Chapter 4 Optimization of Insulin Resistance Model

Transcription

1 Background Type-II diabetes is a condition characterized by insulin resistance followed by progressive decrease in insulin secretion. The epidemiological evidence points to the fact that most type-2 diabetic individuals are generally obese and have abnormal lipid profile. Many reports establish the link between changed food habits such as intake of high fat, high carbohydrate food regardless of need and a sedentary lifestyle (Kahn and Flier, 2000, Nathan et al., 2007, Diamond, 2011). To develop and use an appropriate animal model, which can mimic the actual human conditions is an important consideration for any pharmacological experiment. Literature search revealed that there have been attempts at developing such models for insulin resistance and using them for different pharmacological experiments (Wright et al., 1983, Srinivasan et al., 2005, Jang et al., 2010, Bell et al., 2000). It is a well-known fact that disease conditions affect the pharmacokinetics and pharmacodynamics of drugs. The same stands valid for herbdrug interactions. In India, transformation of lifestyle from traditional to western culture with a drastic change in food habits and lack of exercise has led to increased prevalence of impaired glucose tolerance and insulin resistance. Obesity is one of the immediate and direct fallouts of changed lifestyle. It has significant effects on the pharmacology of the drugs(blouin and Warren, 1999). Considering all these observations, we attempted to optimize an appropriate animal model, which can simulate the actual clinical scenario of obesity associated with impaired glucose tolerance and insulin resistance Materials and Methods Materials All the materials required for different experiments were procured from the manufacturers/ suppliers through stores, MCOPS. The selection of the materials required for the experiments was based on the methods employed, literature search and pilot experiments. The detailed information on the materials employed is presented in the following tables. 46

2 Food supplements, Chemicals and Reagents Chemicals Purity/Grade Manufacturer High Carbohydrate Diet DL-Methionine 99% Loba Chemie, Mumbai, India Casein Extra pure Suvedhanath laboratories, Baroda, India Cellulose Extra pure Sigma-Aldrich Pvt. Ltd., Bangalore, India Cholesterol 95% Sigma-Aldrich Pvt. Ltd., Bangalore, India Choline bitartrate Extra pure Sigma-Aldrich Pvt. Ltd., Bangalore, India Lard NA Local Sources, Udupi, India Mineral mix Ostovet, Veterinary Formulation Virbac Animal Health India Pvt Ltd, Mumbai, India Normal pellet diet NA Amrut Agro Industries, Pune, India Sucrose Pure Shree Renuka Sugars Ltd, Belgaum India Vitamin mix Veterinary Formulation Virbac Animal Health India Pvt Ltd, Mumbai, India Model Optimization experiments Fructose Extra pure Suvedhanath laboratories, Baroda, India Sucrose Pure Shree Renuka Sugars Ltd, Belgaum India Streptozotocin 98% Sigma-Aldrich Pvt. Ltd., Bangalore, India Diagnostic Kits AST ALT Glucose Rat Insulin Elisa Total Cholesterol Triglycerides Aspen Laboratories, New Delhi, India Aspen Laboratories, New Delhi, India Aspen Laboratories, New Delhi, India Mercodia Diagnostics, Sweeden Aspen Laboratories, New Delhi, India Aspen Laboratories, New Delhi, India Animal Anesthesia Anesthetic ether LR Grade Nice Chemicals, Cochin, India Ketamine Injectable Neon Labs, Ahmedabad, India Histopathology Formalin LR Grade Suvedhanath laboratories, Baroda, India 47

