CBI 7335
No. of Pages 9, Model 5G
13 April 2015 Chemico-Biological Interactions xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
4
Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes
7
S. Ashwini, Zachariah Bobby ⇑, Manoj Joseph
8
Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research [JIPMER], Pondicherry 605 006, India
3
9 10 1 2 2 5 13 14 15 16 17 18 19 20 21 22 23 24
a r t i c l e
i n f o
Article history: Received 30 December 2014 Received in revised form 1 April 2015 Accepted 7 April 2015 Available online xxxx Keywords: Mild hypothyroidism Overt hypothyroidism Type 2 diabetes Glucose tolerance Oxidative stress
a b s t r a c t Background: Co-existence of type 2 diabetes and hypothyroidism is an emerging trend observed in clinical practice. Although the effects of isolated type 2 diabetes and hypothyroidism are well known, there are limited studies addressing the metabolic complications when these two conditions co-exist. The aim of the present study was to assess the interaction between type 2 diabetes and hypothyroidism with respect to glucose tolerance, dyslipidemia and redox balance in a state were these two diseases coexist. Methods: Sixty male Wistar Albino rats were randomised into six groups. Group 1: control, Group 2: overt hypothyroidism, Group 3: mild hypothyroidism, Group 4: type 2 diabetes, Group 5: mild hypothyroidism + type 2 diabetes, Group 6: overt hypothyroidism + type 2 diabetes. Experimental hypothyroidism was created by the administration of propyl-2-thiouracil and type 2 diabetes by feeding rats with 60% fructose (w/w). The duration of the study was 6 weeks. All the parameters were estimated at the start (basal) and the end of the study. Intraperitoneal glucose tolerance test was carried out and area under curve (AUC) calculated to assess the glucose tolerance. Thyroid profile, lipid profile and oxidative stress parameters were also measured. Results: Plasma TSH level was elevated 3-fold in the mild hypothyroid group and 15.2-fold in the overt hypothyroid group in comparison to the control group. Thyroid profile was found to be normal in type 2 diabetic group. There was no significant difference between hypothyroidism and hypothyroidism + diabetes groups with respect to thyroid profile. Among the six groups the degree of glucose intolerance was found to be maximum for overt hypothyroidism + diabetes group, followed by diabetes group and overt hypothyroidism group. An interesting finding was that glucose intolerance was significantly reduced in mild hypothyroidism + diabetes group (increase in AUC: 48.04%) in comparison with isolated diabetes group (increase in AUC: 71.63%). Similar results were obtained with parameters of oxidative stress. Oxidative stress was observed in overt hypothyroidism + diabetes, diabetes, and overt hypothyroidism groups with severity decreasing in that order. Coexistence of mild hypothyroidism with diabetes decreased oxidative stress in comparison with isolated diabetes group. There was no statistical difference in lipid profile between mild hypothyroidism + diabetes and isolated diabetes group. Conclusion: Presence of mild hypothyroidism in type 2 diabetes confers a protective effect with respect to glucose tolerance and redox balance whereas presence of overt hypothyroidism in type 2 diabetes has a deleterious effect. The increased incidence of hypothyroidism in diabetes, especially subclinical hypothyroidism, could be a reflection of a physiological attempt by the body to mitigate damage wrought by diabetes. Ó 2015 Published by Elsevier Ireland Ltd.
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
58 59
1. Introduction
60
Diabetes mellitus is the most common endocrinopathy encountered in clinical practice. It encompasses a spectrum of disorders which share the common feature of hyperglycemia. The number of people suffering from diabetes has seen a significant increase
61 62 63
⇑ Corresponding author. Tel.: +91 94436 02996, +91 413 2273078; fax: +91 0413 2272067/66. E-mail address: [email protected] (Z. Bobby).
