Journal of the American College of Nutrition, Vol. 18, No. 1, 83-87 (1999)
Published by the American College of Nutrition
Dietary Fructose but Not Starch is Responsible for Hyperlipidemia Associated with Copper Deficiency in Rats: Effect of High-Fat Diet
Meira Fields, PhD, FACN and
Charles G. Lewis, PhD, FACN
Nutrient Requirements and Functions Laboratory, Beltsville Human Nutrition Research Center, ARS, United States Department of Agriculture, Beltsville, Maryland
Address reprint requests to: Meira Fields, PhD, FACN, USDA, ARS, BHNRC, NRFL; Bldg. 307, Rm. 330, BARC-East; Beltsville, MD 20705-2350
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ABSTRACT
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Objective: To test the hypothesis that copper deficiency in rats may be hyperlipidemic only when the diets consumed contain nutrients which contribute to blood lipids such as fructose and high fat.
Methods: Weanling male Sprague Dawley rats were fed diets which contained either starch or fructose as their sole carbohydrate source. The diets were either inadequate (0.6 µg Cu/g) or adequate (6.0 µg Cu/g) in copper and contained either high (300 g/kg) or low (60 g/kg) fat. At the end of the 4th week the rats were killed. Livers were analyzed for copper content. Plasma was analyzed for cholesterol and triglyceride concentrations.
Results: High-fat diet did not increase blood lipids in rats fed a copper-deficient diet containing starch. In contrast, the combination of high-fat diet with fructose increased blood triglycerides and fructose with copper deficiency resulted in a significant increases in blood cholesterol.
Conclusions: Hyperlipidemia of copper deficiency in rats is dependent on synergistic effects between dietary fructose and copper deficiency and fructose and amount of dietary fat. Hyperlipidemia does not develop if starch is the main source of dietary carbohydrate in a copper-deficient diet even if a high-fat diet is fed.
Key words: copper deficiency, starch, fructose, high-fat, cholesterol, triglycerides
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INTRODUCTION
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Numerous investigators including ourselves have reported that in rats, copper deficiency is an inducer of hypercholesterolemia and hypertriglyceridemia [1–5]. However, we have repeatedly reported that copper deficiency by itself does not result in a uniform effect on blood lipids. The ability to elevate blood lipids and the magnitude of lipemia are dependent on the type of dietary carbohydrate consumed. The consumption of a copper-deficient diet containing simple sugars induces high levels of blood lipids but the consumption of a copper-deficient diet containing starch does not [5–7]. What is the reason for this lack of a uniform hypercholesterolemic effect of copper deficiency? We suggest that unless the diet contains a nutrient that has the ability to raise blood lipids, hyperlipidemia of copper deficiency will not develop.
The majority of diets that are used to induce copper deficiency in experimental animals contain simple sugars [1–8]. Simple sugars, compared to complex carbohydrates such as starch, have the ability to increase blood lipids even in a copper-adequate animal. It is reasonable to assume that the mechanism responsible for hypercholesterolemia of copper deficiency is dependent on hyperlipidemic properties of simple sugars [9–11]. This hypothesis seems logical since starch, a non-lipogenic nutrient, which does not have the ability to induce lipemia in copper-adequate rats will have only a limited ability to raise blood lipids in copper-deficient rats. It is therefore suggested that in order to potentiate hyperlipidemia in starch-fed, copper-deficient rats, a lipogenic nutrient has to be fed.
High levels of dietary fat are associated with high blood lipids [12]. If our hypothesis is correct and a lipogenic nutrient is prerequisite for hyperlipidemia of copper deficiency in rats, then the consumption of a copper-deficient diet high in fat which contains starch should result in high blood lipid concentration. This hypothesis was tested in the present study. Levels of dietary fat used in this study were similar to those consumed by the average American [13,14].
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MATERIALS AND METHODS
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Forty weanling male Sprague-Dawley rats were randomly divided into eight dietary groups based on levels of fat [60 g/kg (low), or 300 g/kg (high)], type of dietary carbohydrate (starch or fructose) and levels of copper [6.0 µg Cu/g (adequate) or 0.6 µg Cu/g (deficient)]. Group 1: starch-fed, copper-adequate (6.0 µg Cu/g), low-fat (60 g/kg). Group 2: starch-fed, copper-deficient (0.6 µg Cu/g), low-fat (60 g/kg). Group 3: fructose-fed, copper-adequate (6.0 µg Cu/g), low-fat (60 g/kg). Group 4: fructose-fed, copper-deficient (0.6 µg CU/g), low-fat (60 g/kg). Group 5: starch-fed, copper-adequate (6.0 µg Cu/g, high fat (300 g/kg). Group 6: starch-fed, copper-deficient (0.6 µg Cu/g), high-fat (300 g/kg). Group 7: fructose-fed, copper-adequate (6.0 µg Cu/g), high-fat (300 g/kg). Group 8: fructose fed, copper-deficient (0.6 µg Cu/g), high-fat (300 g/kg). The composition of the diets is described in Table 1.