3 Instruments Instruments Model Make Analytical Balance BT 124 S Sartorius, Germany Auto analyzer Star-21 SEAC, India Chemiluminometer FLx 800 BioTek, USA Deep freezer (-20 o C) Carrier Carrier, India Electronic Weighing Balance Essae DS65 Essae-Teraoka Limited, India Glucometer and Strips Accucheck, Sensor Comfort Roche, Germany Micropipettes Eppendorf Eppendorf, Germany Micro plate reader ELx-800 BioTek, USA Mixer and Grinder Pulsar Sumeet Research & Holding Ltd, Chennai, India Misc. Glassware NA Borosil/Qualigens, Mumbai, India Orbital Shaker ph meter ph System 361 Systronics, India Refrigerator (2-8) NA Samsung, India Refrigerated Centrifuge Mikro Hettich, Germany Vortexer Spinix Spinix, India Consumables Sterile Consumables Manufacturer 96 Well micro plates Tarsons, India Centrifuge Tubes Tarsons, India Disposable syringes/needles Dispovan, India Sterile Gloves Surgen, India Microcentrifuge Tubes Eppendorf, Germany Micropipette Tips Tarsons, India Methods Animal selection and grouping All the animal experiments were performed at Central Animal Research Facility, Manipal University, Manipal. Animal care and handling was done according to the guidelines put forth by committee for the purpose of control and supervision of experiments on animals (CPCSEA), issued by the Ministry of Environment and Forests, Government of India. The protocol for the animal experiments were prepared as per CPCSEA directive and submitted to Institutional Animal Ethics Committee (IAEC). The animal studies were initiated after obtaining written approval from IAEC (IAEC/KMC/07/ ). Healthy, inbred, post weaned, male albino rats of Wistar strain weighing around 30 to 35g were selected for the study. They were maintained under controlled conditions of temperature (23±2 C), humidity (50±5%) and day / night cycles (14 hour light and 10 hour dark). The animals were put on custom-made high carbohydrate diet (HCD),except for the control 48

4 group, which was put on regular normal pellet diet (NPD) and water ad libitum. Two rats were housed in each autoclave-sterilized polypropylene cage containing autoclave sterilized paddy husk as bedding. The animals were maintained for about six months with scheduled monitoring of food, water intake and body weight measurement. Rodents selected for the optimization experiments were put on high carbohydrate diet except for the group of six, which were put on normal pellet diet. The group of rats put on normal pellet diet served as control group. The environmental conditions were maintained uniformly for all the rats for the next six months. After ensuring reasonable stabilization of the body weight, the optimization experiments were initiated Composition and preparation of High Carbohydrate Diet High carbohydrate diet preparation was adapted from the earlier experiments (Wright et al., 1983, Srinivasan et al., 2005) with some modification, mainly to include the component of fat (1.5% Lard) with high carbohydrate (55% sucrose) in to the diet. Composition of the HCD is presented in table Method of HCD preparation Required quantities of all the raw materials were weighed. Normal Pellet (rat feed) and sugar were ground separately to make powder. Lard was warmed over water bath and Cholesterol and choline were mixed in to it Powdered sugar, casein, cellulose and DL-methionine were mixed properly by adding distilled water mixed with vitamin and mineral mix to make a semisolid preparation. The mixture of lard, choline and cholesterol was added to the above blend and mixed thoroughly. To the mixture, normal pellet powder was added, mixed well and allowed to soak for about 10 minutes. Water was added quantity sufficient, suitable enough to make pellets. The pellets were kept in freezer to make the preparation harder. 49

5 Table 2.1: Composition of High Carbohydrate Diet (HCD) Ingredients % w/w For 10 Kg Cholesterol g Lard g Choline g Casein g Cellulose g Vitamin mix g Mineral mix g Sucrose g Powdered NPD g Methionine g Measurement of food, water intake and body weight The measurements of food, water intake and body weight were done every fortnightly. One day before, known amounts of food (in g) and water (100 ml) were provided for each cage. Next day at the same time, the remaining food was weighed, water was measured and recorded. The difference was calculated for each cage, which gives the consumption. On the same day, the body weight was also recorded. Graphs were plotted with mean change in the parameter versus time Model optimization experiment Treatment: The control group was continued with normal pellet diet (NPD). The remaining animals were randomized to five different groups. Following are the details. Table 2.2: Animal Grouping Sl. No. Group Treatment No of animals 01 Normal Control NPD + Water HCD HCD + Water HCD + Sucrose HCD + 30% sucrose solution HCD + Fructose HCD + 25% fructose solution HCD + STZ-1 HCD + Water + STZ (35 mg/kg, i.p) HCD + STZ-2 HCD + Water + STZ (20 mg/kg, i.p) 06 All the groups were given water ad libitum except for group 03 and 04, which received 30% sucrose solution and 25% fructose solution, respectively in place of water. Groups 05 and 06 received streptozotocin (STZ) at the doses of 35 and 20 mg/kg respectively through intraperitoneal (i.p.) route, whereas the control group received i.p. injection of only citrate buffer (Srinivasan et al., 2005). 50