over the last half a century despite rapid advances in our understanding of this metabolic disorder. About 8% of the world adult population, approximately 380 million individuals, were estimated to suffer from diabetes in 2013 [1]. India is home to one in every six adults suffering from diabetes. Type 2 Diabetes is characterised by hyperglycemia in the setting of a peripheral resistance to insulin and a relative decrease in the amount of circulating insulin. It constitutes nearly 90% of all cases of diabetes. Thyroid disorders constitute another major group of endocrinopathies. Hypothyroidism is the most common form of thyroid
http://dx.doi.org/10.1016/j.cbi.2015.04.007 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: S. Ashwini et al., Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.04.007
64 65 66 67 68 69 70 71 72 73
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dysfunction seen in the general population. One in ten adults in India suffer from hypothyroidism [2]. Hypothyroidism is divided into overt and subclinical based on the levels of circulating hormones. Subclinical disease is a mild form of disease in which the peripheral thyroid hormone levels are within the normal range but levels of thyroid stimulating hormone (TSH) are abnormally high. Overt disease on the other hand is characterised by deranged peripheral thyroid hormone levels also. Co-existence of type 2 diabetes and hypothyroidism is an emerging trend observed in clinical practice. A strong link has been reported between these two diseases in the recent years [3]. Epidemiological studies reveal that type 2 diabetic patients are at a greater risk for development of hypothyroidism in comparison with normal subjects [4,5]. The Fremantle Diabetes Study found subclinical hypothyroidism to be a common incidental finding in women with type 2 diabetes [6]. Thyroid hormones play a major role in regulation of energy metabolism. Abnormalities in levels of thyroid hormones can lead to a dysregulation of glucose homeostasis thus complicating the management of diabetes. Patients with coexisting hypothyroidism and diabetes have been found to require earlier insulin therapy and need more endocrine attention [7]. Hypothyroidism is known to play a causal role in some of the classical risk factors of diabetes such as dyslipidaemia and hypertension. Conversely, abnormalities in thyroid hormone profile have been found in patients with diabetes. The nocturnal peak in levels of TSH is blunted and levels of serum T3 is reduced in patients with poorly controlled diabetes [8]. Although the effects of isolated type 2 diabetes and hypothyroidism are well known, there are few studies addressing the complications of co-existing type 2 diabetes and hypothyroidism. The pathogenic processes in both diabetes and hypothyroidism are involved in an intricate interplay which is yet to be fully elucidated. The present study was designed to assess the interaction between type 2 diabetes and hypothyroidism with respect to glucose tolerance, dyslipidemia and redox balance in a state were these two diseases coexist, using an animal model.
111
2. Materials and methods
112
2.1. Chemicals
113
119
6-Propyl-2-thiouracil (PTU), 2-thiobarbituric acid (TBA), I-glutathione reduced (GSH), hydrogen peroxide (H2O2), 5,50 -dithiobis 2-nitrobenzoicacid (DTNB), sodium sulphate, 2-4dinitrophenylhydrazine(DNPH) & 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) of molecular grade were purchased from Sigma Aldrich (USA). All other chemicals were of analytical grade obtained from SRL (India).
120
2.2. Animal experiments
121
The study was conducted in the Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India after obtaining due approval from Scientific Advisory Committee and the Institutional Animal Ethics Committee. Five month old male Wistar Albino rats were obtained from Institute Central Animal House and maintained in polycarbonate cages under a 12-h light/12-h dark cycle with food and water available ad libitum. Totally sixty rats were used in this study. They were randomised into six groups with ten rats in each group. The six groups are as follows:
74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
114 115 116 117 118
122 123 124 125 126 127 128 129 130 131 132 133
Group 1: control [C] Group 2: overt hypothyroidism [OH]
Group Group Group Group
3: 4: 5: 6:
mild hypothyroidism [MH] type 2 diabetes [D] mild hypothyroidism + diabetes [MH + D] overt hypothyroidism + diabetes [OH + D]
134 135 136 137 138
Group 1 did not receive any intervention throughout the experimental period. Group 2 was given 0.05% (w/v) propylthiouracil in drinking water for 6 weeks to induce overt hypothyroidism [9]. Group 3 was given 0.005% (w/v) propylthiouracil in drinking water for 6 weeks to induce mild hypothyroidism. Group 4 was given 60% (w/w) fructose supplemented rat chow to induce type 2 diabetes [10]. Group 5 was given 0.005% (w/v) propylthiouracil in drinking water and 60% (w/w) fructose supplemented rat chow for 6 weeks to induce a state of co-existing mild hypothyroidism and type 2 diabetes. Group 6 was given 0.05% (w/v) propylthiouracil in drinking water and 60% fructose supplemented rat chow for 6 weeks to induce a state of co-existing overt hypothyroidism and type 2 diabetes.
