U. Santoso1, S. Ishikawa2 and K. Tanaka2
1Faculty of Agriculture, Bengkulu University, Bengkulu - Indonesia. 2Division of Bioresources and Bioproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-0809 - Japan Corresponding E-mail: [email protected] Received January 26, 2010; Accepted July 28, 2010 ABSTRACT
The present study was conducted to evaluate the effect of fermented chub mackerel extract (FCME) on lipid metabolism in diabetic rats. Four week-old male Wistar rats were divided into threegroups based on weight. All rats were induced with diabetes mellitus by single intraperitoneal injectionof streptozotocin at 45 mg/kg body weight. Thereafter, they were randomly distributed to threetreatments with 7 rats assigned to each treatment. One group was the control with no additive, and two-treatment groups were given the purified diets supplemented with 1% or 2% FCME. Experimentalresults showed that in comparison to the control, diabetic rats fed FCME increased feed intake (P<0.01)and body weight gain (P<0.05). FCME inclusion significantly reduced the activities of acetyl-CoAcarboxylase (P<0.01) and fatty acid synthetase (P<0.05) in diabetic rats. FCME significantly increasedcholesterol 7 -hydroxylase with no effect on HMG-CoA reductase activity. FCME had no effect onhepatic triglyceride, free cholesterol and phospholipid. FCME inclusion at 1% level significantlyreduced serum triglyceride. FCME significantly increased HDL-cholesterol (P<0.05) with no effect onLDL + VLDL-cholesterol, and significantly reduced atherogenic index. FCME did not significantlyaffect serum insulin and glucose concentration. In conclusion, FCME supplementation altered lipidmetabolism in diabetic rats. FCME supplementation reduced the risk of atherosclerosis in diabetic rats. Keywords: fermented chub mackerel extract, lipid metabolism, diabetic rat INTRODUCTION
and a significant higher LDL/HDL-c ratio.
Furthermore, they stated that the difference in Diabetic mellitus is a state of nutritional state lipoprotein profile per se may be the cause of of nutritional starvation which frequently results increased atherosclerosis in diabetic swines. In in severe metabolic imbalance and pathological addition, the lack of insulin action combined with change in many tissues (Inui et al., 2000). In the effect of glucocorticoids also the LDL relation to lipid metabolism, diabetic condition is receptor in the liver (Schneider, 1991) resulting characterized by increases both fatty acid and lower removing the major proportion of IDL and cortisol. Consequently this could account for why VLDL secretion is increased in ketotic type I hypercholesterolemia. Duchateau et al. (2000) diabetes, which is caused by insulin deficiency found that there was significantly increases in (Brindley, 1991). Hypersecretion of VLDL cause plasma apo L level, cholesterol and triglyceride in hypertriglyceridemia. Other dyslipidemia includes low HDL-cholesterol, and small, dense LDL. The Some investigations have been conducted to incidence of atherosclerosis is 3-4 times greater in lower obesity in diabetic animals. Dixon et al. diabetics than non-diabetics at comparable plasma (2002) found that atorvastatin (80 mg/day) total cholesterol. Beyond total cholesterol inclusion protected diabetic swine against concentration, lipid abnormalities in the plasma of coronary artery atherosclerosis. This protection diabetics include elevated triglyceride, decrease was in part caused by reduced plasma triglyceride HDL-c levels, and the presence of small dense concentration since other lipid parameters, LDL. Dixon et al. (2002) found that the diabetic swine had a higher and broader IDL/LDL peak affected. Georgopoulos et al. (1998) found that a J.Indonesian Trop.Anim.Agric. 35(3) September 2010 high monounsaturated fatty-acid enriched diet is not preferable to a high-carbohydrate diet in patients with type 1 Diabetes mellitus with regard Kanzaki Company, Ltd., Takamatsu, Japan. The to the occurrence of postprandial lipemia. Higashi main constituents of this extract are peptides with et al. (2002) found that oleic acid enriched diet 20-50 chain-length amino acids. This product was associated with increased formation of post- contains 39,6% moisture, 51.1% crude protein, prandial chylomicron remnants compared with the 0.0% crude fat, 0.0% crude fiber, 8.7% crude ash linoleic acid enriched diet in patients with type 2 and 0.6% nitrogen free extract (NFE). Amino acid Diabetes mellitus. We recently found that fermented chub mackerel extract was effective to reduce lipid profiles in growing chicks (Tanaka et Blood samples were drawn from tail arterial al., 1990), broiler chicks (Tanaka et al., 1992) and under ether narcosis before and every weeks after rats (Santoso et al., 2000; 2001). This FCME was given streptozotocin, and left in an ice water to known to be rich in peptides. Peptides were prevent glucose degradation. The serum was cholesterol-enriched cells, activate the plasma centrifugation. Simultaneously glucose excretion LCAT and to protect against atherosclerosis to urine confirmed by test paper (Testepe, (Garber et al., 2001). Therefore, it was assumed that FCME inclusion to the diet would reduce At the end of experimental period, all rats lipid profiles in diabetic animals. Therefore, the were weighed individually. Thereafter, blood present study was conducted to evaluate effect of samples were drawn from heart and removed the FCME on lipid metabolism in diabetic rats.