6 Administration of STZ: STZ is a light sensitive compound. Hence, all the procedures including weighing, preparation of dose and administration were performed under dim light. The weighed quantity of STZ was dissolved in freshly prepared citrate buffer (50 mm sodium citrate at ph 4.4) maintained at 2 o C to 8 o C and injected via intra peritoneal route. The drinking water was replaced with 10% sucrose solution for the next 12 hours and the animals were monitored for the subsequent 07 days for mortality due to hypoglycemia or hyperglycemia. The extent of induction of diabetes was assessed on 7 th day. Oral glucose tolerance test (OGTT) Blood Sampling, and Biochemistry: OGTT and Blood Sampling: The rats were fasted overnight and were administered with glucose 2g/Kg orally as 40% solution. Blood samples were collected by retro orbital puncture method before the administration of glucose solution (0 min) and at 15, 30, 60, 90 and 120 minutes. The collected samples were centrifuged and the plasma was separated and refrigerated at 2-8 o C until the estimation of glucose and insulin. Additional blood samples were collected for the estimation of total cholesterol, triglycerides, and liver function tests Glucose estimation: The plasma glucose estimation was performed by glucose oxidaseperoxidase method using commercially available kits Calculation: Insulin estimation: Quantitative plasma insulin estimation was performed by ELISA (Solid phase two-site enzyme immune assay, based on direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule) by commercially available kit. Unknown insulin concentration was calculated by regression analysis. Estimation of lipid profile: Total cholesterol in plasma was estimated by cholesterol oxidaseperoxidase method using commercially available kits. Triglycerides were estimated using GPO-POD (Glycerokinase, peroxidase) method. Calculation for Cholesterol and triglycerides: Liver function tests: AST and ALT in plasma were estimated using modified IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) method by using commercially available kits. 51

7 Histopathology: The rats were killed and thereafter pancreas, spleen, liver, kidney and heart were removed and preserved in neutral buffered 10% formalin. Later, paraffin sections of 5 micron were made and stained with haematoxylin and eosin for histo-pathological evaluation Data Evaluation and Statistical Analyses Glucose excursion, AUC were analyzed with Winnonlin Professional version 5.3, Pharsight Corporation, St.Louis, USA. The statistical tests of significance were applied with GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA. The obtained glucose values were normalized to percentage values. The data analyses were performed for both actual and percentage normalized data. Two-Way analysis of variance (2- way ANOVA) followed by Bonferroni post-test was applied for OGTT-profile assessment. For rest of the assessments, one way ANOVA with Dunnett s test was used. NPD was used as comparator (control group) for glucose excursion, and AUC analyses of non-stz groups, whereas HCD was used as comparator for STZ treated groups. The obtained data was presented with descriptive statistics (Mean, SD, SEM and n) or graphically. 52

8 4.3. Results Food, water intake and body weight The post-weaned rats (about 30 days old) were taken in to the study and were put on HCD as per the allocation. Food, water intake and body weights were measured and recorded periodically up to 27 weeks. The food intake was relatively higher in HCD fed rats as compared to the rats on NPD. However, the difference was not significant statistically. The water intake in both the groups did not differ much. For the first three weeks, the weight gain was similar in both groups, however, there was a significant weight gain in HCD fed diet from seventh week onwards with mean body weight of ±2.47 grams as compared to NPD fed rats with mean body weight of ±12.00 grams. The difference became statistically significant (p<0.05) after 11th week with mean body weight of ±3.07 g and ±13.53 g for HCD and NPD fed rats respectively. The details are presented in table 2.3 and figure 2.1 to 2.3. Table 2.3: Food, Water intake and body weight measurement Duration Food Intake (g) Water Intake (ml) Body Weight (g) (Weeks) NPD HCD NPD HCD NPD HCD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.07* ± ± ± ± ± ±3.08* ± ± ± ± ± ±3.82* ± ± ± ± ± ±4.01* *p<0.05; ANOVA followed by Dunnet s post test; n= 64 for HCD rats; n=6 for NPD rats 53