139
2.3. Sample collection
152
The duration of the study was 6 weeks. All the parameters were estimated at the start (basal) and at the end of the study. Blood was collected from retro-orbital plexus under mild ether anaesthesia. At the end of the experimental period the animals were sacrificed, liver was excised, snap frozen in liquid nitrogen and stored at 80 °C for subsequent analysis.
153
2.4. Assessment of glucose tolerance
159
Intraperitoneal glucose tolerance test (IPGTT) was carried out to assess glucose tolerance. IPGTT was performed as described by Yuan et al. [11]. Blood samples were collected from the rats after they were maintained in a fasting state overnight (15 h). Rats were then injected 2.0 g/kg body weight of glucose (in 1.5 ml saline) intraperitoneally. Blood samples were collected for the estimation of plasma glucose at 30, 60 and 120 min after the glucose injection. Plasma glucose was estimated by glucose oxidase–peroxidase method (Reckon diagnostics, Vadodara, India) in a fully automated clinical chemistry analyser (AU-400, OLYMPUS, Essex, UK).
160
2.5. Lipid profile
170
The following biochemical parameters were analysed in fasting plasma sample using a fully automated clinical chemistry analyser (AU-400, OLYMPUS, Essex, UK). Total cholesterol was measured using cholesterol oxidase–peroxidase method (Genuine Bio-systems, Chennai, India), triglycerides (TG) using an enzymatic glycerol phosphate oxidase peroxidase method (Agappe Diagnostics, Kerala, India), and high-density lipoprotein (HDL) cholesterol by the cholesterol oxidase–peroxidase method (Lab-Care Diagnostics, Mumbai, India). Very low density lipoprotein (VLDL) cholesterol and low-density lipoprotein (LDL) cholesterol in plasma were calculated using Friedwald’s formula [12].
171
2.6. Thyroid profile
182
Plasma rat free T3, free T4 and TSH were measured using ELISA kits from CUSABIO, Japan.
183
2.7. Oxidative stress
185
All the oxidative stress parameters were estimated using standardized spectrophotometric methods. Whole blood reduced glutathione was measured by method described by Beutler et al. [13]. Catalase activity in erythrocytes was determined by the
186
Please cite this article in press as: S. Ashwini et al., Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.04.007
140 141 142 143 144 145 146 147 148 149 150 151
154 155 156 157 158
161 162 163 164 165 166 167 168 169
172 173 174 175 176 177 178 179 180 181
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method described by Aebi [14]. Glutathione Peroxidase (GPx) activity in erythrocytes was measured by method described by Wendel et al. [15]. Plasma MDA level was determined by the method described by Yagi et al. [16]. Total antioxidant potential in plasma was measured by the method described by Benzie and Strain [17].
