High fructose feeding of magnesium deficient rats is associated with increased plasma triglyceride concentration and increased oxidative stress

Magnesium Research. Volume 16, Number 1, 7-12, March 2003, ORIGINAL ARTICLE

Author(s) : Jérôme Busserolles, Elyett Gueux, Edmond Rock, Andrzej Mazur and Yves Rayssiguier , Centre de Recherche en Nutrition Humaine d‘Auvergne, Unité des Maladies Métaboliques et Micronutriments, INRA, Theix, 63122 Saint‐Genès‐Champanelle, France. .

Summary : The purpose of this study was to assess whether dietary carbohydrate could differentially influence the consequences of magnesium deficiency with particular emphasis on lipid metabolism and oxidative stress. Rats were fed a sucrose based or starch based diet either adequate or deficient in magnesium for two weeks. Magnesium deficient rats, as compared with rats fed magnesium adequate diets, displayed the usual decrease in plasma magnesium concentration. The classic symptoms of inflammation including hyperaemia, increased number of blood leukocytes and enlarged spleen weight were observed in these rats. Plasma TG and plasma apo B concentrations were also significantly increased. In addition, magnesium‐deficient animals presented an increased susceptibility to lipid peroxidation of heart and liver tissues as shown by TBARS concentration. Regardless of magnesium status, sucrose feeding did not affect the magnesium plasma level and inflammatory parameters. Feeding rats the sucrose diets induced hypertriglyceridaemia and increased plasma apo B concentration. Heart and liver susceptibility to lipid peroxidation were significantly increased in rats fed the sucrose diets as compared with those fed the starch diets. Sucrose feeding in magnesium deficient rats was associated with higher plasma triglycerides concentration and higher tissue susceptibility to peroxidation as compared with magnesium deficient rats fed the starch diet. The results emphasised the potential detrimental and additional effect of sucrose feeding and magnesium deficiency on cardiovascular risk. Since the intake of magnesium has been reduced appreciably in industrialised countries while fructose consumption has been rapidly increased, the impact of this eating pattern should be clarified in humans.

Keywords : Magnesium, fructose, triglyceride, oxidative stress, cardiovascular risk

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ARTICLE

Auteur(s) : Jérôme Busserolles, Elyett Gueux, Edmond Rock, Andrzej Mazur and Yves Rayssiguier

Centre de Recherche en Nutrition Humaine d'Auvergne, Unité des Maladies Métaboliques et Micronutriments, INRA, Theix, 63122 Saint-Genès-Champanelle, France.

Address for correspondence: Y. Rayssiguier, Unité des Maladies Métaboliques et Micronutriments, INRA-Theix, 63122 St-Genes-Champanelle, France. Phone + (33) 473 62 42 30; Fax + (33) 473 62 46 38;
email: yves.rayssiguier@clermont.inra.fr

Introduction

The implication of magnesium in lipid metabolism and in cardiovascular diseases has received particular attention. We previously reported that magnesium deficiency in rats induces hyperlipaemia [1] and oxidative stress [2] leading to increased cardiovascular risk. Recent observations suggest that oxidative stress and hypertriglyceridaemia in this experimental model are the results of the inflammatory response that occurs during magnesium deficiency [3]. D-Fructose is asugar that exists in foods as a simple sugar and as a component of disaccharide sucrose, consistingof one molecule of glucose and one of fructose. Because of the use of high fructose corn sweeteners and of sucrose in manufactured foods, the dietary consumption of fructose has increased several-fold over what is present in natural foods [4]. Although there is little evidence that modest amounts of fructose have detrimental effects on carbohydrate and lipid metabolisms, larger doses of fructose have been associated with numerous metabolic abnormalities in humans and laboratory animals, suggesting that high fructose consumption induces adverse effects for health [4, 5]. High-sucrose and high-fructose diets were used in animal models to induce the metabolic changes observed in syndrome X, a disorder in which insulin resistance, hypertension, dyslipaemia and high incidence of cardiovascular diseases are described [6]. The underlying mechanisms for the detrimental consequences of a high fructose diet in animal models are not clear. However, recent experiments indicate that fructose feeding facilitates oxidative damage [7]. Several studies suggesting an interaction between dietary magnesium and dietary carbohydrate have been previously published: fructose as compared with starch exacerbated some consequences of experimental magnesium deficiency such as hyperlipaemia [1], nephrocalcinosis [8, 9], while the role of dietary magnesium on fructose-induced insulin insensitivity is still unclear [10, 11]. Since the intake of magnesium has been reduced appreciably in industrialised countries [12, 13] while fructose consumption has been rapidly increasing, the impact of this eating pattern should be clarified. The purpose of this study was to assess whether carbohydrate could differentially influence the consequences of experimental magnesium deficiency with particular emphasis on lipid metabolism and oxidative stress. Sucrose was chosen as a carbohydrate source to study the effect of a high fructose diet as compared with a starch diet.

