial state, may be an independent risk factor for cardiovascular disease that has received much attention. The coefficient of variation for fasting plasma glucose (CV-FPG) is also an independent predictor of cardiovascular mortality in type 2 diabetes [8]. Brun et al. [9] found that a CV-FPG < 15% was associated with the lower cardiovascular mortality rate, whereas a CV-FPG > 25% predicted the higher cardiovascular mortality rate. The result indicated that high FPG instability seems to be associated with an endothelial dysfunction which may increase the risk of cardiovascular morbidity and mortality.

The mechanisms by which glucose fluctuation damages the cells remain uncertain. Recently, there is growing evidence that an acute increase of plasma glucose is accompanied by ROS generation that may contribute to the endothelial dysfunction. It is of extreme importance that ROS generation has been implicated in the etiology of several diseases. In a normal cellular environment, ROS is essential to life, while in case of overproduction or exhaustion of antioxidants it might become deleterious. The ROS designation comprehends not only free radicals, such as superoxide radical (O2·−), hydroxyl radical (OH·), but also non-radicals, namely hydrogen peroxide (H2O2) and singlet oxygen (1O2). The generation of ROS is one major factor in the development of diabetes and its complications [10]. Of all the ROS, O2·− is the most important one and it is the source of other ROS. Clinical evidence [11] suggests that in vivo glucose fluctuations may be dangerous for endothelial cells and that this effect may be mediated by the overproduction of ROS.

ROS possesses one or more unpaired electrons, its short lifetime and instability make it difficult to be detected. The fluorescence methodology, associated with the use of suitable probes, is an excellent approach to measure ROS because of its high sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques [12, 13]. DHE has been used as a fluorescent probe for detecting O2·− due to its reported relative specificity for this ROS [14, 15]. The product is a red fluorescent compound (probably ethidium). DHR123 is a non-fluorescent molecule that diffuses across cell membranes and is oxidized by ROS to the fluorescent rhodamine 123 within cells.

In this study, we employed a cellular model in which PIECs were exposed to constant high glucose or intermittent high glucose, a condition that partly mimics glucose fluctuation actually happening in vivo in patients with diabetes. We used DHE and DHR123 as ROS captures to detect ROS by fluorescent microscopy and flow cytometry, respectively. In agreement with previous studies, we found that constant high glucose concentrations produced increased generation of ROS. However, ROS level in intermittent high glucose group was higher than that in constant high glucose group. In our study, the wider the glucose level fluctuated, the more ROS the PIECs produced. This result suggested that fluctuating hyperglycemia may induce more severe damage in endothelial cells. Our result is consistent with what Quagliaro [1618] has verified that intermittent high glucose enhanced ICAM-1, VCAM-1 and E-selectin expression and apoptosis related to oxidative stress in human umbilical vein endothelial cells (HUVECs). Previous studies and our present in vitro cell-based study demonstrated that fluctuating hyperglycemia resulted in more toxic effects than constant high glucose did probably due to more severe oxidative stress. Studies have shown that temporary hyperglycemia induces adhesion of monocyte to endothelial cells compared with consistent hyperglycemia in SD rats [3, 4]. A previous research [5] using Kakizaki rat thoracic aorta also indicated that repetitive fluctuations in blood glucose enhance monocyte adhesion to the endothelium.

Mitochondrion is the main source of O2·− in many types of cells. In addition to mitochondrial sources, O2·− can be generated by several enzymes including the vascular isoforms of NADPH oxidase, which is thought to be the most important enzymatic source [19, 20]. The vascular NAD(P)H oxidase complex is an important source of O2·− in vascular endothelial cells. Schleicher et al. [21] verified that ROS production in endothelial cells is mediated by activation of NADPH oxidase because inhibition of this enzyme completely prevents the high glucose-induced endothelial ROS formation. The increased NADPH oxidase activity is closely correlated with diabetic complications [22]. A recent study showed that suppression of NADPH oxidase might be a promising therapeutic strategy because localized adventitial delivery of an NADPH oxidase inhibitor reduced overall vascular O2·− and neointima formation [23]. In the present study, we found that high glucose resulted in an increase in NADPH oxidase activity in PIECs, and intermittent high glucose induced higher activity of NADPH oxidase as compared with constant high glucose. Our result suggested that the endothelial ROS formation induced by high glucose was mainly mediated by NADPH oxidase activation. Our previous study [24] also verified that apocynin, an inhibitor of NADPH oxidase, at a concentration of 10 μM, reduced, but did not completely inhibit, ROS produced by high glucose. The higher level of ROS induced by intermittent high glucose as compared with constant high glucose was probably due to more significant activation of NADPH oxidase.

In this study, NADPH oxidases and ROS levels in the mannitol-treated groups were slightly increased, and the elevation showed no statistical significance as compared with the control group. It means that glucose uptake and metabolism, but not high osmolality, are required for high glucose-induced ROS generation. So, it is more likely that glucose itself or its reaction product plays the role. Some metabolic changes and glucose toxicities may act in concert. Glucose fluctuation may weaken the adaption and cause much stronger toxicities.

In conclusion, high glucose may be involved in the development of oxidative stress and endothelial cells injury, and glucose fluctuation seems to exert a more dangerous effect. The clinical outcomes might be improved by decreasing the glucose fluctuations in diabetic patients.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No.30872727). This study greatly acknowledges the support from department of cell biology, Shanghai Jiaotong University School of Medicine. The authors should also give many thanks to Professor Ji-shou Hou, Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine.

Copyright information

© Springer-Verlag 2009