196
2.8. In vitro resistance to lipid peroxidation
197
From all the six experimental groups, stored liver tissues were taken and homogenised. 10% liver homogenate was prepared using 0.1 M ice cold Tris–HCl buffer (0.1 M, PH: 7.5). The crude homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. The protein content in the supernatant was estimated by method described by Lowry et al. The supernatant was used for the determination of lipid peroxidation. Lipid peroxidation was initiated in vitro by addition of 0.1 ml of FeSO4 (25 lm), 0.1 ml of ascorbate (100 lm) and 0.1 ml of KH2Po4 (10 mm) to 1 ml of the homogenate [18]. The final volume was made up to 3 ml with distilled water and incubated at 37 °C for 1 h. After the incubation period the degree of lipid peroxidation in the sample was assessed by measuring the malondialdehyde (MDA) levels by method described by Ohkawa et al. [19]. The results were expressed as lMol of MDA/mg of protein. Percentage increase in lipid peroxidation for each of the experimental group (with respect to control group):
190 191 192 193 194
198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215
= [MDA of control group MDA of each of the other five experimental group (OH, MH, D, OH + D, MH + D)/MDA of control group] * 100
216 217 218 219 220 221
Percentage inhibition of lipid peroxidation for each of the experimental group (with respect to OH + D group): = [MDA of OH + D group MDA of each of the other five experimental group (C, OH, MH, D, OH + D)/MDA of OH + D group] * 100
2.9.1. Statistical analyses All values are expressed as mean ± standard deviation. The difference in mean values among the groups were analysed using two way repeated measures analysis of variance (ANOVA) with Bonferroni post hoc test using PRISM 5 software (GraphPad Software, San Diego, CA [www.graphpad.com]). ‘Time’ (two time points – basal, end of study) was considered to be factor 1 and experimental ‘group’ (six groups – C, OH, MH, D, MH + D, OH + D) was considered to be factor 2. The main effect and interaction effect of factors 1&2 were assessed. Results of in vitro lipid peroxidation assay were analysed using one way ANOVA with Bonferroni post hoc test. A ‘p’ value less than 0.05 was considered to be statistically significant.
226
3. Results
239
3.1. Thyroid profile
240
Hypothyroidism was successfully induced in the experimental animals (Table 1). Hypothyroid rats showed significant decreases in plasma free T3 and free T4 levels with concomitant increase in TSH level. Diabetic rats did not show any significant difference in thyroid profile in comparison with control. In overt hypothyroid rats plasma free T3 and free T4 levels were decreased 17.85-fold and 14.84-fold respectively; further plasma TSH level was elevated 15.26-fold in comparison with control. There was no significant difference between overt hypothyroidism and overt hypothyroidism + diabetes groups in thyroid profile. In mild hypothyroid rats plasma free T3 and free T4 levels were decreased 1.51-fold and 1.46-fold respectively, further plasma TSH level was elevated 3.04-fold in comparison with control. There was no significant difference between mild hypothyroidism and mild hypothyroidism + diabetes groups in thyroid profile.
241
3.2. Oxidative stress parameters
256
There was no significant difference in oxidative stress parameters between the various groups at the start of the study (Tables 2 and 3). At the end of the study glutathione peroxidase (GPX) activity, reduced glutathione (GSH) level and Total antioxidant
257
227 228 229 230 231 232 233 234 235 236 237 238
242 243 244 245 246 247 248 249 250 251 252 253 254 255
222 223
2.9. Plasma insulin
224
Plasma rat insulin was measured using ELISA kits from Crystal Chem, USA.