Abbreviations: Mg, magnesium, TG, triacylglycerol; apo B, apolipoprotein B; VLDL, very low-density lipoprotein; TBARS, thiobarbituric acid-reactive substances; LPL, lipoprotein lipase; TGRLP, triglyceride-rich lipoproteins; SOD, superoxyde dismutase; GPX, glutathione peroxidase.

Materials and methods

Experimental design

Male Wistar rats, weighing 201 ± 2 g (mean ± SEM) were derived from the colony of laboratory animals of the National Institute of Agronomic Research (INRA, Clermont-Ferrand/Theix, France). Rats were housed in wired-bottomed cages in a temperature-controlled room (22°C) with a 12-h light-dark cycle. They were randomly divided into 4 groups based on the source of carbohydrate and magnesium status: group 1: starch, magnesium adequate; group 2: starch, magnesium deficient; group 3: sucrose, magnesium adequate; group 4: sucrose, magnesium deficient. Animals were fed the appropriate diets for two weeks. The synthetic diets contained (g/kg): 200 casein, 650 starch or sucrose, 50 corn oil, 50 alphacel, 3 DL-methionine, 2 choline bitartrate, 35 modified AIN-76 mineral mix and 10 AIN-76A vitamin mix (ICN Biomedicals, Orsay, France). The MgO was omitted from the mineral mix in the Mg-deficient diets. The Mg concentrations of the diets, determined by flame atomic spectrophotometric analysis, were 45 and 950 g/Kg for magnesium-deficient and magnesium adequate diets respectively. Diets, offered as powder, and deionised water were provided ad libitum through the experiment. The redness of the ears was recorded to evaluate the occurrence and intensity of the clinical signs of the inflammation. We used the score of Nishio et al. [14] based on the following criteria: scores from 0 to 4 (score 0: no hyperaemia, score 1: hyperaemia at the base of the ears, score 2: hyperaemia over half of the ears, score 3: hyperaemia at over three quarters to the ears, score 4: hyperaemia over the entire ears). All procedures were in accord with institute's guide for care and use of laboratory animals.

Sample collections

Non fasted rats were weighed, anaesthetised with sodium pentobarbital (40 mg/kg body weight, intraperitoneally) and killed. Blood was collected from the abdominal aorta into heparinised tubes. The number of total white cells was recorded by a cell counter (Cobas, Hoffmann, La Roche). Plasma was obtained after low speed centrifugation (2000 g, 15 min). Spleen was removed and weighed. The heart and liver were rapidly removed, washed in ice cold saline (9g NaCl/L), placed in liquid N₂ and stored at – 80°C until analysis.

Analytical procedures

Magnesium was determined in plasma by flame atomic absorption spectrometry (Perkin Elmer 800). The susceptibility of heart and liver to peroxidation was determined in tissue homogenates after lipid peroxidation was induced with FeSO4 (2µM)-ascorbate (50µM) for 30 min in a water bath at 37°C, using a standard of 1,1,3,3-tetrahethoxypropane, as previously described [2]. Triacylglycerol (TG) (Biotrol, Paris, France) was determined in plasma by enzymatic procedures. Plasma apolipoprotein B (apo B) was determined by radial immunodiffusion using sheep anti-rat apo B antiserum, as previously described [15].

Statistical analysis

Statistical analysis were performed using “Statview” (Abacus Concepts, Inc.) software package. Results were expressed as means ± SEM. Two-way analysis of variance (ANOVA), defined as p < 0.05, was adopted to determine the main effects (carbohydrate and magnesium status) and interaction. All data were subjected to one-way analysis of variance (ANOVA). When significant F ratios were found, the individual means were compared by PLSD-Student test (p < 0.05).

