Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis Juan Li a, Minglian Huang b, Xingping Shen b,⁎ a b
Department of Emergency, Zhongshan Hospital Xiamen University, Xiamen 361004, Fujian, China Department of Endocrinology, Zhongshan Hospital Xiamen University, Xiamen 361004, Fujian, China
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 20 May 2014 Accepted 11 June 2014 Available online xxxx Keywords: Hyperglycemic crises Oxidative stress Pro-inflammatory cytokines
a b s t r a c t Aims: To investigate the relationship between oxidative stress and serum levels of pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis. Methods: Seventy-three patients presenting to hospital with diabetic ketoacidosis or non-ketotic hyperglycemia were studied. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, total antioxidant capacity (TAC), 8-iso-prostaglandin F2α (8-iso-prostaglandinF2α, 8-iso-PGF2α), tumor necrosis factor receptor-I (TNF-RI), interleukin -1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured in all patients. The patients were then given an intravenous infusion of insulin 0.1U • kg-1 • h-1, as well as fluids, symptomatic therapy and parenteral and intravenous nutrition. Results: Before treatment, SOD and TAC were significantly lower (P b 0.05), whereas MDA and 8-iso-PGF2α were significantly higher (P b 0.05) in patients with hyperglycemic crises compared to controls. After treatment, SOD and TAC significantly increased (P b 0.05), while MDA and 8-iso-PGF2α significantly decreased (P b 0.05). 2. TNF-RI, IL-1β, TNF-α and IL-6 were significantly higher, both before and after treatment, in patients with hyperglycemic crises compared to controls (P b 0.05). 3. Before treatment, IL-6 and TNF-α were positively correlated with 8-iso-PGF2α (r = 0.32, r = 0.36, P b 0.05) in patients with hyperglycemic crises. After treatment, IL-6 and SOD were negatively correlated within patients (r = − 0.33, P b 0.05). Multiple regression analysis indicated that 8-iso-PGF2α affects the level of serum IL-6. Conclusion: Patients with hyperglycemic crises have significantly increased oxidative stress and dysregulated serum pro-inflammatory cytokines that can be effectively treated by intensive insulin therapy. 1.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction Hyperglycemic crisis is one of the most common acute complications in type 2 diabetes (T2DM). It is defined as an acute elevation of blood glucose and, during treatment, is often accompanied by wide fluctuations in blood glucose levels. Major clinical manifestations of T2DM include absolute or relative lack of insulin, increased levels of counter-regulatory hormones, and dysregulation of electrolytes and protein, as well as fat and carbohydrate metabolism (Kitabchi, Umpierrez, Miles, & Fisher, 2009). Basic and clinical studies have
Conflicts of interest: The authors declare that they have no conflict of interest. Funding: Xiamen Key Fund for Medical Research (third period) Fujian Provincial Natural Science Foundation (2012D040) Fujian Medical Innovation Fund (2009-CXB-60) ⁎ Corresponding author. Tel.: +86 592 2590250; fax: +86 21 64085875. E-mail address: [email protected] (X. Shen).
shown that those with a history of repeated hyperglycemic crises are at increased risk for myocardial infarction, heart failure and cardiogenic shock. Furthermore, acute hyperglycemia can accelerate the inflammatory immune response. Therefore, acute hyperglycemia is now considered to be a risk factor for myocardial infarction (Ceriello, 2008). T2DM is often associated with a "low-grade inflammatory status” accompanied by insulin resistance. Hyperglycemia can induce monocytes to produce pro-inflammatory cytokines and chemokines and is characterized by increased levels of serum acute phase proteins and many cytokines (such as TNFα, lL-6, lL-1β). This causes a change in insulin sensitivity and subsequently affects glucose metabolism (Vaarala & Yki-Jarvinen, 2012). In addition, pro-inflammatory cytokines are closely related to the intracellular redox state (Goldberg, 2009). It has been shown that insulin not only regulates glucose and lipid metabolism, but also has anti-inflammatory effects. A large number of studies have demonstrated (Aljada, Ghanim, Mohanty,
http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008 1056-8727/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
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J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Kapur, & Dandona, 2002) that insulin exerts its anti-inflammatory effect via multiple pathways. Insulin induces the release of nitric oxide (NO), increases the expression of endothelial NO, and inhibits the expression of NF-κB and MCP-1, each of which are thought to limit the progression of atherosclerosis. Free radicals are positively correlated with diabetic macrovascular and microvascular complications. High blood sugar can induce oxidative stress and increased glucose levels induce ROS in the mitochondria and cause intracellular oxidative stress, which leads to insulin resistance (Dandona, Chaudhuri, Ghanim, & Mohanty, 2006; Stentz, Umpierrez, Cuervo, & Kitabchi, 2004). In addition, wide fluctuations in blood glucose decrease the expression of radical-scavenging toxic genes, and cause oxidative stress (Chaudhuri & Umpierrez, 2012). Our previous studies have shown that T2D patients with hyperglycemia crises tend to have increased oxidative stress. In obese and non-diabetic patients, insulin therapy significantly inhibits levels of reactive oxygen species (ROS), plasminogen activator inhibitor and ICAM-1, indicating that insulin exhibits comprehensive anti-oxidant and anti-inflammatory actions (Ceriello et al., 2008). Insulin and fluid treatments alleviate the hyperglycemic crises and decrease the amount of oxidative stress (Shen, Li, Zou, Wu, & Zhang, 2012). Because hyperglycemia is an inducer of both oxidative stress and inflammation (Dandona, Mohanty, Chaudhuri, Garg, & Aljada, 2005), this study aims to examine the changes in, and correlation between, oxidative stress and blood levels of pro-inflammatory cytokines in T2D patients with hyperglycemic crises before and after intensive insulin treatment.