225
Table 1 thyroid profile in rats with isolated and co-existing type 2 diabetes and hypothyroidism. S. No
Name of the group
Time period
Rat free T3 (pMol/L)
Rat free T4 (pMol/L)
Rat TSH (lIU/ml)
1
Control [C]
2
Overt hypothyroidism [OH]
3
Mild hypothyroidism [MH]
4
Diabetes [D]
5
Mild hypothyroidism + diabetes [MH + D]
6
Overt hypothyroidism + diabetes [OH + D]
End of Basal End of Basal End of Basal End of Basal End of Basal End of Basal F p F p F p
4.57 ± 0.53 4.61 ± 0.59 0.27 ± 0.08ax 4.97 ± 0.88 3.02 ± 0.15abx 4.73 ± 0.52 4.72 ± 0.72bc 4.80 ± 0.77 2.99 + 0.27abdx 4.65 ± 0.68 0.28 + 0.08acdex 4.69 ± 0.70 420
No. of Pages 9, Model 5G
13 April 2015 Chemico-Biological Interactions xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
4
Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes
7
S. Ashwini, Zachariah Bobby ⇑, Manoj Joseph
8
Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research [JIPMER], Pondicherry 605 006, India
3
9 10 1 2 2 5 13 14 15 16 17 18 19 20 21 22 23 24
a r t i c l e
i n f o
Article history: Received 30 December 2014 Received in revised form 1 April 2015 Accepted 7 April 2015 Available online xxxx Keywords: Mild hypothyroidism Overt hypothyroidism Type 2 diabetes Glucose tolerance Oxidative stress
a b s t r a c t Background: Co-existence of type 2 diabetes and hypothyroidism is an emerging trend observed in clinical practice. Although the effects of isolated type 2 diabetes and hypothyroidism are well known, there are limited studies addressing the metabolic complications when these two conditions co-exist. The aim of the present study was to assess the interaction between type 2 diabetes and hypothyroidism with respect to glucose tolerance, dyslipidemia and redox balance in a state were these two diseases coexist. Methods: Sixty male Wistar Albino rats were randomised into six groups. Group 1: control, Group 2: overt hypothyroidism, Group 3: mild hypothyroidism, Group 4: type 2 diabetes, Group 5: mild hypothyroidism + type 2 diabetes, Group 6: overt hypothyroidism + type 2 diabetes. Experimental hypothyroidism was created by the administration of propyl-2-thiouracil and type 2 diabetes by feeding rats with 60% fructose (w/w). The duration of the study was 6 weeks. All the parameters were estimated at the start (basal) and the end of the study. Intraperitoneal glucose tolerance test was carried out and area under curve (AUC) calculated to assess the glucose tolerance. Thyroid profile, lipid profile and oxidative stress parameters were also measured. Results: Plasma TSH level was elevated 3-fold in the mild hypothyroid group and 15.2-fold in the overt hypothyroid group in comparison to the control group. Thyroid profile was found to be normal in type 2 diabetic group. There was no significant difference between hypothyroidism and hypothyroidism + diabetes groups with respect to thyroid profile. Among the six groups the degree of glucose intolerance was found to be maximum for overt hypothyroidism + diabetes group, followed by diabetes group and overt hypothyroidism group. An interesting finding was that glucose intolerance was significantly reduced in mild hypothyroidism + diabetes group (increase in AUC: 48.04%) in comparison with isolated diabetes group (increase in AUC: 71.63%). Similar results were obtained with parameters of oxidative stress. Oxidative stress was observed in overt hypothyroidism + diabetes, diabetes, and overt hypothyroidism groups with severity decreasing in that order. Coexistence of mild hypothyroidism with diabetes decreased oxidative stress in comparison with isolated diabetes group. There was no statistical difference in lipid profile between mild hypothyroidism + diabetes and isolated diabetes group. Conclusion: Presence of mild hypothyroidism in type 2 diabetes confers a protective effect with respect to glucose tolerance and redox balance whereas presence of overt hypothyroidism in type 2 diabetes has a deleterious effect. The increased incidence of hypothyroidism in diabetes, especially subclinical hypothyroidism, could be a reflection of a physiological attempt by the body to mitigate damage wrought by diabetes. Ó 2015 Published by Elsevier Ireland Ltd.
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
58 59
1. Introduction
60
Diabetes mellitus is the most common endocrinopathy encountered in clinical practice. It encompasses a spectrum of disorders which share the common feature of hyperglycemia. The number of people suffering from diabetes has seen a significant increase
61 62 63
⇑ Corresponding author. Tel.: +91 94436 02996, +91 413 2273078; fax: +91 0413 2272067/66. E-mail address: [email protected] (Z. Bobby).