liver under ether narcosis. The serum stored at–30oC until the determination of glucose, insulin, MATERIALS AND METHODS
total cholesterol, HDL-cholesterol and lipidfractions.
described (Santoso et al., 1995). The activities of experiment were purchased from Japan SLC Inc (Hamamatsu, Shizuoka, Japan). They were then weighed individually and divided into three Coenzyme A carboxylase (E.C. activity groups based on weight. All rats were induced was assayed by H14CO3-fixation method (Qureshi with diabetes mellitus by single intraperitoneal et al., 1980). Fatty acid synthetase (FAS) activity injection of streptozotocin at 45 mg/kg body incorporation method (HSU et al., 1965). The 3- carbonyl)amino]-D-glucopyranose (given Sigma) hydroxy-3-methylglutaryl-CoA reductase activity at 0.1 M citrate buffer solution, pH 4.5}.
was assayed by the method of Shefer et al. Thereafter, they were randomly distributed to (1973). The protein content of the solution used three treatments with 7 rats assigned to each for enzyme assay was determined by the method treatment. One group was the control with no of Lowry et al. (1951) using bovine serum additive, and two-treatment groups were given the albumin as the standard. ACC and FAS activities purified diets supplemented with 1% or 2% FCME. The rats were raised to 7 weeks of age in converted to product per minute per milligram of individual cages in an air-conditional room protein at 37oC. 3-hydroxy-3-methylglutaryl-CoA (temperature 22+2oC with humidity 50 to 60%) reductase activity was expressed as picomole of with the light on from 08:00 to 20:00. Rats were substrate converted to product per minute per mg fed a commercial nonpurified diet (type CE-2, protein at 38oC. Cholesterol 7-hydroxylase was Japan Clea) for a week before the initiation of the expressed as nmol/hour/mg protein. Samples were experiment with purified diets. The composition of experimental diets is shown in Table 1. Feed The lipid fractions were separated by thin- and water were provided for ad libitum layer chromatography on silica gel chromarod consumption. To confirm the induction of diabetes using hexane-diethylether-formic acid (60:10:1) mellitus, the serum glucose level in the fasting and hexane-benzene (1:1) as developing solvent state (16-hours starvation) was determined by and quantified by IATROSCAN TH-10 TLC/FID using a commercial kit (Glucose CII-Test Wako Analyzer (Iatron Laboratories, Inc., Tokyo, Lipid Metabolism of Diabetic Rat (U. Santoso, et al.) Table 1. Composition of Experimental Diet (%) 1) Supplied 650.0 g CaHP O4, 160.0 g NaCl, 140.0 g K2CO3, 32.7 g MgCO3, 10.0 g FeSO4. 7H2O, 3 .0 g Mn SO4 . H2 O, 1 .0 g CoCl2 . 6 H2 O, 1 .0 g CuSO4 , 2 .0 g Zn CO3 , 0 .1 g KI an d 0 .2 g NaF p er 1 kg.
2) Supplied 0.10 g retinyl acet at e, 0.00005 g cholecalciferol and 0.8995 g corn starch per 1 gram mixture.
3) Supplied 0.083 g thiamine-HCl, 0.233 g riboflavine, 0.833 g niacin, 0.75 g Ca-pantothenate, 0.1 g p yrido xin e-HCl, 0 .0 58 g fo lic acid, 1 5 g in osit o l, 1 .6 6 7 g p -amino ben zo ic acid, 0 .00 5 g bio t in , 0 .0 0 4 g cy an o co balamin, 3 3 .3 3 3 g ch o lin e-HCl, 0 .33 3 g menadio ne and 4 7.5 99 co rn st rach p er 10 0 g mix t ure.
fatty acid synthetase were significantly lower in Concentrations of serum total cholesterol, diabetic rats fed FCME, whereas cholesterol 7 HDL-cholesterol were measured with commercial kits (Cholesterol E Test Wako Kit and HDL- (P<0.01). The activity of HMG-CoA reductase cholesterol E Test Wako Kit from Wako Junyaku was not significantly affected (Table 4).
Kogyo Co. LTD). The difference between the total FCME had no effect on hepatic triglyceride, cholesterol and HDL-cholesterol was assumed to triglyceride was significantly lower in rats fed diet Fudamoto, 1995). An atherogenic index was with FCME (P<0.01), but total cholesterol and measured using equation published by Nishizawa free cholesterol were not significantly influenced.
increased (P<0.05) with lower atherogenic index response variables using one-way ANOVA in (P<0.05). Mackerel extract had no effect on which the overall treatment differences were LDL+VLDL-cholesterol concentration (Table 5).
represented by single orthogonal contrasts Dietary FCME had no effect on serum insulin.
between control and treatment groups (Shinjo, However, 2% FCME inclusion tended to increase insulin concentration at 24.4% level.