9 Water intake (ml) Food intake (gm) Chapter 4 50 Food intake 40 HCD NPD Duration (weeks) Figure 2.1: Graphical representation of food intake. The data represents mean ± S.E.M 100 Water intake 80 HCD NPD Duration (weeks) Figure 2.2: Graphical representation of water intake. The data represents mean ± S.E.M 54

10 Body weight (gm) Chapter 4 Body Weight * * * * HCD NPD Duration (weeks) Figure 2.3: Graphical representation of weight gain. The data represents mean ± S.E.M Oral glucose tolerance test (OGTT) After an overnight fast, a known amount of glucose (2g/Kg as 40% solution) was given and the glucose levels were measured before glucose administration and after the glucose administration at scheduled time intervals. The details are presented in table 2.4 and figures 2.4 to Table 2.4: Plasma Glucose levels (mg/dl) Treatment NPD HCD HCD+Fructose Time (min) Mean SD SE Mean SD SE Mean SD SE * * * * * * * Treatment HCD+Sucrose HCD+STZ (20mg/Kg) HCD+STZ (35 mg/kg) Time (min) Mean SD SE Mean SD SE Mean SD SE * * * * *p<0.05 Vs NPD; p<0.05 Vs HCD; n=6 for all the groups except HCD+STZ (35 mg/kg) where n=4; 2-way ANOVA followed by Bonferroni test. 55

11 Figure 2.4: OGTT profile of control group (NPD fed rats). The data represents the profile of individual rats Figure 2.5: OGTT profile of HCD group. The data represents the profile of individual rats 56

12 Figure 2.6: OGTT profile of HCD+Fructose group. The data represents the profile of individual rats Figure 2.7: OGTT profile of HCD+Sucrose group. The data represents the profile of individual rats 57

13 Figure 2.8: OGTT profile of HCD+STZ (20 mg/kg) group. The data represents the profile of individual rats Figure 2.9: OGTT profile of HCD+STZ (35 mg/kg) group. The data represents the profile of individual rats 58

14 Figure 2.10 (a): Mean OGTT profile of all groups. The data represents mean ± S.E.M Figure 2.10 (b): Mean OGTT profile (STZ treatment). The data represents mean ± S.E.M 59

15 The obtained values were tabulated with descriptive statistics, giving mean, SD and SEM for each group. HCD and HCD+ Sucrose in comparison with NPD showed significant difference (p<0.05) in the OGTT profile from 30 minutes onwards for all time points. HCD+ Fructose group showed significant difference in the OGTT from 60 minute onwards. The STZ treated groups were compared against HCD instead of NPD and HCD+ STZ (20 mg/kg) did not show any significant change in the profile with respect to HCD whereas HCD+STZ (35 mg/kg) showed marked difference (p<0.05). The glucose (mg/dl) excursion observed (mean±sem) were ±8.718, ±5.915, ±6.672, ± and ±18.708, respectively for HCD, HCD+Fructose, HCD+Sucrose, HCD+STZ (20 mg/kg) and HCD+STZ (35 mg/kg) respectively as compared to excursion in NPD group, ±1.905 mg/dl respectively. The fructose supplemented group and the group treated with STZ at the dose of 20 mg/kg did not show appreciable difference as compared to their respective control groups. HCD+STZ (35 mg/kg) did show prompt induction of diabetes, which appeared to be type-i as the animals were entirely dependent on external insulin for the survival. We also observed that the animals eventually lost body weight and became very lean. HCD+STZ (35 mg/kg) resulted in substantial mortality with 30 deaths out of 34 animals. Autopsy revealed fluid accumulation in the intestine and inflamed kidneys. The observation of the group treated with HCD+STZ (20 mg/kg) revealed that the group initially showed hyperglycemia by 7 th day and eventually turned normoglycaemic by 15 th day. The variability was very high with standard deviation ranging from to as compared to HCD alone or HCD with fructose and glucose supplementation, which remained at to , to and to respectively. Statistical evaluation of percentage normalized OGTT data showed that HCD+ Sucrose group significantly affected the OGTT from 30 min onwards at all the time points with values (% increase) 56.02±7.133, 63.55±8.913, 55.26±7.544, 58.45±7.844 as compared to NPD values of 45.83±3.559, 31.62±3.868, 19.57±3.077, 9.50±3.205 respectively. Percentage normalized OGTT values of HCD+STZ (20 mg/kg) did not vary significantly from respective control group. It can be observed that in both the cases of comparison, HCD+ Sucrose group demonstrated significant disruption of glucose tolerance. The AUC of OGTT profile was calculated by applying linear trapezoidal rule for each group and compared with one-way ANOVA followed by Dunnett s test. NPD served as control group for all sugar supplementation groups and HCD for both the STZ add-on groups. 60