over the last half a century despite rapid advances in our understanding of this metabolic disorder. About 8% of the world adult population, approximately 380 million individuals, were estimated to suffer from diabetes in 2013 [1]. India is home to one in every six adults suffering from diabetes. Type 2 Diabetes is characterised by hyperglycemia in the setting of a peripheral resistance to insulin and a relative decrease in the amount of circulating insulin. It constitutes nearly 90% of all cases of diabetes. Thyroid disorders constitute another major group of endocrinopathies. Hypothyroidism is the most common form of thyroid
http://dx.doi.org/10.1016/j.cbi.2015.04.007 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: S. Ashwini et al., Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.04.007
64 65 66 67 68 69 70 71 72 73
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No. of Pages 9, Model 5G
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dysfunction seen in the general population. One in ten adults in India suffer from hypothyroidism [2]. Hypothyroidism is divided into overt and subclinical based on the levels of circulating hormones. Subclinical disease is a mild form of disease in which the peripheral thyroid hormone levels are within the normal range but levels of thyroid stimulating hormone (TSH) are abnormally high. Overt disease on the other hand is characterised by deranged peripheral thyroid hormone levels also. Co-existence of type 2 diabetes and hypothyroidism is an emerging trend observed in clinical practice. A strong link has been reported between these two diseases in the recent years [3]. Epidemiological studies reveal that type 2 diabetic patients are at a greater risk for development of hypothyroidism in comparison with normal subjects [4,5]. The Fremantle Diabetes Study found subclinical hypothyroidism to be a common incidental finding in women with type 2 diabetes [6]. Thyroid hormones play a major role in regulation of energy metabolism. Abnormalities in levels of thyroid hormones can lead to a dysregulation of glucose homeostasis thus complicating the management of diabetes. Patients with coexisting hypothyroidism and diabetes have been found to require earlier insulin therapy and need more endocrine attention [7]. Hypothyroidism is known to play a causal role in some of the classical risk factors of diabetes such as dyslipidaemia and hypertension. Conversely, abnormalities in thyroid hormone profile have been found in patients with diabetes. The nocturnal peak in levels of TSH is blunted and levels of serum T3 is reduced in patients with poorly controlled diabetes [8]. Although the effects of isolated type 2 diabetes and hypothyroidism are well known, there are few studies addressing the complications of co-existing type 2 diabetes and hypothyroidism. The pathogenic processes in both diabetes and hypothyroidism are involved in an intricate interplay which is yet to be fully elucidated. The present study was designed to assess the interaction between type 2 diabetes and hypothyroidism with respect to glucose tolerance, dyslipidemia and redox balance in a state were these two diseases coexist, using an animal model.
111
2. Materials and methods
112
2.1. Chemicals
113
119
6-Propyl-2-thiouracil (PTU), 2-thiobarbituric acid (TBA), I-glutathione reduced (GSH), hydrogen peroxide (H2O2), 5,50 -dithiobis 2-nitrobenzoicacid (DTNB), sodium sulphate, 2-4dinitrophenylhydrazine(DNPH) & 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) of molecular grade were purchased from Sigma Aldrich (USA). All other chemicals were of analytical grade obtained from SRL (India).
120
2.2. Animal experiments
121
The study was conducted in the Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India after obtaining due approval from Scientific Advisory Committee and the Institutional Animal Ethics Committee. Five month old male Wistar Albino rats were obtained from Institute Central Animal House and maintained in polycarbonate cages under a 12-h light/12-h dark cycle with food and water available ad libitum. Totally sixty rats were used in this study. They were randomised into six groups with ten rats in each group. The six groups are as follows:
74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
114 115 116 117 118
122 123 124 125 126 127 128 129 130 131 132 133
Group 1: control [C] Group 2: overt hypothyroidism [OH]
Group Group Group Group
3: 4: 5: 6:
mild hypothyroidism [MH] type 2 diabetes [D] mild hypothyroidism + diabetes [MH + D] overt hypothyroidism + diabetes [OH + D]
134 135 136 137 138
Group 1 did not receive any intervention throughout the experimental period. Group 2 was given 0.05% (w/v) propylthiouracil in drinking water for 6 weeks to induce overt hypothyroidism [9]. Group 3 was given 0.005% (w/v) propylthiouracil in drinking water for 6 weeks to induce mild hypothyroidism. Group 4 was given 60% (w/w) fructose supplemented rat chow to induce type 2 diabetes [10]. Group 5 was given 0.005% (w/v) propylthiouracil in drinking water and 60% (w/w) fructose supplemented rat chow for 6 weeks to induce a state of co-existing mild hypothyroidism and type 2 diabetes. Group 6 was given 0.05% (w/v) propylthiouracil in drinking water and 60% fructose supplemented rat chow for 6 weeks to induce a state of co-existing overt hypothyroidism and type 2 diabetes.