2.2.2. Blood sample collection and analysis Blood samples were collected from patients with hyperglycemic crisis at admission and 72 h after remission. Blood glucose was measured by glucose oxidase; glycosylated hemoglobin (HbAlc) was measured using high performance liquid chromatography; leukocyte count (WBC) was determined by the five classification instrument method; creatinine (Cr), blood urea nitrogen (BUN) and electrolytes were analyzed using the alkaline picric acid method, enzyme coupling rate method, and the enzymatic assay, respectively. Islet cell antibodies (ICA) and glutamic acid decarboxylase antibody (GADAb) were measured by enzyme-linked immunosorbent assay (ELISA) (provided by Niomerica, US); C peptide was measured by electrochemiluminescent immunoassay; 8-iso-prostaglandinF2α (8-iso -PGF2α) was determined by ELISA (ADL, US); and superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (TAC) were measured with kits provided by Nanjing Jiancheng Bioengineering Institute. Tumor necrosis factor receptor 1 (TNF-RI), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using ELISA (Wuhan Boster Biological Engineering Limited, China).
2. Materials and methods
2.2.4. Statistical analysis Data are presented as mean ± standard deviation (x ± s). The statistical software SPSS 11.5 was used for data analysis. A paired t test was used for before and after treatment comparisons. The groups were compared using the Student's t-test. A Pearson correlation analysis and multiple regression analysis were performed to determine the relationship among variables within each group.
2.1. Study population Seventy three TD2M patients (45 males and 28 females) with diabetic ketoacidosis (DKA) or non-ketotic hyperglycemia (NKH) were studied between June 2005 and February 2008. DKA patients were defined as having an admission blood glucose level N 13.9 mmol/L, blood pH b 7.3, blood HCO3 b 18 mmol/L, an anion gap N 15 mmol/L, and urine ketone positive (Dandona et al., 2006; Kitabchi, Umpierrez, Murphy, & Kreisberg, 2006). NKH patients were defined as having an admission blood glucose N 22.4 mmol/L, blood pH N 7.3, blood HCO3 N 18 mmol/L, and urine ketone negative. Patients with hyperglycemic crises had no signs of infection or other known preexisting diseases that would induce DKA or NKH. Patients with gastrointestinal bleeding, fever, endocrine disease, myocardial infarction, heart failure, heart disease, chronic obstructive pulmonary disease (COPD), renal insufficiency, and those who were pregnant or who smoked, were excluded. The control group included 33 healthy subjects (19 males and 14 females) with a mean age of 52.11 ± 7.63 years. Subjects in the control group had no known heart, brain, kidney, endocrine or metabolic disease. A 5 ml fasting blood sample was collected from each control subject, centrifuged to obtain plasma and then stored at 70 °C. Xiamen University Zhongshan Hospital Institutional Review Board and ethics committee approved the study, and all subjects signed an informed consent. 2.2. Methods 2.2.1. Treatment Central venous access was established in all patients with hyperglycemic crisis after admission to the hospital. Patients were given insulin (0.1 U/kg/h) via micro pump. Fluids and symptomatic treatments were also administered. DKA and NKH patients received enteral nutrition with identical calorie and/or intravenous nutritional support. Patients were considered to be in remission when they were fully conscious with a glucose level b 13.9 mmol/L, a blood pH N 7.35, blood HCO3 N 18 mmol/L, and an anion gap 8 ~ 15 mmol/L.
2.2.3. Body mass index (BMI) and waist/hip ratio (WHR) When a patient's condition had stabilized, BMI and WHR were measured. The measurements were recorded in kg and cm, to an accuracy of 0.1 kg and 0.1 cm, respectively. BMI = weight (kg)/height (m) 2, WHR = waist/hip.
3. Results 3.1. General clinical data Before treatment, the WBC was significantly higher in patients with hyperglycemic crises (DKA and NKH groups) compared to controls (P b 0.05) but after control of the hyperglycemic crisis returned to levels similar to controls (Table 1). All other clinical parameters were similar between the control group and the hyperglycemic group at the time of crisis and after insulin treatment. 3.2. Biochemical data Before treatment, levels of blood glucose, HbA1c, and C-Peptides were significantly higher than controls in both the DKA and NKH hyperglycemic patients (Table 2). In addition, the DKA patients were ketonic, had a greater anion gap, and lower HCO3 levels than controls (P b 0.05). After insulin treatment, all biochemical parameters (HbA1c was not re-measured) returned to control levels except the level of C Peptides, which remained low in both groups of patients (P b 0.05). SOD and TAC were significantly lower in the hyperglycemic patients prior to the insulin treatment, and remained lower after treatment, though rising significantly in response to treatment (Table 3). MDA, 8-iso-PGF2α, IL-1B, TNF-a, and IL-6 were all significantly higher before treatment in both groups of patients in hyperglycemic crises compared with controls (P b 0.05). These proinflammatory cytokine levels were significantly reduced upon remission (P b 0.05) but remained significantly higher than control levels (P b 0.05).
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Table 1 Clinical characteristics of diabetic hyperglycemia at admission (x ± s). Group
Case
Age
BMI (kg/m2)
(male/female) Control DKA before treatment after treatment NKH before treatment after treatment
WHR
Body temperature
Systolic blood pressure
Diastolic blood pressure
# of Neutrophils
(°C)
(mm Hg)
(mm Hg)
(White blood cell × 106)
76 ± 8
33 (19/14)
52.11 ± 7.63
24.20 ± 4.01
0.86 ± 0.07
36.95 ± 5.12
122 ± 15
6.25 ± 0.80
37 (22/15)
53.22 ± 10.04
25.21 ± 6.01 36.80 ± 8.19
0.84 ± 0.08 120 ± 16
37.22 ± 7.30 76 ± 10
124 ± 18
78 ± 9 6.50 ± 2.64b
14.28 ± 3.55a
36 (23/13)
54.50 ± 11.22
25.77 ± 7.77 37.11 ± 6.39
0.85 ± 0.09 128 ± 15
37.08 ± 5.58 78 ± 9
126 ± 19
77 ± 9 5.66 ± 3.41b
15.55 ± 4.92a
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia; blank: N/A. a P b 0.05 vs. control. b P b 0.05 before treatment vs. after treatment.