The salt mixture was prepared in our laboratory and formulated to omit copper carbonate. The copper-adequate diets were prepared by adding copper carbonate to the copper-deficient diets. The concentration of copper in all diets was analyzed by atomic absorption spectrometry. In order to increase total fat from 60 g to 300 g/kg, both protein and carbohydrates had to be reduced. As a result, total protein was reduced from 200 g/kg to 180 g/kg. This level of protein, however, is somehow lower than the recommended level for AIN-93G but much higher than 140 g/kg recommended for AIN-93M [15].
Rats were fed their respective diets for 4 weeks. They were then killed following an overnight fast. Livers, pancreata and hearts were removed and weighed. The concentrations of copper were measured in diets and livers as previously described [5]. Certified Reference Materials were digested and analyzed along with tissues and diets to assure accuracy. Blood was collected into heparinized test tubes for the measurements of cholesterol and triglycerides by the enzymatic automated procedure of the Centrifichem. Hematocrit was measured by conventional methods.
All data were expressed as mean±standard error of the mean (SEM) and analyzed by analysis of variance ANOVA with two levels of copper, two types of dietary carbohydrates and two levels of dietary fat. The independent effects of copper, carbohydrate and fat and the interactions between them were examined. Differences at p<0.05 were considered statistically significant.
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RESULTS
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Body mass, relative organ sizes, hematocrit and liver copper concentrations are presented in Table 2. Rats fed the fructose diet exhibited reduced body mass compared with rats fed starch. Copper deficiency was also responsible for a reduced body mass. The consumption of the high-fat diet reduced body mass in rats fed the starch-based diet. Relative liver size was increased by fructose compared with starch. The largest liver size was noted in copper-deficient rats fed fructose. The consumption of high-fat diet reduced liver size. Similarly, the consumption of a high-fat diet caused a reduction in pancreas size. The combination of copper deficiency with fructose resulted in the smallest pancreata. Fructose feeding and copper deficiency were responsible for heart hypertrophy. Copper-deficient rats fed fructose were anemic. High fat diet had no effect on hematocrit in rats fed fructose. It did, however, lower hematocrit in rats fed starch. In addition, high-fat diet reduced hematocrit in copper-deficient rats.
The concentration of liver copper is also presented in Table 2. As expected, the consumption of a low-copper diet resulted in a reduced concentration of hepatic copper compared to copper-adequate controls. Starch-fed rats exhibited higher liver copper concentration than fructose-fed rats. The consumption of a high-fat diet was responsible for elevating liver copper concentration. Rats that consumed the copper-adequate diet containing high-fat and starch exhibited the highest hepatic copper concentration.
The concentrations of plasma cholesterol and triglycerides are presented in Table 3. The type of dietary carbohydrate and levels of dietary copper affected plasma cholesterol concentrations. The consumption of diets low in copper which contained fructose resulted in the highest concentration of plasma cholesterol. Blood cholesterol was not affected by the level of dietary fat. The consumption of a starch-based diet with either copper deficiency or high fat diet did not increase blood cholesterol. Plasma triglycerides were elevated by fructose feeding, by copper deficiency and dietary fat. Copper-deficient rats which consumed the fructose-based diets exhibited higher triglyceride concentration than copper-deficient rats fed starch. The highest concentration of triglyceride in plasma developed in rats fed the diet which was inadequate in copper and contained fructose and high fat. The high fat diet did not increase blood triglycerides when the diet consumed contained starch.
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DISCUSSION
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Data of the present study show that the type of dietary carbohydrate determined whether copper deficiency or high-fat diet will induce hyperlipidemia. Dietary fructose but not starch, in copper-deficient rats, induced hypercholesterolemia as well as hypertriglyceridemia when combined with a high fat diet.