139
2.3. Sample collection
152
The duration of the study was 6 weeks. All the parameters were estimated at the start (basal) and at the end of the study. Blood was collected from retro-orbital plexus under mild ether anaesthesia. At the end of the experimental period the animals were sacrificed, liver was excised, snap frozen in liquid nitrogen and stored at 80 °C for subsequent analysis.
153
2.4. Assessment of glucose tolerance
159
Intraperitoneal glucose tolerance test (IPGTT) was carried out to assess glucose tolerance. IPGTT was performed as described by Yuan et al. [11]. Blood samples were collected from the rats after they were maintained in a fasting state overnight (15 h). Rats were then injected 2.0 g/kg body weight of glucose (in 1.5 ml saline) intraperitoneally. Blood samples were collected for the estimation of plasma glucose at 30, 60 and 120 min after the glucose injection. Plasma glucose was estimated by glucose oxidase–peroxidase method (Reckon diagnostics, Vadodara, India) in a fully automated clinical chemistry analyser (AU-400, OLYMPUS, Essex, UK).
160
2.5. Lipid profile
170
The following biochemical parameters were analysed in fasting plasma sample using a fully automated clinical chemistry analyser (AU-400, OLYMPUS, Essex, UK). Total cholesterol was measured using cholesterol oxidase–peroxidase method (Genuine Bio-systems, Chennai, India), triglycerides (TG) using an enzymatic glycerol phosphate oxidase peroxidase method (Agappe Diagnostics, Kerala, India), and high-density lipoprotein (HDL) cholesterol by the cholesterol oxidase–peroxidase method (Lab-Care Diagnostics, Mumbai, India). Very low density lipoprotein (VLDL) cholesterol and low-density lipoprotein (LDL) cholesterol in plasma were calculated using Friedwald’s formula [12].
171
2.6. Thyroid profile
182
Plasma rat free T3, free T4 and TSH were measured using ELISA kits from CUSABIO, Japan.
183
2.7. Oxidative stress
185
All the oxidative stress parameters were estimated using standardized spectrophotometric methods. Whole blood reduced glutathione was measured by method described by Beutler et al. [13]. Catalase activity in erythrocytes was determined by the
186
Please cite this article in press as: S. Ashwini et al., Mild hypothyroidism improves glucose tolerance in experimental type 2 diabetes, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.04.007
140 141 142 143 144 145 146 147 148 149 150 151
154 155 156 157 158
161 162 163 164 165 166 167 168 169
172 173 174 175 176 177 178 179 180 181
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method described by Aebi [14]. Glutathione Peroxidase (GPx) activity in erythrocytes was measured by method described by Wendel et al. [15]. Plasma MDA level was determined by the method described by Yagi et al. [16]. Total antioxidant potential in plasma was measured by the method described by Benzie and Strain [17].