3.3. Relationship among variables: correlation and multiple regression analysis Before insulin treatment, IL-6 and TNF-α were significantly, positively correlated with 8-iso-PGF2α within patients experiencing hyperglycemic crises (r = 0.32, r = 0.36, respectively; P b 0.05). After insulin treatment, a significant, negative correlation was observed between IL-6 and SOD (r = − 0.33, P b 0.05). IL-6 and MDA levels were positively correlated with each other in patients with hyperglycemic crises both before and after treatment (r = 0.36, P b 0.05). A multiple stepwise regression analysis was performed using TNF-RI, IL-1β, TNF-α and IL-6 as dependent variables and age, BMI, WHR, body temperature, systolic blood pressure, diastolic blood pressure, blood cell count, blood glucose, HbA1c, APH, HCO3, effective osmolality, anion gap, C peptide, SOD, MDA, TAC and 8-iso-PGF2α as independent variables. When IL-6 was the dependent variable, 8-iso-PGF2α was one of the independent variables. The standard regression coefficient was − 0.36 (P b 0.05). 4. Discussion A wide variety of antioxidants exists within the body that influences each other, and accurate measurement of specific antioxidant content is difficult. Therefore, multiple parameters are used to evaluate oxidative stress. Hyperglycemia is an inducer of oxidative stress and inflammation (Dandona et al., 2005). The results of the current study suggest that T2DM patients with hyperglycemic crises exhibit dysregulated levels of 8-iso-PGF2α, SOD, MDA, and TAC, which is consistent with previous studies as well as our preliminary data (Ceriello, 2005; Chaudhuri & Umpierrez, 2012; Dandona et al., 2006; Shen et al., 2012). Patients with hyperglycemic crises have a decreased level of TAC partially due to lack of storage for the raw materials used to generate TAC. Hyperglycemia promotes neutrophils and monocytes to produce
more superoxide anions, thus increasing the expression of the p47phox subunit in the NADPH oxidase complex, leading to increased opportunity for oxygen conversion to superoxide anions (Dandona, Chaudhuri, Ghanim, & Mohanty, 2009). Acute hyperglycemia can also decrease the expression of free radicals, while fluctuations in blood glucose levels can cause oxidative stress (Chaudhuri & Umpierrez, 2012; Monnier et al., 2006). Reactive oxygen species (ROS) are generated by oxygen and NADPH oxidase. In obese and non-diabetic patients, ROS, plasminogen activator inhibitor and ICAM-1 are decreased after treatment with insulin, indicating that insulin has comprehensive antioxidant and rapid anti-inflammatory effects (Ceriello et al., 2008; Dandona et al., 2001). SOD, MDA, TAC and 8-iso-PGF2α levels were significantly improved in patients recovering from hyperglycemic crisis 72 h after insulin treatment. This may be due to the fact that insulin reduces the expression of the p47phox subunit in the NADPH oxidase enzyme complex, directly inhibiting NADPH oxidase, leading to a reduction in NFκB expression (Dandona et al., 2001; Ji et al., 2010) and subsequently ROS. However, although all parameters indicating oxidative stress had improved in patients 72 h after treatment, oxidative stress indicators were still significantly below levels seen in healthy controls. These data suggest that insulin has certain antioxidant effects. The oxidative stress induced by hyperglycemia is not only related to diabetes itself, but is also associated with a variety of biochemical mechanisms and fluctuations in blood glucose during treatment (Chaudhuri & Umpierrez, 2012). Because fluctuations in blood glucose are a critical indicator of patient mortality (Krinsley, 2008; Krinsley & Keegan, 2010), fluctuations in blood glucose must be minimized when treating hyperglycemic crises. Our research suggests that T2DM patients with hyperglycemic syndrome have increased oxidative stress that remains significantly elevated in remission, albeit at a reduced level following insulin infusion. Acute hyperglycemia can significantly increase the inflammatory and immune cytokines in the peripheral circulation that play
Table 2 Biochemical characteristics of diabetic hyperglycemia before and after treatment (x ± s). Group Control DKA Before treatment After treatment NKH Before treatment After treatment
Blood glucose (mmol/L)
HbA1c (%)
pH (mmol/L)
HCO3−
Ketone (mmol/L)
Effective osmotic pressure (mmol/L)
Anion gap (nmol/L)
C peptides
5.36 ± 1.02
5.80 ± 1.26
7.38 ± 1.68
23.78 ± 3.90
N
286.29 ± 13.38
11.46 ± 3.90
0.79 ± 0.09
20.19 ± 8.60a 7.55 ± 3.21b
15.27 ± 6.90a
7.20 ± 2.48a 7.36 ± 1.97b
11.50 ± 2.20a 22.63 ± 3.23
P N
279.90 ± 15.70 280.62 ± 13.33
28.70 ± 5.16a 12.48 ± 4.57b
0.36 ± 0.06a 0.35 ± 0.07a
27.03 ± 10.45a 7.53 ± 2.76b
13.62 ± 7.63a
7.39 ± 2.39 7.38 ± 3.01
24.06 ± 3.05 24.11 ± 4.17
N N
293.51 ± 16.25 288.309 ± 18.11
11.79 ± 4.62 12.54 ± 5.03
0.45 ± 0.08a 0.40 ± 0.09a
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia; blank: N/A. a P b 0.05 vs. control. b P b 0.05 before treatment vs. after treatment.