Results

Magnesium deficient rats as compared with rats fed magnesium adequate diets displayed the usual decrease in plasma magnesium concentration and growth retardation. The classical symptoms of inflammation including hyperaemia, increased number of blood leukocytes and enlarged spleen weight were observed in these rats (table I). Liver weight, plasma TG and plasma apo B concentrations were found significantly increased (table II). In addition, magnesium deficient animals presented an increased susceptibility to lipid peroxidation of heart and liver tissues as shown by thiobarbituric acid-reactive substances (TBARS) concentration (table III).

Table I. Plasma magnesium level, body weight, hyperaemia, number of blood leukocytes and relative spleen weight in rats consuming starch or sucrose diet either adequate or deficient in magnesium.

2.4 ± 0.2
Leukocytes (10⁹ cells/L)	4.4 ± 0.3a	17.3 ± 3.8b	7.0 ± 0.4a	15.7 ± 2.0b	NS	< 0.001	NS
Relative spleen weight (g/100g BWt)	0.45 ± 0.02a	0.58 ± 0.05b	0.42 ± 0.02a	0.58 ± 0.02b	NS	< 0.001	NS

Results are means ± SEM (eight rats per group). NS, not significant. Means in the same row with different superscripts are significantly (p < 0.05) different (PLSD Fisher post-ANOVA). ^‡ p value, two-way ANOVA.
Ch, carbohydrate.

Table II. Relative liver weight, plasma triacylglycerol and apolipoprotein B concentrations in rats consuming starch or sucrose diet either adequate or deficient in magnesium

	Starch		Sucrose		Two-way ANOVA ^‡
	Adequate	Deficient	Adequate	Deficient	Ch	Mg	Ch x Mg
Relative liver weight (g/100 g BWt)	4.62 ± 0.04a	5.10 ± 0.12b	5.42 ± 0.10c	5.57 ± 0.12c	< 0.001	< 0.005	NS
TG (mM)	1.21 ± 0.12a	2.63 ± 0.27b	2.78 ± 0.36b	4.20 ± 0.39c	< 0.001	< 0.001	NS
ApoB (mg/L)	19.8 ± 0.4a	29.6 ± 1.3b	23.8 ± 0.8c	29.8 ± 0.8b	< 0.05	< 0.001	< 0.05

Table III. Heart and liver tissue susceptibility to peroxidation in rats consuming starch or sucrose diet either adequate or deficient in magnesium

	Starch		Sucrose		Two-way ANOVA ^‡
	Adequate	Deficient	Adequate	Deficient	Ch	Mg	Ch x Mg
Heart TBARS (nmol/g)	39 ± 2a	63 ± 5b	93 ± 8c	123 ± 8d	< 0.001	< 0.001	NS
Liver TBARS (nmol/g)	32 ± 3a	62 ± 7b	40 ± 2a	93 ± 10c	< 0.005	< 0.001	NS

While sucrose feeding resulted in higher body weight, it did not affect the magnesium plasma level and inflammatory parameters (table I). Feeding rats the sucrose diets induced higher liver weight, hypertriglyceridaemia and increased plasma apo B concentration (table II). Heart and liver susceptibility to lipid peroxidation were significantly increased in rats fed the sucrose as compared with the starch diets (table III).
No differences were observed in body weight (table I) of magnesium-deficient rats fed sucrose or starch however, sucrose feeding in magnesium deficient rats was associated with higher liver weight, higher plasma triglycerides concentration (table II) and higher tissue susceptibility to peroxidation (table III) than in magnesium deficient rats fed the starch diet.