196
2.8. In vitro resistance to lipid peroxidation
197
From all the six experimental groups, stored liver tissues were taken and homogenised. 10% liver homogenate was prepared using 0.1 M ice cold Tris–HCl buffer (0.1 M, PH: 7.5). The crude homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. The protein content in the supernatant was estimated by method described by Lowry et al. The supernatant was used for the determination of lipid peroxidation. Lipid peroxidation was initiated in vitro by addition of 0.1 ml of FeSO4 (25 lm), 0.1 ml of ascorbate (100 lm) and 0.1 ml of KH2Po4 (10 mm) to 1 ml of the homogenate [18]. The final volume was made up to 3 ml with distilled water and incubated at 37 °C for 1 h. After the incubation period the degree of lipid peroxidation in the sample was assessed by measuring the malondialdehyde (MDA) levels by method described by Ohkawa et al. [19]. The results were expressed as lMol of MDA/mg of protein. Percentage increase in lipid peroxidation for each of the experimental group (with respect to control group):
190 191 192 193 194
198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215
= [MDA of control group MDA of each of the other five experimental group (OH, MH, D, OH + D, MH + D)/MDA of control group] * 100
216 217 218 219 220 221
Percentage inhibition of lipid peroxidation for each of the experimental group (with respect to OH + D group): = [MDA of OH + D group MDA of each of the other five experimental group (C, OH, MH, D, OH + D)/MDA of OH + D group] * 100
2.9.1. Statistical analyses All values are expressed as mean ± standard deviation. The difference in mean values among the groups were analysed using two way repeated measures analysis of variance (ANOVA) with Bonferroni post hoc test using PRISM 5 software (GraphPad Software, San Diego, CA [www.graphpad.com]). ‘Time’ (two time points – basal, end of study) was considered to be factor 1 and experimental ‘group’ (six groups – C, OH, MH, D, MH + D, OH + D) was considered to be factor 2. The main effect and interaction effect of factors 1&2 were assessed. Results of in vitro lipid peroxidation assay were analysed using one way ANOVA with Bonferroni post hoc test. A ‘p’ value less than 0.05 was considered to be statistically significant.
226
3. Results
239
3.1. Thyroid profile
240
Hypothyroidism was successfully induced in the experimental animals (Table 1). Hypothyroid rats showed significant decreases in plasma free T3 and free T4 levels with concomitant increase in TSH level. Diabetic rats did not show any significant difference in thyroid profile in comparison with control. In overt hypothyroid rats plasma free T3 and free T4 levels were decreased 17.85-fold and 14.84-fold respectively; further plasma TSH level was elevated 15.26-fold in comparison with control. There was no significant difference between overt hypothyroidism and overt hypothyroidism + diabetes groups in thyroid profile. In mild hypothyroid rats plasma free T3 and free T4 levels were decreased 1.51-fold and 1.46-fold respectively, further plasma TSH level was elevated 3.04-fold in comparison with control. There was no significant difference between mild hypothyroidism and mild hypothyroidism + diabetes groups in thyroid profile.
241
3.2. Oxidative stress parameters
256
There was no significant difference in oxidative stress parameters between the various groups at the start of the study (Tables 2 and 3). At the end of the study glutathione peroxidase (GPX) activity, reduced glutathione (GSH) level and Total antioxidant
257
227 228 229 230 231 232 233 234 235 236 237 238
242 243 244 245 246 247 248 249 250 251 252 253 254 255
222 223
2.9. Plasma insulin
224
Plasma rat insulin was measured using ELISA kits from Crystal Chem, USA.
225
Table 1 thyroid profile in rats with isolated and co-existing type 2 diabetes and hypothyroidism. S. No
Name of the group
Time period
Rat free T3 (pMol/L)
Rat free T4 (pMol/L)
Rat TSH (lIU/ml)
1
Control [C]
2
Overt hypothyroidism [OH]
3
Mild hypothyroidism [MH]
4
Diabetes [D]
5
Mild hypothyroidism + diabetes [MH + D]
6
Overt hypothyroidism + diabetes [OH + D]
End of Basal End of Basal End of Basal End of Basal End of Basal End of Basal F p F p F p
4.57 ± 0.53 4.61 ± 0.59 0.27 ± 0.08ax 4.97 ± 0.88 3.02 ± 0.15abx 4.73 ± 0.52 4.72 ± 0.72bc 4.80 ± 0.77 2.99 + 0.27abdx 4.65 ± 0.68 0.28 + 0.08acdex 4.69 ± 0.70 420