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
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J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Table 3 Oxidative stress markers and pro-inflammatory cytokines in diabetic hyperglycemia before and after treatment (x ± s). Group
SOD (kU/L)
Control DKA Before treatment After treatment NKH Before treatment After treatment
87.32 ± 12.68
MDA (μmol/L) 4.92 ± 1.17 a
TAC (kU/L)
8-iso-PGF2α (μg/L)
28.66 ± 10.70 a
a
6.12 ± 1.65
TNF-RI (ng/L)
IL-1β (ng/L)
365.89 ± 82.63 a
TNF-α (ng/L)
2.13 ± 0.60 a
50.11 ± 16.72 70.69 ± 13.04ab
10.06 ± 2.36 6.23 ± 1.71ab
19.23 ± 9.85 23.24 ± 8.37ab
20.31 ± 3.63 13.92 ± 5.23ab
537.12 ± 132.05 410.22 ± 108.38
53.10 ± 13.84a 69.22 ± 12.43ab
9.63 ± 1.39a 6.28 ± 2.06ab
19.64 ± 11.77a 22.86 ± 10.73ab
19.19 ± 4.55a 12.29 ± 4.36ab
596.85 ± 121.62a 450.59 ± 150.24ab
ab
IL-6 (ng/L)
22.47 ± 4.93 a
6.85 ± 1.87 a
9.15 ± 1.66 4.82 ± 1.85ab
90.35 ± 13.82 33.29 ± 11.07ab
12.88 ± 1.63a 9.12 ± 2.87ab
11.13 ± 2.63a 5.07 ± 1.49ab
86.21 ± 16.42a 31.73 ± 12.55ab
13.57 ± 2.26a 10.59 ± 1.99ab
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia. a P b 0.05 vs. control; bP b 0.05 before treatment vs. after treatment.
important roles in diabetic immune activation (Ceriello, 2005). Elevated IL-6 and TNF-α can result in insulin resistance by affecting the signaling pathway of the insulin receptor in adipose tissue and skeletal muscle, TNF-RI (Hube & Hauner, 2000). Because TNF-RI has a longer half-life in peripheral circulation, it is recognized as a marker of TNF system activation (Arias et al., 2008; Bullo, Garcia-Lorda, & Salas-Salvado, 2002; Good et al., 2006). IL-1β dysregulates insulin signaling, which in turn decreases insulin sensitivity via excessive autocrine and paracrine signaling in macrophages and the surrounding tissue (Su et al., 2009). We observed that TNF-RI, IL-1β, TNF-α and IL-6 were significantly elevated in patients with hyperglycemic crises and were significantly decreased after insulin treatment, although levels in remission remained significantly higher than in controls, which is in line with previous studies and our earlier report (Esposito et al., 2002; Kolb & Mandrup-Poulsen, 2005). While diabetic patients are in an acute hyperglycemic state, the increase in circulating cytokines is mainly due to non-circulating cells, such as adipocytes and endothelial cells (Pickup, Chusney, Thomas, & Burt, 2000). High blood sugar can affect the activation of NADPH oxidase and PKC and increase the impact of pro-inflammatory cytokines. PKC activation can phosphorylate p38 MAPK subfamilies and ERK1/2, thereby activating NF-κB, which, in turn, enhances monocytes to secrete various pro-inflammatory cytokines (Dasu, Devaraj, & Jialal, 2007; Pickup, 2004). Oxidative stress, soluble advanced glycation end products, and lipid peroxide production caused by hyperglycemia, may jointly activate the key upstream kinases of proinflammatory cytokines, while the superoxide anion can simultaneously activate redox-sensitive pro-inflammatory transcription factors such as NFκB, activator protein-1, and hypoxia -inducible factor α, so that the inflammatory process is amplified and sustained. In addition, fluctuation in blood glucose during the course of treatment is another important factor that induces the production of pro-inflammatory cytokines. In the current study, metabolic stress caused by low-grade inflammation was significantly enhanced during the hyperglycemic crisis in patients with T2DM. Increased levels of TNF-RI, IL-1β, TNF-α and IL-6 levels in patients with hyperglycemic crises affect insulin sensitivity via a variety of mechanisms, such as decreased expression of PPAR-γ (Pickup, 2004) and directly interfere with phosphorylation of insulin receptors in peripheral tissue. This affects insulin signaling, indirectly interferes with leptin release in lipid cells, neurons and other cells, dysregulates activation of the hypothalamic–pituitary–adrenal axis which decreases insulin sensitivity (Kolb & Mandrup-Poulsen, 2005), and increases the expression of cell adhesion molecules that are involved in endothelial cell damage. The current study demonstrates that IL-6 is closely related to oxidative stress. In cases of hyperglycemia, increased oxidative stress and activated PKC-α/β promote IL-6 secretion mediated by NF-Κb (Devaraj, Venugopal, Singh, & Jialal, 2005), though the exact mechanism is unknown. The pro-inflammatory cytokines were significantly decreased in patients with hyperglycemic crises after insulin treatment, indicating that insulin plays an anti-inflammatory role in hyperglycemic crises. It is likely that insulin plays an antiinflammatory role by inhibiting NFκB in monocyte nuclei, where it
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Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
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The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis Juan Li a, Minglian Huang b, Xingping Shen b,⁎ a b
Department of Emergency, Zhongshan Hospital Xiamen University, Xiamen 361004, Fujian, China Department of Endocrinology, Zhongshan Hospital Xiamen University, Xiamen 361004, Fujian, China
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 20 May 2014 Accepted 11 June 2014 Available online xxxx Keywords: Hyperglycemic crises Oxidative stress Pro-inflammatory cytokines