16 AUC (min*g/dl) Glucose excursion (mg/dl) Chapter 4 HCD+Sucrose group caused significant difference for peak glucose value. The results are presented in table 2.5 and figure 2.11 Table 2.5: Analysis of OGTT profile Treatment NPD HCD Units Mean SD SE Mean SD SE PGL mg/dl AUC min*mg/dl * TPGL [median ( range)] min 15 (15-15) 30 (30-60) Treatment HCD+Fructose HCD+Sucrose Units Mean SD SE Mean SD SE PGL mg/dl AUC min*mg/dl 15675* * TPGL [median (range)] min 15 (15-60) 15 (15-120) Treatment STZ20 mg/kg STZ35 mg/kg Units Mean SD SE Mean SD SE PGL mg/dl AUC min*mg/dl TPGL [median (range)] min 15 (15-60) 60 (60-60) *p<0.05 Vs NPD; p<0.05 Vs HCD; One way ANOVA with Dunnett s test; PGL: Peak Glucose Level; AUC: Area Under the Curve; TPGL: Time of PGL OGTT-AUC analysis 800 OGTT-Glucose excursion NPD HCD HCD+Fructose HCD+Sucrose HCD+STZ20 HCD+STZ NPD HCD HCD+Fructose HCD+Sucrose HCD+STZ20 HCD+STZ35 0 Treatment 0 Treatment Figure 2.11: AUC analysis and glucose excursion. The data represents mean ± S.E.M (n=6) 61

17 Plasma Insulin and OGTT Insulin estimation was performed for all the groups with OGTT challenge. A six point calibration curve was constructed for the estimation of insulin. The calibration range was 0.2 µg/l to 10 µg/l. The calibration curve was accurate and precise with coefficient of determination (r 2 ) being Linearity obtained for insulin estimation is presented in Figure 2.12 O.D. at 450 nm y = x R² = Insulin levels (µg/l) OD (450 nm) Figure 2.12: Linearity for insulin estimation The obtained insulin levels were tabulated and group-wise comparison was performed with two-way analysis of variance followed by Bonferroni post-test. The rats fed with NPD served as control group for all group comparisons. HCD served as control group to perform a differential assessment of effect with sucrose and fructose supplementation. All the groups, except the group with add-on STZ (35 mg/kg) treatment, showed significant rise in degree of insulin release as compared to NPD group. The group with STZ had lower insulin levels as compared to NPD group. The differential assessment for sucrose and fructose supplementation groups revealed that the group with sucrose supplementation fared better than HCD alone. The data are summarized in table 2.6 and figure 2.13 Table 2.6: Insulin Levels (µg/l) Time NPD HCD HCD+ Fructose HCD+ Sucrose HCD+ STZ ± ±0.055* 1.239±0.038* 1.346±0.047* 0.381± ± ±0.162* 3.831±0.132* 5.135±0.208* 0.983± ± ±0.071* 3.418±0.159* 4.152±0.166* 0.760± ± ±0.123* 2.997±0.144* 2.824±0.092* 0.661± ± ±0.079* 2.527±0.121* 2.609±0.100* 0.637± ± ±0.079* 3.104±0.116* 3.946±0.148* 0.562±0.038 *p<0.05 Vs NPD; p<0.05 Vs HCD; 2-way ANOVA followed by Bonferroni test; n=3 62

