Chapter 5 Effect of tender coconut water on carbohydrate metabolism in rats fed high fructose diet

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1 Chapter 5 Effect of tender coconut water on carbohydrate metabolism in rats fed high fructose diet In the previous chapter, the effect of administration of tender coconut water on peroxidation was discussed. TCW administration was found to be beneficial in increasing the antioxidant status and decreasing the lipid peroxide formation. In addition to hypercholesterolemia and oxidative stress, abnormalities of glucose utilization were also reported in fructose fed hypertensive rats (Reaven, 1991 ). Glucose intolerance and insulin resistance are reported to play a key role in the pathogenesis of essential hypertension (Vasdev and Gill, 2005). Insulin resistance is characterized by an inadequate glucose uptake in peripheral tissues at a given concentration of plasma insulin and impairement of the non oxidative pathway of intracellular glucose metabolism (Ferrannini etap, 1987). Rats fed a high fructose diet provide an animal model of insulin resistance (Iyer and Katovich, 1996; Galipeau et at, 2002; Song et- al, 2004 ). Studies have demonstrated low levels of insulin- stimulated glucose oxidation in the liver (Huxtable, 1992), skeletal muscle (Banks etal, 1992) and adipose tissue (Evans et ap, 2002) in fructose fed rats. In addition hepatic metabolism of fructose leads to alterations in the activities of key enzymes of glucose metabolism (Southgate, 1995) and activation of stress sensitive pathways that may desensitize insulin signalling (Kelley ef-.j, 2004). Changes in the structure and 131

2 functions of proteins caused by the process of non-enzymatic glycation and advanced glycation end products (AGEs) are reported in respect of fructose fed rats. Collagen, the most abundant connective tissue protein, contains several dibasic aminoacids and has slow turnover rate which makes it highly susceptible to modification by glycation (Anitha etlil, 2005). AGEs is formed by reactive metabolic intermediates which bind proteins, DNA and other molecules and disrupt their structure and functions which lead to different diseases such as vascular complications. Chemical analysis of TCW indicate that it is a rich source of free ammo acid L-Arginine as supported by previous studies (Anurag and Rajamohan, 2003; Sandhya and Rajamohan, 2008). Rainer and Stefanie (2001) reported that L-Arginine possesses vasodilator function. Studies have reported that L-Arginine stimulates the release of pancreatic insulin (Schmidt et ap, 1992) and restores the defective - insulin mediated vasodilation (Wascher elal, 1997). In view of these reports, the present study examined whether TCW has beneficial effects on impaired carbohydrate metabolism in high fructose fed rats. 5.1 Materials and Methods Experimental groups Male albino rats (Sprague Dawley strain ) weighing g, were used for the study. The rats were divided into 4 groups of six each and fed the following diet. 132

3 Group 1 Control rats Group 2 Control rats+ TCW(4 ml/loog body weight) Group 3 High fructose fed (hypertensive) rats Group 4 High fructose fed (hypertensive) rats + TCW Rats were fed the respective diet and maintained for 5 weeks. All other experimental conditions were the same as described in chapter 3. At the end of experimental period they were sacrificed and blood and tissues were collected in ice cold containers. 5.2 Results Following biochemical parameters were studied: Levels of glucose, insulin, glycosylated haemoglobin (Hb A 1c ) and Homeostasis Model Assessment Score (HOMA) The concentration of blood glucose, plasma insulin, glycosylated haemoglobin and insulin resistance index (HOMA) were significantly elevated in fructose fed rats as compared to control rats. TCW treatment to fructose fed rats prevented the increase. The degree of insulin resistance as measured by HOMA was higher in fructose fed rats while in tender coconut water treated rats the values were normal (Table 15). 133

4 Table 15 Levels of blood glucose, insulin, Hb Ale and HOMA in plasma Groups Glucose Insulin HbA1c HOMA* {mg/di) (µu/ml) (%) ± 7.8 b 21.1± 1.4 b 1.61 ± 0.07 b 4.45±0.41 b ± 9.6 b 22.3± 1.3 b 1.73± 0.04 b 4.96±0.47 b ± 11.2 a 46.5± 2.l a 3.94 ± o.os a 17.23±1.27 a ± IO.S b 27.1± 1.4 b 1.96 ± 0.02 b 7.44±1.70 b F ratio Values are mean ± SD for six rats. * fasting plasma insulin(µ U/ml) x glucose (mmol/l)/ P<0.05, indicates that the results are significantly different from group l, b indicates that the results are significantly different from group Activities of hexokinase, phosphoglucomutase, pyruvate kinase and glycogen content in liver The activities of hexokinase, phosphoglucomutase, pyruvate kinase and hepatic glycogen significantly decreased in fructose fed hypertensive rats. Tender coconut water administration to fructose fed rats significantly increased the enzyme activities and hepatic glycogen (Table 16). 134