a b s t r a c t Aims: To investigate the relationship between oxidative stress and serum levels of pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis. Methods: Seventy-three patients presenting to hospital with diabetic ketoacidosis or non-ketotic hyperglycemia were studied. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, total antioxidant capacity (TAC), 8-iso-prostaglandin F2α (8-iso-prostaglandinF2α, 8-iso-PGF2α), tumor necrosis factor receptor-I (TNF-RI), interleukin -1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured in all patients. The patients were then given an intravenous infusion of insulin 0.1U • kg-1 • h-1, as well as fluids, symptomatic therapy and parenteral and intravenous nutrition. Results: Before treatment, SOD and TAC were significantly lower (P b 0.05), whereas MDA and 8-iso-PGF2α were significantly higher (P b 0.05) in patients with hyperglycemic crises compared to controls. After treatment, SOD and TAC significantly increased (P b 0.05), while MDA and 8-iso-PGF2α significantly decreased (P b 0.05). 2. TNF-RI, IL-1β, TNF-α and IL-6 were significantly higher, both before and after treatment, in patients with hyperglycemic crises compared to controls (P b 0.05). 3. Before treatment, IL-6 and TNF-α were positively correlated with 8-iso-PGF2α (r = 0.32, r = 0.36, P b 0.05) in patients with hyperglycemic crises. After treatment, IL-6 and SOD were negatively correlated within patients (r = − 0.33, P b 0.05). Multiple regression analysis indicated that 8-iso-PGF2α affects the level of serum IL-6. Conclusion: Patients with hyperglycemic crises have significantly increased oxidative stress and dysregulated serum pro-inflammatory cytokines that can be effectively treated by intensive insulin therapy. 1.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction Hyperglycemic crisis is one of the most common acute complications in type 2 diabetes (T2DM). It is defined as an acute elevation of blood glucose and, during treatment, is often accompanied by wide fluctuations in blood glucose levels. Major clinical manifestations of T2DM include absolute or relative lack of insulin, increased levels of counter-regulatory hormones, and dysregulation of electrolytes and protein, as well as fat and carbohydrate metabolism (Kitabchi, Umpierrez, Miles, & Fisher, 2009). Basic and clinical studies have
Conflicts of interest: The authors declare that they have no conflict of interest. Funding: Xiamen Key Fund for Medical Research (third period) Fujian Provincial Natural Science Foundation (2012D040) Fujian Medical Innovation Fund (2009-CXB-60) ⁎ Corresponding author. Tel.: +86 592 2590250; fax: +86 21 64085875. E-mail address: [email protected] (X. Shen).
shown that those with a history of repeated hyperglycemic crises are at increased risk for myocardial infarction, heart failure and cardiogenic shock. Furthermore, acute hyperglycemia can accelerate the inflammatory immune response. Therefore, acute hyperglycemia is now considered to be a risk factor for myocardial infarction (Ceriello, 2008). T2DM is often associated with a "low-grade inflammatory status” accompanied by insulin resistance. Hyperglycemia can induce monocytes to produce pro-inflammatory cytokines and chemokines and is characterized by increased levels of serum acute phase proteins and many cytokines (such as TNFα, lL-6, lL-1β). This causes a change in insulin sensitivity and subsequently affects glucose metabolism (Vaarala & Yki-Jarvinen, 2012). In addition, pro-inflammatory cytokines are closely related to the intracellular redox state (Goldberg, 2009). It has been shown that insulin not only regulates glucose and lipid metabolism, but also has anti-inflammatory effects. A large number of studies have demonstrated (Aljada, Ghanim, Mohanty,
http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008 1056-8727/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
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J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Kapur, & Dandona, 2002) that insulin exerts its anti-inflammatory effect via multiple pathways. Insulin induces the release of nitric oxide (NO), increases the expression of endothelial NO, and inhibits the expression of NF-κB and MCP-1, each of which are thought to limit the progression of atherosclerosis. Free radicals are positively correlated with diabetic macrovascular and microvascular complications. High blood sugar can induce oxidative stress and increased glucose levels induce ROS in the mitochondria and cause intracellular oxidative stress, which leads to insulin resistance (Dandona, Chaudhuri, Ghanim, & Mohanty, 2006; Stentz, Umpierrez, Cuervo, & Kitabchi, 2004). In addition, wide fluctuations in blood glucose decrease the expression of radical-scavenging toxic genes, and cause oxidative stress (Chaudhuri & Umpierrez, 2012). Our previous studies have shown that T2D patients with hyperglycemia crises tend to have increased oxidative stress. In obese and non-diabetic patients, insulin therapy significantly inhibits levels of reactive oxygen species (ROS), plasminogen activator inhibitor and ICAM-1, indicating that insulin exhibits comprehensive anti-oxidant and anti-inflammatory actions (Ceriello et al., 2008). Insulin and fluid treatments alleviate the hyperglycemic crises and decrease the amount of oxidative stress (Shen, Li, Zou, Wu, & Zhang, 2012). Because hyperglycemia is an inducer of both oxidative stress and inflammation (Dandona, Mohanty, Chaudhuri, Garg, & Aljada, 2005), this study aims to examine the changes in, and correlation between, oxidative stress and blood levels of pro-inflammatory cytokines in T2D patients with hyperglycemic crises before and after intensive insulin treatment.