18 Insulin/Glucose (ng/ml) Insulin/Glucose (ng/ml) Insulin/Glucose (ng/ml) Insulin/Glucose (ng/ml) Insulin/Glucose (ng/ml) Chapter 4 OGTT: Glucose and Insulin OGTT: Plasma Glucose and insulin NPD OGTT: Plasma Glucose and insulin HCD Glucose Insulin Glucose Insulin Time (min) Time (min) OGTT: Plasma Glucose and insulin HCD+Fructose OGTT: Plasma Glucose and insulin HCD+Sucrose Glucose Insulin Glucose Insulin Time (min) Time (min) OGTT: Plasma Glucose and insulin HCD+STZ Glucose Insulin Time (min) Figure 2.13: A comparative representation of insulin levels and corresponding plasma glucose levels (ng/ml) (n=6) 63

19 AST (IU/L) ALT (IU/L) Tryglycerides (mg/dl) Total cholesterol (mg/dl) Chapter Biochemical Evaluation Other biochemical investigations including triglycerides (TG), total cholesterol (TC), AST and ALT were performed along with insulin estimation. There was a clear difference in TG levels across different diet groups and add-on STZ treatment.tg levels were significantly high (p<0.05) in both fructose and sucrose supplementation groups as compared to NPD. The group with STZ showed decrease in TG levels, although TC levels were increased significantly (p<0.05) as compared to NPD. No statistically significant changes were observed in rest of the groups with respect to TC levels. Both AST and ALT levels were high in all HCD treated groups. Fructose, sucrose and add-on STZ groups showed significant (p<0.05) increases in the liver enzymes as compared to NPD. The rise in the liver profile was highest with STZ treatment. The results are presented in table 2.7 and figure 2.14 Table 2.7: Biochemical parameters Treatment (Diet manipulation) Triglycerides (mg/dl) Total Cholesterol (mg/dl) AST (IU/L) ALT (IU/L) NPD 123.9± ± ± ±1.82 HCD 155.9± ± ± ±2.23 HCD+Fructose 190.6±4.48* 105.7± ±3.18* 66.2±3.36* HCD+Sucrose 194.1±28.16* 89.4± ±6.46* 74.0±3.31* HCD+STZ (35 mg/kg) 138.4± ±11.38* 160.7±6.18* 79.5±2.01* *p<0.05 Vs NPD, One-Way ANOVA with Dunnett s test; n=6 250 Triglyceride Levels 150 Total Cholesterol 200 * * * NPD HCD HCD+Fructose HCD+Sucrose 50 HCD NPD HCD+Fructose HCD+Sucrose HCD+STZ (20 mg/kg) HCD+ STZ (20 mg/kg) 0 Treatment 0 Treatment 200 AST 100 ALT 150 * * * * * * Treatment NPD HCD HCD+Fructose HCD+Sucrose HCD+STZ Treatment NPD HCD HCD+Fructose HCD+Sucrose HCD+STZ Figure 2.14: Effect of diet and additional STZ treatment on biochemical parameters. The data represents mean ± S.E.M 64

20 Histopathology Histopathological evaluation was performed for HCD+Sucrose group and compared with HCD alone and age-matched normal control group. Sections of adipose, pancreas, liver, kidney, spleen and cardiac muscle were obtained from the respective group and pathological changes were observed. Sections from heart, spleen, kidney and pancreas did not show any significant pathology across all the groups examined. The section of adipose tissue showed enlarged adipocytes in HCD fed rats. The degree of enlargement was more with HCD+Sucrose group as compared to HCD alone. Significant fatty changes were observed in the liver of HCD treated rats. The lesions were more pronounced in HCD+sucrose rats. The liver sections of HCD+Sucrose group showed ballooning degeneration, congested blood vessels with perivascular lymphatic infiltrates. The photomicrographs are presented in figures 2.15 to The magnification used was 20x. Figure 2.15: Adipose: NPD Group showing normal adipocytes 65

21 Figure 2.16: Adipose: HCD Group showing enlarged adipocytes Figure 2.17: Adipose: HCD + Sucrose Group showing enlarged adipocytes 66

22 Figure 2.18: Pancreas: Normal Group. No significant observation Figure 2.19: Pancreas: HCD Group. No significant observation 67

23 Figure 2.20: Pancreas: HCD+ Sucrose Group. No significant observation Figure 2.21: Liver: Normal Group. No significant observation. 68