5 Table 16 Activities of hexokinase, phosphoglucomutase, pyruvate kinase and glycogen content in liver Groups Hexokinase a Phosphoglucomutase Pyruvate kinase 1 Glycogen ± 0.33 b 1.43±0.46 b ± 0.65 b l.53±0.23 b ± 0.83 a 3.47±0.39 a ± 0.44 b 2.94±0.18 b F ratio ±0.12 b 25.23±3.7 b 0.61±0.17 b 25.0l±0.9 b 0.29±0.09 a 15.98±0.9 a 0.30±0.03 b 22.96±0.9 b Values are mean± SD for six rats. a mg of glucose phosphorylated I hour/g protein. P µ M of Pi liberated/ hour/g protein. 1 units/ g protein. 6 mg/loog wet tissue. P<0.05, indicates that the results are significantly different from group l, b indicates that the results are significantly different from group Activities of glycogen phosphorylase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in liver The activities of glycogen phosphorylase, glucose-6-phosphatase, fructose 1, 6 bisphosphatase in fructose fed rats showed significant increase whereas tender coconut water treatment to fructose fed rats restored the enzyme activities (Table 17). 135

6 Table 17 Activities of glycogen phosphorylase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in liver Groups Glycogen Glucose-6- Fructose 1,6 phosphorylase a phosphatase a bisphosphatase P ±0.93 b 3.47± 0.59 b 5.27± 0.42 b ±0.25 b 3.60± 0.52 b 5.01± 0.95 b ±0.22 a 6.62± l.o a 12.64± 0.39 a ±0.38 b 4.50± 0.50 b 6.28± 0.27 b F ratio Values are mean± SD for six rats. a µ M of Pi liberated I hour/g protein. µ M of Pi liberated I minute/g protein. P<0.05, indicates that the results are significantly different from group I, b indicates that the results are significantly different from group Protein bound carbohydrates in serum Serum hexosamine and sialic acid were elevated by high fructose diet. TCW treatment to fructose fed rats reduced the levels significantly (Table 18). Table 18 Protein bound carbohydrates in serum Groups Hexosamine Sialic acid (mg/di) (mg/di) ±1.36 b 9.68±0.93 b ±1.5i 9.60±0.74 b ±2.58 a 13.03±0.32 a ±1.48 b 1 l.54±0.43 b F ratio Values are mean± SD for six rats. P<0.05, indicates that the results are significantly different from group I, b indicates that the results are significantly different from group

7 5.2.5 Total collagen in tail tendon Total collagen content in tail tendon was elevated in high fructose fed rats compared to normal rats. TCW supplementation reduced the levels of total collagen in fructose fed rats (Fig. 18). Fig 18 Concentration of total collagen in tail tendon Total collagen 120 Cl) 100 ::, Ill Ill 80 a 5.3 Discussion E 60 b ,. "a. E,I',/' 20,l' rl",i'... 0 rl',c,c Group 1 Group 2 Group 3 Group 4 Values are mean± SD for six rats. P<0.05, indicates that the results are significantly different from group 1, b indicates that the results are significantly different from group 3. In the study TCW administration ameliorated the high blood glucose levels and insulin raised by high fructose diet. These findings are consistent with those of other investigators (Salihu et af, 2009). High fructose diet is reported to produce hyperglycemia and insulin resistance (Thorburn et al, 1989; Dai ef a:v, 1994). Fructose feeding has been reported to decrease the efficacy of insulin extraction by the liver, and to retard insulin clearance from the circulation which results in insulin resistance (Suga el af, 2000). Insulin exerts an anti-natriuretic effect. It also has a direct vasodilatory effect on 137

8 vascular smooth muscle (Anderson et al, 1991). Insulin regulates the vascular tone by regulating intracellular calcium of vascular smooth muscle (Zemel, 1995). There is substantial evidence in both humans and animals that an increase in plasma triglyceride concentration is the expected consequence of insulin resistance (Reaven et al, 1991) which may be due to over production of VLDL and impairement in clearance (Vasdev etaf, 1994; Sambandam et al, 1997). The liver glycogen content was significantly lower in fructose-fed rats whereas TCW supplementation increased the glycogen content. Studies have reported that hyperglycemia impairs the ability of liver to synthesize glycogen. Decreased glycogen content in tissues could be due to the reduction in glycogen synthase activity (Vrana et al, 1978). Reduced hepatic glycogen synthase activity has been found in fructose-fed rats (Vaag et JJI., 1992). Thorburn et af.. ( 1991) have suggested a gluconeogenic source for the elevated hepatic glucose output because fasting liver glycogen levels decreased in fructose- fed rats compared with controls. The results indicate that the increase in the concentration of glycogen in the liver of the rats given TCW may be due to increased glycogenesis. It may also be due to decreased degradation of glycogen in treated rats as evident from the decreased activity of glycogen phosphorylase. There was decreased gluconeogenesis as evident from the lower activity of glucose-6-phosphatase and fructose-i, 6-diphosphatase in TCW treated rats. The utilization of glucose 138