2.2.2. Blood sample collection and analysis Blood samples were collected from patients with hyperglycemic crisis at admission and 72 h after remission. Blood glucose was measured by glucose oxidase; glycosylated hemoglobin (HbAlc) was measured using high performance liquid chromatography; leukocyte count (WBC) was determined by the five classification instrument method; creatinine (Cr), blood urea nitrogen (BUN) and electrolytes were analyzed using the alkaline picric acid method, enzyme coupling rate method, and the enzymatic assay, respectively. Islet cell antibodies (ICA) and glutamic acid decarboxylase antibody (GADAb) were measured by enzyme-linked immunosorbent assay (ELISA) (provided by Niomerica, US); C peptide was measured by electrochemiluminescent immunoassay; 8-iso-prostaglandinF2α (8-iso -PGF2α) was determined by ELISA (ADL, US); and superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (TAC) were measured with kits provided by Nanjing Jiancheng Bioengineering Institute. Tumor necrosis factor receptor 1 (TNF-RI), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using ELISA (Wuhan Boster Biological Engineering Limited, China).
2. Materials and methods
2.2.4. Statistical analysis Data are presented as mean ± standard deviation (x ± s). The statistical software SPSS 11.5 was used for data analysis. A paired t test was used for before and after treatment comparisons. The groups were compared using the Student's t-test. A Pearson correlation analysis and multiple regression analysis were performed to determine the relationship among variables within each group.
2.1. Study population Seventy three TD2M patients (45 males and 28 females) with diabetic ketoacidosis (DKA) or non-ketotic hyperglycemia (NKH) were studied between June 2005 and February 2008. DKA patients were defined as having an admission blood glucose level N 13.9 mmol/L, blood pH b 7.3, blood HCO3 b 18 mmol/L, an anion gap N 15 mmol/L, and urine ketone positive (Dandona et al., 2006; Kitabchi, Umpierrez, Murphy, & Kreisberg, 2006). NKH patients were defined as having an admission blood glucose N 22.4 mmol/L, blood pH N 7.3, blood HCO3 N 18 mmol/L, and urine ketone negative. Patients with hyperglycemic crises had no signs of infection or other known preexisting diseases that would induce DKA or NKH. Patients with gastrointestinal bleeding, fever, endocrine disease, myocardial infarction, heart failure, heart disease, chronic obstructive pulmonary disease (COPD), renal insufficiency, and those who were pregnant or who smoked, were excluded. The control group included 33 healthy subjects (19 males and 14 females) with a mean age of 52.11 ± 7.63 years. Subjects in the control group had no known heart, brain, kidney, endocrine or metabolic disease. A 5 ml fasting blood sample was collected from each control subject, centrifuged to obtain plasma and then stored at 70 °C. Xiamen University Zhongshan Hospital Institutional Review Board and ethics committee approved the study, and all subjects signed an informed consent. 2.2. Methods 2.2.1. Treatment Central venous access was established in all patients with hyperglycemic crisis after admission to the hospital. Patients were given insulin (0.1 U/kg/h) via micro pump. Fluids and symptomatic treatments were also administered. DKA and NKH patients received enteral nutrition with identical calorie and/or intravenous nutritional support. Patients were considered to be in remission when they were fully conscious with a glucose level b 13.9 mmol/L, a blood pH N 7.35, blood HCO3 N 18 mmol/L, and an anion gap 8 ~ 15 mmol/L.
2.2.3. Body mass index (BMI) and waist/hip ratio (WHR) When a patient's condition had stabilized, BMI and WHR were measured. The measurements were recorded in kg and cm, to an accuracy of 0.1 kg and 0.1 cm, respectively. BMI = weight (kg)/height (m) 2, WHR = waist/hip.
3. Results 3.1. General clinical data Before treatment, the WBC was significantly higher in patients with hyperglycemic crises (DKA and NKH groups) compared to controls (P b 0.05) but after control of the hyperglycemic crisis returned to levels similar to controls (Table 1). All other clinical parameters were similar between the control group and the hyperglycemic group at the time of crisis and after insulin treatment. 3.2. Biochemical data Before treatment, levels of blood glucose, HbA1c, and C-Peptides were significantly higher than controls in both the DKA and NKH hyperglycemic patients (Table 2). In addition, the DKA patients were ketonic, had a greater anion gap, and lower HCO3 levels than controls (P b 0.05). After insulin treatment, all biochemical parameters (HbA1c was not re-measured) returned to control levels except the level of C Peptides, which remained low in both groups of patients (P b 0.05). SOD and TAC were significantly lower in the hyperglycemic patients prior to the insulin treatment, and remained lower after treatment, though rising significantly in response to treatment (Table 3). MDA, 8-iso-PGF2α, IL-1B, TNF-a, and IL-6 were all significantly higher before treatment in both groups of patients in hyperglycemic crises compared with controls (P b 0.05). These proinflammatory cytokine levels were significantly reduced upon remission (P b 0.05) but remained significantly higher than control levels (P b 0.05).
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
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Table 1 Clinical characteristics of diabetic hyperglycemia at admission (x ± s). Group
Case
Age
BMI (kg/m2)
(male/female) Control DKA before treatment after treatment NKH before treatment after treatment
WHR
Body temperature
Systolic blood pressure
Diastolic blood pressure
# of Neutrophils
(°C)
(mm Hg)
(mm Hg)
(White blood cell × 106)
76 ± 8
33 (19/14)
52.11 ± 7.63
24.20 ± 4.01
0.86 ± 0.07
36.95 ± 5.12
122 ± 15
6.25 ± 0.80
37 (22/15)
53.22 ± 10.04
25.21 ± 6.01 36.80 ± 8.19
0.84 ± 0.08 120 ± 16
37.22 ± 7.30 76 ± 10
124 ± 18
78 ± 9 6.50 ± 2.64b
14.28 ± 3.55a
36 (23/13)
54.50 ± 11.22
25.77 ± 7.77 37.11 ± 6.39
0.85 ± 0.09 128 ± 15
37.08 ± 5.58 78 ± 9
126 ± 19
77 ± 9 5.66 ± 3.41b
15.55 ± 4.92a
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia; blank: N/A. a P b 0.05 vs. control. b P b 0.05 before treatment vs. after treatment.