24 Figure 2.22: Liver: HCD Group: The micrographs show ballooning degeneration, congested blood vessels with perivascular lymphatic infiltrates Figure 2.23: Liver: HCD+ Sucrose Group. The micrographs show ballooning degeneration, congested blood vessels with perivascular lymphatic infiltrates 69

25 Figure 2.24: Kidney: NPD Group. No significant observation Figure 2.25: Kidney: HCD Group. No significant observation 70

26 Figure 2.26: Kidney: HCD+Sucrose Group. No significant observation Figure 2.27: Spleen: NPD Group. No significant observation 71

27 Figure 2.28: Spleen: HCD Group. No significant observation Figure 2.29: Spleen: HCD+Sucrose Group. No significant observation 72

28 Figure 2.30: Cardiac Muscle: NPD Group. No significant observation Figure 2.31: Cardiac muscle: HCD Group. No significant observation. 73

29 Figure 2.32: Cardiac muscle: HCD +Sucrose Group. No significant observation. 74

30 4.4. Discussion Considering the epidemiological and etiological aspects and the risk factors for human type-2 diabetes, it becomes very important that we choose appropriate animal model, which can mimic the actual pathophysiological condition for the experimentation with the anti-diabetic drugs, used in pre-diabetics and initial stages of type-2 diabetes patients. According to the available information on pathogenesis of IGT and insulin resistance, it was necessary to induce obesity, which further will initiate a chain of events. Increased visceral adipose tissue raises circulating FFA due to insulin refractory adipocytes associated with decreased adiponectin. FFAs in turn get deposited in liver and muscles disturbing glucose uptake.(kahn and Flier, 2000, Kahn et al., 2001) We started our work with assessment of existing models for insulin resistance, so that we can optimize the best-suited model for the subsequent experiments with glibenclamide and metformin. We chose two reported models as the basis for this experiment, sucrose-induced insulin resistance model by Wright et al., and high fat diet+ low dose STZ model for type-2 diabetes by K. Srinivasan et al. We carefully studied the reported diet composition and modified to incorporate both high carbohydrate and high fat components. We raised the sucrose content to 55% and optimized fat content to 1.5% of the total diet composition. Another important modification was the selection of animals and duration of feeding. We selected post-weaned rodents and put them on HCD diet for 6 months. This was to ensure that the animals were exposed to the altered food habit early in their life so that food avoidance problem could be eliminated and, at the same time, they were exposed to HCD for a reasonable length of their lifetime. Two rats per cage (12 X 8 X 8 inches) were maintained since the start of the experiment. Cages allowed enough mobility initially but eventually as the rats grew in size, mobility of the rats got restricted making them more sedentary. We felt that these were some important considerations to mimic the actual disease progression. As a part of the optimization experiment, once the body weights were reasonably high and stabilized indicating reasonable degree of obesity, the rats were subjected to supplementation with 30% sucrose solution, 25% fructose solution as substitute to drinking water in addition to HCD for one month. Simultaneously, two separate groups were administered with STZ injection at the doses of 20 mg and 35 mg per kg. The former sets of trials were done to inflict profound stress on beta cells to create additional insulin demand, which mimics condition of insulin resistance. The low dose STZ administration was done to partially damage the beta cells, 75