Discussion

The classic signs of magnesium deficiency, including growth retardation, were observed in magnesium-deficient rats. Even if fructose feeding may induce alteration in magnesium absorption and magnesium retention, the mechanisms by which fructose may affect magnesium balance are largely unknown [16, 17]. Fructose feeding did not systematically affect magnesium status [18] and in the present experiment plasma magnesium concentration was not altered by the type of carbohydrate. Dietary magnesium deficiency in rats gives rise after a few days to a characteristic allergy-like crisis, the first visible symptom being a peripheral vasodilatation of the ears. Blood leukocyte response, as shown in the present study, is also a consequence of magnesium deficiency [19] and the greater spleen size in the magnesium deficient rats is believe to be due to infiltration of the spleen with polymorphonuclear cells and macrophages [20]. The underlying mechanism for the activation of inflammatory cells of magnesium deficiency remains unclear. The pathophysiological response of the immune stress includes activation of several processes which are dependant of cytosolic activation. Magnesium frequently acts as a natural calcium antagonist [21] and several recent studies suggest that calcium is implicated in the inflammatory response in the magnesium deficient pattern [22]. In agreement with previous data, the carbohydrate source did not affect the inflammatory response in magnesium-deficient rats [18]. Moreover in the present experiment, sucrose feeding had no significant effect on total blood leukocyte counts. Thus the characteristics of the inflammatory response of severe magnesium deficiency investigated in the present study were not aggravated by fructose.
In the present work, both sucrose feeding and magnesium deficiency have a significant hypertriglyceridaemic effect. Moreover, these two combined nutritional factors have additional effects on plasma TG level. Increased plasma triglyceride-rich lipoprotein (TGRLP) concentration could result from enhanced hepatic lipogenesis, overproduction of very low-density lipoproteins (VLDL) triacylglycerol and decreased peripheral catabolism. In magnesium deficient rats, the increase in apo B concentration is consistent with the increase in TGRLP. Other experiments in magnesium deficient rats indicated a complex pattern of alteration in lipid metabolism and apoprotein and suggested a defect in the catabolism of TGRLP as a major factor underlying the altered lipoprotein profile [23]. Alteration in lipoprotein metabolism has been related to the inflammatory response since similar changes such as those observed in magnesium deficient rats have been described in other inflammatory conditions. Inflammatory response increases serum TG levels by decreasing TG clearance, several mechanisms contribute to these alterations such as decreased activities in lipoprotein lipase (LPL), hepatic lipoprotein lipase and lecithin cholesterol acetyl transferase. Moreover inflammation is a potent stimulus for producing oxidation of serum lipoproteins [24, 25]. Concerning the effect of fructose, it has been widely accepted that, in both animals and humans, although changes in LPL activity partially account for the changes in blood lipids, hypertriglyceridaemia has been essentially related to increased synthesis of TG and their release into the plasma in the form of VLDL [26, 27]. In the present experiment, the increased plasma TG concentration in magnesium deficient rats fed the sucrose diet as compared with the starch diet was not accompanied by an increased plasma apo B concentration. A reduced capacity of the liver to synthesise apo B may explain the increased ratio of TG/apo B in the plasma of Mg deficient rats fed the sucrose diet as previously suggested [27]. Consequently, one can hypothesize that the combined effects of high fructose feeding and magnesium deficiency on hypertriglyceridaemia may involve an additional effect of fructose on the lower VLDL clearance of magnesium deficient rats.
Consistent with previous data showing the harmful effect of magnesium deficiency on lipid peroxidation in the cardiovascular system [2], tissue homogenates from magnesium deficient animals were more susceptible to lipid peroxidation than animals fed diets adequate in magnesium [2]. In the present experiment, animals fed magnesium adequate diets with sucrose as the carbohydrate source also had higher susceptibility to peroxidation of heart tissue whereas no differences were observed in liver tissue. The high total antioxidant potential [28], and high superoxyde dismutase (SOD), glutathione peroxidase (GPX) and catalase activities [29] found in the liver in rats may explain this result. Combined magnesium deficiency and sucrose feeding results in additive effects on lipid susceptibility to peroxidation in tissue homogenates. Altogether these results emphasise the additional effect of fructose feeding and magnesium deficiency on vascular risk as shown by hyperlipaemia, and heart susceptibility to free radical-mediated injury. These results are consistent with other studies showing the detrimental effect of a high fructose diet in animals models when antioxidant defences are reduced. For instance, the interaction of dietary fructose with copper has received considerable attention and dietary fructose when compared with starch enhances the severity of the signs of copper deficiency in this experimental model of oxidative stress [30].

Conclusion

Diets in the industrialised part of the world contain less magnesium that the daily recommendation of 6 mg/kg body weight [31] and the consumption of fructose is increasing [4]. The consequences on public health is still uncertain, however additional studies are needed to determine the potential consequences of this eating pattern in relation to the development of cardiovascular diseases.

Acknowledgement

The authors would like to thank the expert technical assistance of C.Lab and D.Bayle.

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2.6 ± 0.2

	Starch		Sucrose		Two-way ANOVA ^‡
	Adequate	Deficient	Adequate	Deficient	Ch	Mg	Ch x Mg
Plasma Mg level (mM)	0.73 ± 0.01a	0.21 ± 0.02b	0.80 ± 0.01c	0.17 ± 0.02b	NS	< 0.001	< 0.005
Body weight (g)	277 ± 5a	258 ± 3bd	296 ± 4c	268 ± 2ad	< 0.001	< 0.001	NS
Inflammation score (AU)	0