3.3. Relationship among variables: correlation and multiple regression analysis Before insulin treatment, IL-6 and TNF-α were significantly, positively correlated with 8-iso-PGF2α within patients experiencing hyperglycemic crises (r = 0.32, r = 0.36, respectively; P b 0.05). After insulin treatment, a significant, negative correlation was observed between IL-6 and SOD (r = − 0.33, P b 0.05). IL-6 and MDA levels were positively correlated with each other in patients with hyperglycemic crises both before and after treatment (r = 0.36, P b 0.05). A multiple stepwise regression analysis was performed using TNF-RI, IL-1β, TNF-α and IL-6 as dependent variables and age, BMI, WHR, body temperature, systolic blood pressure, diastolic blood pressure, blood cell count, blood glucose, HbA1c, APH, HCO3, effective osmolality, anion gap, C peptide, SOD, MDA, TAC and 8-iso-PGF2α as independent variables. When IL-6 was the dependent variable, 8-iso-PGF2α was one of the independent variables. The standard regression coefficient was − 0.36 (P b 0.05). 4. Discussion A wide variety of antioxidants exists within the body that influences each other, and accurate measurement of specific antioxidant content is difficult. Therefore, multiple parameters are used to evaluate oxidative stress. Hyperglycemia is an inducer of oxidative stress and inflammation (Dandona et al., 2005). The results of the current study suggest that T2DM patients with hyperglycemic crises exhibit dysregulated levels of 8-iso-PGF2α, SOD, MDA, and TAC, which is consistent with previous studies as well as our preliminary data (Ceriello, 2005; Chaudhuri & Umpierrez, 2012; Dandona et al., 2006; Shen et al., 2012). Patients with hyperglycemic crises have a decreased level of TAC partially due to lack of storage for the raw materials used to generate TAC. Hyperglycemia promotes neutrophils and monocytes to produce
more superoxide anions, thus increasing the expression of the p47phox subunit in the NADPH oxidase complex, leading to increased opportunity for oxygen conversion to superoxide anions (Dandona, Chaudhuri, Ghanim, & Mohanty, 2009). Acute hyperglycemia can also decrease the expression of free radicals, while fluctuations in blood glucose levels can cause oxidative stress (Chaudhuri & Umpierrez, 2012; Monnier et al., 2006). Reactive oxygen species (ROS) are generated by oxygen and NADPH oxidase. In obese and non-diabetic patients, ROS, plasminogen activator inhibitor and ICAM-1 are decreased after treatment with insulin, indicating that insulin has comprehensive antioxidant and rapid anti-inflammatory effects (Ceriello et al., 2008; Dandona et al., 2001). SOD, MDA, TAC and 8-iso-PGF2α levels were significantly improved in patients recovering from hyperglycemic crisis 72 h after insulin treatment. This may be due to the fact that insulin reduces the expression of the p47phox subunit in the NADPH oxidase enzyme complex, directly inhibiting NADPH oxidase, leading to a reduction in NFκB expression (Dandona et al., 2001; Ji et al., 2010) and subsequently ROS. However, although all parameters indicating oxidative stress had improved in patients 72 h after treatment, oxidative stress indicators were still significantly below levels seen in healthy controls. These data suggest that insulin has certain antioxidant effects. The oxidative stress induced by hyperglycemia is not only related to diabetes itself, but is also associated with a variety of biochemical mechanisms and fluctuations in blood glucose during treatment (Chaudhuri & Umpierrez, 2012). Because fluctuations in blood glucose are a critical indicator of patient mortality (Krinsley, 2008; Krinsley & Keegan, 2010), fluctuations in blood glucose must be minimized when treating hyperglycemic crises. Our research suggests that T2DM patients with hyperglycemic syndrome have increased oxidative stress that remains significantly elevated in remission, albeit at a reduced level following insulin infusion. Acute hyperglycemia can significantly increase the inflammatory and immune cytokines in the peripheral circulation that play
Table 2 Biochemical characteristics of diabetic hyperglycemia before and after treatment (x ± s). Group Control DKA Before treatment After treatment NKH Before treatment After treatment
Blood glucose (mmol/L)
HbA1c (%)
pH (mmol/L)
HCO3−
Ketone (mmol/L)
Effective osmotic pressure (mmol/L)
Anion gap (nmol/L)
C peptides
5.36 ± 1.02
5.80 ± 1.26
7.38 ± 1.68
23.78 ± 3.90
N
286.29 ± 13.38
11.46 ± 3.90
0.79 ± 0.09
20.19 ± 8.60a 7.55 ± 3.21b
15.27 ± 6.90a
7.20 ± 2.48a 7.36 ± 1.97b
11.50 ± 2.20a 22.63 ± 3.23
P N
279.90 ± 15.70 280.62 ± 13.33
28.70 ± 5.16a 12.48 ± 4.57b
0.36 ± 0.06a 0.35 ± 0.07a
27.03 ± 10.45a 7.53 ± 2.76b
13.62 ± 7.63a
7.39 ± 2.39 7.38 ± 3.01
24.06 ± 3.05 24.11 ± 4.17
N N
293.51 ± 16.25 288.309 ± 18.11
11.79 ± 4.62 12.54 ± 5.03
0.45 ± 0.08a 0.40 ± 0.09a
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia; blank: N/A. a P b 0.05 vs. control. b P b 0.05 before treatment vs. after treatment.