9 by glycolytic and citric acid cycle and also by the pentose phosphate pathway may be increased as evident from the increased activity of some of the enzymes involved in these pathways. Thus the hypoglycemic action oftcw may be due to increased utilization of glucose in the liver for glycogen synthesis, decreased degradation of glycogen and also due to decreased gluconeogenesis. The decreased activity of glycolytic enzyme hexokinase showed lesser utilization of glucose by glycolysis causing blood glucose level to increase. The hypoglycemic action of TCW may partly be due to the aminoacid L- Arginine which is reported to restore the defective insulin mediated vasodilation thereby decreasing hyperglycemia. L-Arginine supplementation represents a potentially novel and useful strategy for the management of hyperglycemia (Mendez and Balderas, 2001; Flynn elaf, 2002). L-Arginine also stimulates the release of pancreatic insulin thereby controlling the hyperglycemia (Schmidt et a! 1992). In addition, TCW contains considerable amounts of vitamin C which is reported to reduce the blood glucose levels (Kurowska et a1, 2000). Hyperglycemia induced vitamin C deficiency is reported to promote endothelial dysfunction (Price et al, 2001 ). An inverse association between plasma vitamin C and glycosylated haemoglobin is also reported (Sargeant et Id, 2000). Vitamin C is also reported to minimize advanced glycation end products (John, 1998). Gluconeogenesis increased since the activity of glucose-6-phosphatase was increased. Fructose 2, 6-bisphosphate may serve as an important regulator 139

10 of carbohydrate metabolism in the liver. The synthesis and degradation of fructose -2, 6-bisphosphate are catalyzed by a single enzyme complex fructose- 2, 6-bisphosphatase. The lowered level of fructose-2, 6-bisphosphate stimulates fructose-i, 6-diphosphatase which favours the conversion of fructose-i, 6- diphosphate to fructose-6-phosphate, an intermediate of gluconeogenesis. An increase in fructose -2, 6-bisphosphate stimulates 6-phosphofructo-1-kinase, which converts fructose-6-phosphate to fructose-i, 6-diphosphate, which favours glycolysis (Van, 1987). Gluconeogenesis from fructose was reported to be greater in liver (Anundi etj, 1987). The protein bound carbohydrates viz hexosamine and sialic acid increased in fructose fed rats whereas TCW supplementation decreased the levels. Studies reported that since fructose-fed rats showed hyperglycemia the protein bound sugars may be altered in these rats. The glycoproteins levels were elevated in plasma of fructose-fed rats (Radhakrishnamoorthy el. al, 1970). Elevated serum sialic acid concentration is reported to be a risk factor for cardiovascular mortality in the general population (Lindberg el-sf, 1991 ). The increased concentration of sialic acid could be due to shedding of sialic acid-non cell membrane glycoconjugates or increased sialyation. Haematological changes and increased levels of acute phase proteins and the altered sialyation have been observed in vascular abnormalities and atherosclerosis (Stuart el:al, 1981 ). 140

11 Hexosamine biosynthesis has been proposed to play a role in mediating the effects of chronic hyperglycemia. Excess flux through the pathway has been shown to result in insulin resistance in cultured cells, tissues and in intact animals (Meclain and Crook, 1996). In vitro studies with collagen showed that fructose is a more potent glycating agent than glucose (Brennan, 1989). Sakai eta9 (2002) reported that fructose enhanced reactive oxygen generation and reduced the digestibility of collagen by collagenase, showing the closer involvement of fructose in crosslinking (Reiser, 1991). MDA, an end product of lipid peroxidation has been reported to react with the free amino groups of collagen and stimulate crosslinking (Fu e/; a.f, 1996). High fructose diet has been shown to cause glycation and crosslinking of skin collagen and promote ageing process in rats (Levi and Werman, 1998). The results indicate that feeding TCW can have significant hypoglycemic effects. TCW treatment was found to be effective in improving insulin sensitivity and metabolic alterations of carbohydrate associated with consumption of high fructose diet. 141