31 compromising the insulin secretion to mimic the phenomenon of inadequate secretion of insulin. Our experiments revealed that the food and water intake did not change significantly and the intake pattern was almost similar to that of the control (NPD). However, there was a significant change in the body weight, which started becoming apparent after seventh week and became statistically significant by eleventh week. This clearly indicates that the initial growth phase was unaffected by diet modification and eventually HCD diet did cause weight gain. The reduced mobility of the rats due to the restricted space might also have contributed to the weight gain. We did not find encouraging results with low dose STZ (35 mg/kg) when administered intraperitonially in HCD fed rats. There was significant mortality (88.23%) associated with the experiment despite monitoring. The rats eventually became insulin dependent within 07 days of STZ injection and lost weight significantly by 11 th day. All the deaths occurred between 4 th day and 11 th day. We tried another dose of STZ (20 mg/kg), much lower than the earlier one. The rats did show the symptoms of STZ intervention, seen as a initial fall in blood glucose levels and subsequent hyperglycemia. On the day 01 of STZ treatment, rats were maintained on 10% glucose solution for initial 12 hours to counter hypoglycemia. The blood glucose levels rose for the next two days. However, by seventh day the rats turned out to be normo-glycemic. The OGTT profile of the group treated with add-on STZ at the dose of 35 mg/kg showed very high glucose values with 233 mg/dl at 0 hr and excursion of 560 mg/dl and by 120 minutes, the levels were still at around 461 mg/dl. Insulin estimation showed that the levels were much lower than the control (NPD) group. The glucose and insulin levels indicated insulin dependent diabetes. The model could not be taken forward because of two important factors. Primary factor was mortality and unpredictable responses to STZ post diet manipulation. STZ at 20 mg/kg was ineffective and 35 mg/kg was toxic in HCD rats. The second factor was progressive loss of body weight making the rats lean precluding the induction of IGT and insulin resistance. Both the STZ treatments failed to achieve the initial objectives of either partial destruction of beta cell mass or insulin resistance. HCD sans sugar supplementation produced adverse changes in glucose tolerance marked by increase in peak glucose levels coupled with significant rise in insulin levels showing the indications of insulin resistance syndrome. To make the signs more pronounced and to bring in reproducible IGT, we chose to introduce additional stress through the supplementation 76

32 with sugar solutions. The sugars, sucrose (30% solution) and fructose (25% solution) in drinking water were provided in place of normal drinking water. The sugar solutions at the chosen strength did not lead to reduced fluid consumption. OGTT performed after one month showed that there was no much change in the peak glucose concentration achieved after glucose challenge at 2g/Kg but the glucose disposition pattern was significantly altered and was at abnormally elevated level as compared to NPD at 60, 90 and 120 minutes despite increased insulin secretion. This observation clearly demonstrated that the peripheral glucose utilization is being crippled because of insulin resistance. This sluggish disposition of the circulating glucose clearly meant the pre-diabetic stage. The degree of OGTT alteration was not the same with both the sugars. OGTT alteration was more with sucrose supplementation than fructose. This was demonstrated when the sugar supplementation groups were compared against HCD group. By considering % glucose excursion, AUC, insulin levels, it can be said that HCD+ 30% sucrose supplementation reasonably answers the objectives of the model optimization experiments for insulin resistance model. Additional biochemical evaluations for TG and TC were performed to understand the extent of lipid abnormalities caused by HCD diet and HCD with add-on interventions. The group with HCD alone showed increased levels of TG and TC as compared to NPD group. However, the rise in TG became statistically significant with add-on sugar supplementations, which was not observed with STZ treated group. The HCD fed animals with sugar supplementation demonstrated all the vital indications of development of IGT and insulin resistance. Increased visceral fat accumulation, abnormally high TG levels, fatty liver as confirmed by histopathology and OGTT establishing abnormal response to glucose load and presence of raised insulin levels supported this. Another observation made in this study was that, in spite of starting experiments in post weaned animals and continuing the treatment for more than 6 months, which is about 1/3 of expected average lifespan of rats, the diet and supplementation, did not cause a rise in FBS. It was clear that HCD and sucrose supplementation could create a condition of hyperinsulinemia, glucose intolerance, hyper triglyceridemia, fatty liver, the conditions classified as insulin resistance syndrome. The literature points towards the fact that in human beings, the transition from impaired glucose tolerance to prompt diabetes is not a rapid process. The progression to type-2 diabetes may take time as less as two years to many years. However, going by the estimates, about 70% of the people with IGT eventually develop diabetes.(nathan et al., 2007). It may also be possible that the species difference and 77

33 effect of duration of study affected the results especially in case of low dose STZ (Rodrigues et al., 1997, Srinivasan and Ramarao, 2007) 4.5. Conclusion High carbohydrate diet (HCD) induced frank obesity with increased circulating triglyceride levels. Sucrose (30%) supplementation inflicted desired stress on the glucose homeostasis resulting in sustained hyperinsulinemia, impaired glucose tolerance and liver abnormalities, simulating the conditions desirable to mimic human insulin resistance syndrome. 78