Please cite this article as: Li, J., et al., The association of oxidative stress and pro-inflammatory cytokines in diabetic patients with hyperglycemic crisis, Journal of Diabetes and Its Complications (2014), http://dx.doi.org/10.1016/j.jdiacomp.2014.06.008
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J. Li et al. / Journal of Diabetes and Its Complications xxx (2014) xxx–xxx
Table 3 Oxidative stress markers and pro-inflammatory cytokines in diabetic hyperglycemia before and after treatment (x ± s). Group
SOD (kU/L)
Control DKA Before treatment After treatment NKH Before treatment After treatment
87.32 ± 12.68
MDA (μmol/L) 4.92 ± 1.17 a
TAC (kU/L)
8-iso-PGF2α (μg/L)
28.66 ± 10.70 a
a
6.12 ± 1.65
TNF-RI (ng/L)
IL-1β (ng/L)
365.89 ± 82.63 a
TNF-α (ng/L)
2.13 ± 0.60 a
50.11 ± 16.72 70.69 ± 13.04ab
10.06 ± 2.36 6.23 ± 1.71ab
19.23 ± 9.85 23.24 ± 8.37ab
20.31 ± 3.63 13.92 ± 5.23ab
537.12 ± 132.05 410.22 ± 108.38
53.10 ± 13.84a 69.22 ± 12.43ab
9.63 ± 1.39a 6.28 ± 2.06ab
19.64 ± 11.77a 22.86 ± 10.73ab
19.19 ± 4.55a 12.29 ± 4.36ab
596.85 ± 121.62a 450.59 ± 150.24ab
ab
IL-6 (ng/L)
22.47 ± 4.93 a
6.85 ± 1.87 a
9.15 ± 1.66 4.82 ± 1.85ab
90.35 ± 13.82 33.29 ± 11.07ab
12.88 ± 1.63a 9.12 ± 2.87ab
11.13 ± 2.63a 5.07 ± 1.49ab
86.21 ± 16.42a 31.73 ± 12.55ab
13.57 ± 2.26a 10.59 ± 1.99ab
Note: DKA: diabetes with ketoacidosis; NKH: non-ketoacidosis hyperglycemia. a P b 0.05 vs. control; bP b 0.05 before treatment vs. after treatment.
important roles in diabetic immune activation (Ceriello, 2005). Elevated IL-6 and TNF-α can result in insulin resistance by affecting the signaling pathway of the insulin receptor in adipose tissue and skeletal muscle, TNF-RI (Hube & Hauner, 2000). Because TNF-RI has a longer half-life in peripheral circulation, it is recognized as a marker of TNF system activation (Arias et al., 2008; Bullo, Garcia-Lorda, & Salas-Salvado, 2002; Good et al., 2006). IL-1β dysregulates insulin signaling, which in turn decreases insulin sensitivity via excessive autocrine and paracrine signaling in macrophages and the surrounding tissue (Su et al., 2009). We observed that TNF-RI, IL-1β, TNF-α and IL-6 were significantly elevated in patients with hyperglycemic crises and were significantly decreased after insulin treatment, although levels in remission remained significantly higher than in controls, which is in line with previous studies and our earlier report (Esposito et al., 2002; Kolb & Mandrup-Poulsen, 2005). While diabetic patients are in an acute hyperglycemic state, the increase in circulating cytokines is mainly due to non-circulating cells, such as adipocytes and endothelial cells (Pickup, Chusney, Thomas, & Burt, 2000). High blood sugar can affect the activation of NADPH oxidase and PKC and increase the impact of pro-inflammatory cytokines. PKC activation can phosphorylate p38 MAPK subfamilies and ERK1/2, thereby activating NF-κB, which, in turn, enhances monocytes to secrete various pro-inflammatory cytokines (Dasu, Devaraj, & Jialal, 2007; Pickup, 2004). Oxidative stress, soluble advanced glycation end products, and lipid peroxide production caused by hyperglycemia, may jointly activate the key upstream kinases of proinflammatory cytokines, while the superoxide anion can simultaneously activate redox-sensitive pro-inflammatory transcription factors such as NFκB, activator protein-1, and hypoxia -inducible factor α, so that the inflammatory process is amplified and sustained. In addition, fluctuation in blood glucose during the course of treatment is another important factor that induces the production of pro-inflammatory cytokines. In the current study, metabolic stress caused by low-grade inflammation was significantly enhanced during the hyperglycemic crisis in patients with T2DM. Increased levels of TNF-RI, IL-1β, TNF-α and IL-6 levels in patients with hyperglycemic crises affect insulin sensitivity via a variety of mechanisms, such as decreased expression of PPAR-γ (Pickup, 2004) and directly interfere with phosphorylation of insulin receptors in peripheral tissue. This affects insulin signaling, indirectly interferes with leptin release in lipid cells, neurons and other cells, dysregulates activation of the hypothalamic–pituitary–adrenal axis which decreases insulin sensitivity (Kolb & Mandrup-Poulsen, 2005), and increases the expression of cell adhesion molecules that are involved in endothelial cell damage. The current study demonstrates that IL-6 is closely related to oxidative stress. In cases of hyperglycemia, increased oxidative stress and activated PKC-α/β promote IL-6 secretion mediated by NF-Κb (Devaraj, Venugopal, Singh, & Jialal, 2005), though the exact mechanism is unknown. The pro-inflammatory cytokines were significantly decreased in patients with hyperglycemic crises after insulin treatment, indicating that insulin plays an anti-inflammatory role in hyperglycemic crises. It is likely that insulin plays an antiinflammatory role by inhibiting NFκB in monocyte nuclei, where it
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