The present study was conducted in order to determine whether hypercholesterolemia and hypertriglyceridemia of copper deficiency was dependent on nutrients which possess the ability to raise blood lipids. We hypothesized that copper deficiency alone was not a strong enough stimulant to increase blood lipids, but the combination of copper-deficiency with fructose feeding was. Dietary fructose possesses hyperlipidemic properties [10,18,19]. It was not surprising, therefore, that copper-deficient rats fed fructose exhibited high concentration of blood lipids. Our hypothesis was further supported by the findings that copper-deficient rats fed starch-based diets did not develop hyperlipidemia.
Diets relatively high in fat content are hyperlipidemic [12]. In order to induce hyperlipemia in rats consuming a starch-based, copper-deficient diet, we fed rats diets high in dietary fat. The consumption of a diet containing starch which was high in fat but low in copper was expected to result in hypercholesterolemia and hypertriglyceridemia because of its high fat content. However, this could not be demonstrated in the present study. Under the present experimental conditions with the diet relatively high in fat, copper-deficient rats fed starch did not develop high blood levels of either cholesterol or triglycerides. However, all rats that had been fed a copper-deficient diet containing fructose developed hypercholesterolemia and hypertriglyceridemia. The combination of fructose with high-fat diet was responsible for a higher magnitude of hypertriglyceridemia. These findings demonstrate that in order to develop hyperlipidemia, dietary fructose but not starch has to be fed.
It has been recently reported that the consumption of high-fat diets deficient in copper had detrimental effects on copper nutritional status and intermediary metabolism [20] and raised blood cholesterol [21]. Examination of the diets used in those studies, however, revealed that they contained a mixture of simple sugars and complex carbohydrates in which the greatest portion of dietary carbohydrate consisted of either fructose or sucrose. Fructose-containing sugars possess hypercholesterolemic properties and have the ability to raise blood lipids even in animals consuming adequate copper [10,18,19]. In addition, in these two separate studies, the experimental diets contained a mixture of saturated and unsaturated fat [20,21]. Saturated fat by itself is hypercholesterolemic [5,18,19]. Based on diet composition, it was impossible to determine whether hypercholesterolemia of copper deficiency was due to high fat, saturated fat or to refined sugars.
It was interesting to note that Petering et al [22] have reported that an inverse relationship occurred between liver copper concentration and levels of serum cholesterol and triglycerides in rats, although the diets that had been used to induce copper-deficiency contained starch. Examination of the diets, however, revealed that dietary iron was approximately six-fold higher than that recommended by AIN for optimal growth of rodents [15–17]. We have recently reported that high dietary iron has the potential to induce hyperlipidemia in copper-deficient rats [23].
What are the mechanisms responsible for the lipemic effects of fructose? Dietary fructose does not stimulate lipoprotein lipase [24]. Consequently, the rate of removal of triglycerides from circulation when fructose is fed might be slower than when starch is fed, leading to higher blood triglyceride levels. A greater conversion of acetate to fatty acids has been reported in fructose-fed rats compared with glucose-fed rats [25]. Activity of hepatic enzymes regulating lipogenesis and gluconeogenesis is increased with diets having fructose in place of starch [9,11]. In addition, redox changes occur after the consumption of fructose [26,27] which may be necessary for hepatic fatty acid synthase gene transcription [4].
The hyperlipidemic effect of a high fat diet, however, depends also on the type of dietary carbohydrate consumed [18,19]. The magnitude of hyperlipidemia following the consumption of dietary fructose was greater in the presence of saturated fat than in the presence of unsaturated fat [5,18,19]. The diets of the present study contained unsaturated fat. The beneficial effects that fat unsaturation exert on hyperlipidemia involve a combination of interactive regulatory mechanisms that include gene expression and adaptive modulation of membrane composition which leads to changes in hormone signaling [28]. Unsaturated fatty acids also reduce the activities of microsomal enzymes involved in fatty acid desaturation and triglyceride synthesis [28].
Data presented in the present study clearly show that the magnitude of hypercholesterolemia and hypertriglyceridemia of copper deficiency is dependent on dietary nutrient interactions between levels and types of nutrients which have the capacity to induce and contribute to hyperlipidemia. These nutrients include fructose, copper and fat.
It should be realized that the present experimental diets contained a higher percentage of fructose and the use of only corn oil as the sole fat does not reflect the average North American diet [29], and the dietary periods were only 4 weeks long. To answer the question of long-term effects of different combinations of fructose, different levels of dietary copper and the type and levels of dietary fats in humans require further studies. However, the present findings and those reported by other investigators suggest that it might be important to limit dietary fructose and levels and type of fat and to increase the levels of current dietary copper [30].
Received June 1, 1998.
Accepted August 1, 1998.
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TOP
ABSTRACT
INTRODUCTION
