DIAB-6209; No. of Pages 7 diabetes research and clinical practice xxx (2014) xxx–xxx
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes D.A. Jones a,*, S.L. Prior a, J.D. Barry b, S. Caplin b, J.N. Baxter b, J.W. Stephens a,b a b
Diabetes Research Group, College of Medicine, Swansea University, Singleton Park, Swansea, UK General Surgery (Upper GI (Obesity) and Endocrinology), Morriston Hospital, Swansea, UK
article info
abstract
Article history:
Aims: In the past 30 years, prevalence of obesity has almost trebled resulting in an increased
Received 12 March 2014
incidence of type 2 diabetes mellitus and other co-morbidities. Visceral adipose tissue is
Received in revised form
believed to play a vital role, but underlying mechanisms remain unclear. Our aim was to
24 June 2014
investigate changes in markers of oxidative damage in human visceral adipose tissue to
Accepted 14 September 2014
determine levels of oxidative burden that may be attributed to obesity and/or diabetes.
Available online xxx
Methods: Visceral adipose tissue samples from 61 subjects undergoing abdominal surgery
Keywords:
3 different markers of oxidative stress. Malondialdehyde (MDA) concentration was mea-
Oxidative stress
sured as a marker of lipid peroxidation, telomere length and Comet assay as markers of
grouped as lean, obese and obese with type 2 diabetes mellitus, were examined using
Obesity
oxidative DNA damage.
Type 2 diabetes mellitus
Results: No significant difference in MDA concentration, telomere length and DNA damage was observed between groups, although longer telomere lengths were seen in the obese with diabetes group compared to the obese group (P < 0.05). Lower MDA concentration and longer telomere length were seen in subjects with diabetes compared to those without (P < 0.05). DNA damage, analysed via Comet assay, was significantly lower in subjects with diabetes compared to those without (P < 0.05). Conclusion: A paradoxical decrease in oxidative stress and DNA damage was observed in samples from subjects with type 2 diabetes mellitus. Further work is required to investigate this further, however this phenomenon may be due to an up regulation of antioxidant defences in adipose tissue. # 2014 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
In the past 30 years the prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes
mellitus and other co-morbidities [1]. Visceral adipose tissue (VAT) plays an important role in these conditions, but the underlying mechanisms remain unclear. A close association exists between obesity, diabetes and oxidative stress [2,3]. Oxidative stress is free radical-mediated damage caused by
* Corresponding author. Tel.: +44 1792 295073; fax: +44 1792 602225. E-mail addresses: [email protected], [email protected] (D.A. Jones). http://dx.doi.org/10.1016/j.diabres.2014.09.054 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Jones DA, et al. Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2014.09.054
DIAB-6209; No. of Pages 7
2
diabetes research and clinical practice xxx (2014) xxx–xxx
excess levels of reactive oxygen species (ROS). In the case of increased levels of ROS, cells have defensive antioxidant systems that scavenge ROS and repair oxidative damage. Increased oxidative stress is associated with obesity and type 2 diabetes and increased DNA damage [4,5]. This association may be attributed to the role of oxidative stress in endothelial dysfunction, which plays a central role in both type 2 diabetes and obesity [6] Over recent years a number of measurements of oxidative stress have been developed, although few hold the reliability and specificity that is required for their use in a more clinical setting. To measure ROS directly is considered unfeasible in vivo due to their highly reactive state and short half-life [7], therefore, common practice is to measure ROS induced damage. Markers of oxidative stress are predominantly measured in blood plasma but few studies look at oxidative stress in target tissues of particular diseases, such as adipose tissue in type 2 diabetes. Therefore, the objective of this study was to attempt to measure markers of oxidative stress and DNA damage in human adipose tissue from three subject groups. Previous studies have observed a significant increase in the generation of ROS and DNA damage in adipocytes subjected to oxidative stress [8] and an acceleration in cell senescence and inflammatory cytokine secretion in obesity [9]. In the case of age-related disease, such as type 2 diabetes, an association with markers of oxidative stress and shortened telomeres have been found [10]. The objective was to examine changes in a biomarkers of oxidative stress in human VAT (hVAT). Two markers of DNA damage (telomere length and Comet assay) and a marker of lipid peroxidation (Thiobarbituric acid reactive substances (TBARS)) were measured in hVAT samples obtained from lean (L), obese (O) and obese patients with type 2 diabetes mellitus (ODM) to look for differences in oxidative burden between the groups that may be associated with obesity and/ or diabetes.
2.
Subjects, materials and methods
2.1.
Samples
hVAT samples were collected from subjects undergoing abdominal surgery that were categorised as being L, (BMI 40, we also grouped according to BMI categories. There were no observed associations between baseline characteristics and obesity classes (obese—30.0–39.9; morbid obesity—>40.0) (Table 2).