FCME inclusion significantly increased feed intake (P<0.01) and body weight gain (P<0.05) from higher feed intake in FCME group. It is FCME had no effect on liver weight (Table 2).
possible that streptozotocin eliminated the advantage of FCME in improving feed conversion concentration of serum glucose (Table 3).
ratio. It is unkown why feed intake of diabetic rats The activities of acetyl-CoA carboxylase and J.Indonesian Trop.Anim.Agric. 35(3) September 2010 Table 2. Effects of Fermented Chub Mackerel Extract on Feed Intake, Body WeightGain and Feed Conversion Ratio of Diabetic Rats Mean+SD for 7 rats * Significantly different (P<0.05) from the control group.
** Significantly different (P<0.01) from the control group.
*** Significantly different (P<0.001) from the control group.
Table 3. Effects of Fermented Chub Mackerel Extract on without cholesterol or those fed high-cholesterol Concentration of Glucos e in the Serum of Diabetic Rats containing diet. A reduced in hepatic fatty acidsynthesis is a major factor which caused lower hepatic triglyceride synthesis (Scorve et al., 1993), and resulting in lower triglyceride secretion into circulation. This may explain lower serum triglyceride in diabetic rats fed 1% FCME.
It is unkown however, although feeding 2% FCME reduced hepatic acetyl-CoA carboxylase and fatty acid synthetase activities it resulted in higher serum triglyceride concentration and no change in hepatic triglyceride. It was known that amajor site of fatty acid synthesis in rats was inadipose tissues. Therefore, it is needed to evaluate correlation between serum glucose and feed the activity of acetyl-CoA carboxylase and fatty intake (r = -0.93), it appear that the higher feed acid synthetase in adipose tissue to elucidate the intake could not fully be explained by lower serum glucose concentration. It was known that triglyceride. In addition, triglyceride clearance FCME rich in glutamic acid, one of an active taste from the circulation by lipoprotein lipase may compounds in feed that may also improve the palatability of diet, and therefore it increased feed intake. As far as growth efficiency concerned, the inclusion of FCME in diabetic rats had no observation that in rats the liver exhibits high beneficial effect. The present results agree with rates of cholesterol synthesis whereas nonhepatic the observation of Santoso et al. (2000) who tissues other than intestine show rates that are less found that FCME inclusion had a little value on than 5% of those in the liver (Balasubramaman et improving feed efficiency in rats fed cholesterol- al., 1976). FCME inclusion did not reduce the activity of hepatic HMG-CoA reductase, a rate Acetyl-CoA carboxylase was suggested as a limiting enzyme in cholesterol synthesis.
rate limiting enzyme in fatty acid synthesis Therefore, FCME inclusion might have no effect (Brindley, 1991). Therefore, the reduction of on hepatic cholesterol synthesis. However, FCME acetyl-CoA carboxylase activity in diabetic rats inclusion might increase hepatic bile acid fed FCME would result in lower fatty acid synthesis as indicated by higher activity of hepatic synthesis. Our previous studies (Santoso et al., cholesterol 7-hydroxylase activity, a rate 2000, 2001) also found that FCME inclusion limiting enzyme in bile acid synthesis. An resulted in lower acetyl-CoA carboxylase and increase in hepatic bile acid synthesis, however, fatty acid synthetase activities of rats fed diet Lipid Metabolism of Diabetic Rat (U. Santoso, et al.) Table 4. Effects of Fermented Chub Mackerel Extract on Activities of Lipogenic Related Acetyl-CoA carboxylase (nmol/min/mg protein Fatty acid synthetase (nmol/min/mg protein Choles terol 7-hydroxylas e (nmol/hr/mg protein 1Mean + SD for 7 rats; *Significantly different (p<0.05) from the control group.
** Significantly different (p<0.01) from the control group.
Table 5. Effects of Fermented Chub Mackerel Extract on the risk of atherosclerosis. In our previous results Various Lipid Fractions and Insulin of Diabetic Rats (Santoso et al., 2000) also showed that whenFCME was supplemented to a high-cholesterol containing diet, an increase in serum HDL- cholesterol with lower LDL-cholesterol without any change in total cholesterol was observed.
Lower atherogenic index found in rats fed FCME indicated that FCME inclusion might reduce the risk of atherosclerosis in diabetic rats. indicated by no effect of FCME on serum glucose CONCLUSION
As far as growth efficiency concerned, the Atherogenix index2 0.50+0.10 0.35+0.05** 0.39+0.07* inclusion of FCME in diabetic rats had no beneficial effect. FCME supplementation reduced the activities of hepatic acetyl-CoA carboxylase 2(Total cholesterol – HDL-cholesterol)/HDL-cholesterol and fatty acid synthetase, but it increased the *Significantly different (p<0.05) from the control group activity of hepatic cholesterol 7-hydroxylase in ** Significantly different (p<0.01) from the control group diabetic rats. FCME supplementation reduced therisk of atherosclerosis as indicated by loweratherogenic index.
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