Comet assay
The Comet assay was used to calculate several parameters as a measure of DNA damage. There was no significant difference between the groups for any Comet parameters measured. No significant difference in parameters was seen between L and O groups, whilst comparing O and ODM groups there was a significant difference found in Comet intensity (1.1 105 [33403.4] v 1.4 105 [38356.4] cdx1000, P < 0.05), head intensity (3.3 104 [8276.1] v 3.9 104 [11731.0] cdx1000, P < 0.05) and tail intensity (7.9 104 [28272.3] v 9.9 104 [28799.1] cdx1000, P < 0.05). There was no significant difference observed in Comet parameters between the L group and obesity group. When comparing the non-diabetes subjects to the diabetes groups (L + O v ODM) the Head intensity for ODM group was significantly higher (3.3 104 [8856.4] v 3.9 104 [11731.0] cdx1000, P < 0.05).
Table 1 – Baseline characteristics compared between groups.
Age (years) Weight (kg) BMI (kg/m2) SBP (mmHg) DBP (mmHg) Glucose (mmol/L)# Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Creatinine (mmol/L) Albumin (mmol/L) DNA (ng/mL) RNA (ng/mL) Males % (n) Current smoker % (n) Hypertension % (n) OSA % (n) Statins % (n) ACE inhibitor % (n)
Lean (n = 20)
Obese (n = 20)
Obese/T2DM (n = 21)
P
55.3 (17.4) 73.5 (13.1) 25 (2.8) 139 (23.4) 80 (13.2) 5.7 (0.6) 5.3 (1.4) 1.5 (0.3) 3.3 (1.1) 1.2 (0.2) 78.2 (17.5) 46.7 (8.1) 15.6 (6.3) 98.0 (61.4) 55.0 (11) 25.0 (5) 20.0 (4) 0 (0) 5.0 (1) 20.0 (4)
49.7 (10.8) 119.6 (36.1) 41 (10.8) 130 (17.0) 79 (11.9) 5.6 (0.3) 5.5 (1.7) 1.2 (0.3) 3.5 (1.6) 1.6 (0.8) 78.7 (12.6) 43.4 (2.4) 15.1 (6.7) 96.7 (51.7) 40.0 (8) 25 0 (5) 25.0 (5) 15.0 (3) 30.0 (6) 10.0 (2)
44.3 (8.6) 136.9 (26.5) 50 (10.7) 144 (19.7) 76 (14.3) 8.1 (1.6) 4.8 (1.2) 1.1 (0.3) 2.6 (0.9) 2.7 (1.4) 66.3 (14.4) 42.2 (4.5) 15.2 (12.1) 77.0 (55.1) 35.0 (7) 14.3 (3) 61.9 (13) 47.6 (10) 42.9 (9) 42.9 (9)
0.03
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes D.A. Jones a,*, S.L. Prior a, J.D. Barry b, S. Caplin b, J.N. Baxter b, J.W. Stephens a,b a b
Diabetes Research Group, College of Medicine, Swansea University, Singleton Park, Swansea, UK General Surgery (Upper GI (Obesity) and Endocrinology), Morriston Hospital, Swansea, UK
article info
abstract
Article history:
Aims: In the past 30 years, prevalence of obesity has almost trebled resulting in an increased
Received 12 March 2014
incidence of type 2 diabetes mellitus and other co-morbidities. Visceral adipose tissue is
Received in revised form
believed to play a vital role, but underlying mechanisms remain unclear. Our aim was to
24 June 2014
investigate changes in markers of oxidative damage in human visceral adipose tissue to
Accepted 14 September 2014
determine levels of oxidative burden that may be attributed to obesity and/or diabetes.
Available online xxx
Methods: Visceral adipose tissue samples from 61 subjects undergoing abdominal surgery
Keywords:
3 different markers of oxidative stress. Malondialdehyde (MDA) concentration was mea-
Oxidative stress
sured as a marker of lipid peroxidation, telomere length and Comet assay as markers of
grouped as lean, obese and obese with type 2 diabetes mellitus, were examined using
Obesity
oxidative DNA damage.
Type 2 diabetes mellitus
Results: No significant difference in MDA concentration, telomere length and DNA damage was observed between groups, although longer telomere lengths were seen in the obese with diabetes group compared to the obese group (P < 0.05). Lower MDA concentration and longer telomere length were seen in subjects with diabetes compared to those without (P < 0.05). DNA damage, analysed via Comet assay, was significantly lower in subjects with diabetes compared to those without (P < 0.05). Conclusion: A paradoxical decrease in oxidative stress and DNA damage was observed in samples from subjects with type 2 diabetes mellitus. Further work is required to investigate this further, however this phenomenon may be due to an up regulation of antioxidant defences in adipose tissue. # 2014 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
In the past 30 years the prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes
mellitus and other co-morbidities [1]. Visceral adipose tissue (VAT) plays an important role in these conditions, but the underlying mechanisms remain unclear. A close association exists between obesity, diabetes and oxidative stress [2,3]. Oxidative stress is free radical-mediated damage caused by
* Corresponding author. Tel.: +44 1792 295073; fax: +44 1792 602225. E-mail addresses: [email protected], [email protected] (D.A. Jones). http://dx.doi.org/10.1016/j.diabres.2014.09.054 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Jones DA, et al. Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes. Diabetes Res Clin Pract (2014), http://dx.doi.org/10.1016/j.diabres.2014.09.054
DIAB-6209; No. of Pages 7
2
diabetes research and clinical practice xxx (2014) xxx–xxx
excess levels of reactive oxygen species (ROS). In the case of increased levels of ROS, cells have defensive antioxidant systems that scavenge ROS and repair oxidative damage. Increased oxidative stress is associated with obesity and type 2 diabetes and increased DNA damage [4,5]. This association may be attributed to the role of oxidative stress in endothelial dysfunction, which plays a central role in both type 2 diabetes and obesity [6] Over recent years a number of measurements of oxidative stress have been developed, although few hold the reliability and specificity that is required for their use in a more clinical setting. To measure ROS directly is considered unfeasible in vivo due to their highly reactive state and short half-life [7], therefore, common practice is to measure ROS induced damage. Markers of oxidative stress are predominantly measured in blood plasma but few studies look at oxidative stress in target tissues of particular diseases, such as adipose tissue in type 2 diabetes. Therefore, the objective of this study was to attempt to measure markers of oxidative stress and DNA damage in human adipose tissue from three subject groups. Previous studies have observed a significant increase in the generation of ROS and DNA damage in adipocytes subjected to oxidative stress [8] and an acceleration in cell senescence and inflammatory cytokine secretion in obesity [9]. In the case of age-related disease, such as type 2 diabetes, an association with markers of oxidative stress and shortened telomeres have been found [10]. The objective was to examine changes in a biomarkers of oxidative stress in human VAT (hVAT). Two markers of DNA damage (telomere length and Comet assay) and a marker of lipid peroxidation (Thiobarbituric acid reactive substances (TBARS)) were measured in hVAT samples obtained from lean (L), obese (O) and obese patients with type 2 diabetes mellitus (ODM) to look for differences in oxidative burden between the groups that may be associated with obesity and/ or diabetes.
2.
Subjects, materials and methods
2.1.
Samples
hVAT samples were collected from subjects undergoing abdominal surgery that were categorised as being L, (BMI 40, we also grouped according to BMI categories. There were no observed associations between baseline characteristics and obesity classes (obese—30.0–39.9; morbid obesity—>40.0) (Table 2).
Comet assay
The Comet assay was used to calculate several parameters as a measure of DNA damage. There was no significant difference between the groups for any Comet parameters measured. No significant difference in parameters was seen between L and O groups, whilst comparing O and ODM groups there was a significant difference found in Comet intensity (1.1 105 [33403.4] v 1.4 105 [38356.4] cdx1000, P < 0.05), head intensity (3.3 104 [8276.1] v 3.9 104 [11731.0] cdx1000, P < 0.05) and tail intensity (7.9 104 [28272.3] v 9.9 104 [28799.1] cdx1000, P < 0.05). There was no significant difference observed in Comet parameters between the L group and obesity group. When comparing the non-diabetes subjects to the diabetes groups (L + O v ODM) the Head intensity for ODM group was significantly higher (3.3 104 [8856.4] v 3.9 104 [11731.0] cdx1000, P < 0.05).
Table 1 – Baseline characteristics compared between groups.
Age (years) Weight (kg) BMI (kg/m2) SBP (mmHg) DBP (mmHg) Glucose (mmol/L)# Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Creatinine (mmol/L) Albumin (mmol/L) DNA (ng/mL) RNA (ng/mL) Males % (n) Current smoker % (n) Hypertension % (n) OSA % (n) Statins % (n) ACE inhibitor % (n)
Lean (n = 20)
Obese (n = 20)
Obese/T2DM (n = 21)
P
55.3 (17.4) 73.5 (13.1) 25 (2.8) 139 (23.4) 80 (13.2) 5.7 (0.6) 5.3 (1.4) 1.5 (0.3) 3.3 (1.1) 1.2 (0.2) 78.2 (17.5) 46.7 (8.1) 15.6 (6.3) 98.0 (61.4) 55.0 (11) 25.0 (5) 20.0 (4) 0 (0) 5.0 (1) 20.0 (4)
49.7 (10.8) 119.6 (36.1) 41 (10.8) 130 (17.0) 79 (11.9) 5.6 (0.3) 5.5 (1.7) 1.2 (0.3) 3.5 (1.6) 1.6 (0.8) 78.7 (12.6) 43.4 (2.4) 15.1 (6.7) 96.7 (51.7) 40.0 (8) 25 0 (5) 25.0 (5) 15.0 (3) 30.0 (6) 10.0 (2)
44.3 (8.6) 136.9 (26.5) 50 (10.7) 144 (19.7) 76 (14.3) 8.1 (1.6) 4.8 (1.2) 1.1 (0.3) 2.6 (0.9) 2.7 (1.4) 66.3 (14.4) 42.2 (4.5) 15.2 (12.1) 77.0 (55.1) 35.0 (7) 14.3 (3) 61.9 (13) 47.6 (10) 42.9 (9) 42.9 (9)
0.03
