To investigate effects of a shifting high fat diet to normal fat diet supplemented with magnesium, zinc and chromium on biochemical parameters in rats with diabetes Aly R. ABDEL-MOEMIN1, Ashraf A. A. EL-MEGEID1, Ebtesam Fath M. OMAR2, Yasmeen F. ABD EL-MONEIM3, Dalia Mohamed TALAAT3 and Sonia Salah ELMARASY1 1Nutrition and Food Science Dep. Faculty of Home Economics, Helwan University, Egypt 2Home Economic Dept. Faculty of Specific Education, Alex. University, Egypt 3Home Economics Dept., Faculty of Specific Education, Fayoum University, Egypt Several minerals play an important role in modulating blood glucose and thyroid hormones. The aim of this study was to investigate the effect of individual and combined minerals; chromium (Cr), magnesium (Mg) and zinc (Zn) on blood glucose, lipid profile, kidney and liver functions, T3, T4 and TSH among obese diabetes rats. The study was carried out on 66 male Wistar rats (150-160g) for 90 days on two stages. First stage included 66 rats (6 of which were control) which were fed on a high fat diet (19% hydrogenated fats and 1% corn oil as a source of essential fatty acids) for 45 days. Second stage of the study included the same 66 rats (6 of which were control) which were injected with (150 mg Alloxan / kg b.w) to induce experimental diabetes and were then fed normal levels of dietary fat supplemented with individual and combined minerals; Cr, Mg and Zn at two levels (high and low). Results indicate that in the positive control rat group (PC) there was elevated cholesterol and triacylglycerol levels. While, rat groups supplemented with combined elements (at low and high levels) led to lowering cholesterol and triacylglycerol significantly (p < 0.05). In particular, Cr had improved triacylglycerol status. The vice versa was noticed in rat groups fed on diets supplemented with individual trace elements. HDL lipoprotein levels were increased in individual supplementation with zinc (20mg) and at higher levels of Mg, Zn and Cr. Cr, at higher levels (200 ^g) led to significant reduction of the VLDL-C compared with negative control (NC) group. Uric acid, urea nitrogen and creatinine were decreased significantly with diets supplemented with individual Zn at higher levels (20mg/ kg b.w). The combination at high levels showed reduction of uric acid, urea nitrogen and creatinine compared to NC. Conclusion: our results indicate that diets supplemented with combined elements led to improving the tested parameters in this study. Key words: diabetes mellitus, rats, alloxan, lipid profile, liver function, thyroid hormones INTRODUCTION Obesity and the incidence of type II diabetes are increasing worldwide. The development of complications in diabetes are well reviewed, with an associated increase in morbidity and mortality. Minerals have been shown to influence hormones at several levels, including hormone secretion and activity, and binding to the target tissue. Moreover, hormones have been shown to influence trace metal metabolism at several levels, including excretion and transport of trace metals (Henkin 1976). Magnesium is a macro mineral which is known to play an important role in carbohydrate metabolism, and its imbalance has been implicated in diabetes mellitus both as a cause and a consequence (American Diabetic Association1992; Mooradian and Morley 1987). Magnesium deficiency has been associated with type II diabetes and may reduce insulin sensitivity and impair glucose tolerance. The aetiology of magnesium depletion in diabetes is unclear. Animal studies suggest that diabetes may impair magnesium absorption; however, there are no published data on magnesium absorption in humans with diabetes (Walti et al. 2003). * Correspondence to: E-mail: dralymoemin@yahoo.co.uk Chromium (Cr) is one of the few trace minerals for which a specific cellular mechanism of action has not been identified. Recent in vitro studies suggest that Cr supplementation may improve insulin sensitivity by enhancing insulin receptor signalling, but this has not been demonstrated in vivo. In a study, investigating the effect of Cr supplementation on insulin receptor signalling in an insulin-resistant rat model, the JCR:LA-corpulent rat, the data suggested that chromium supplementation of obese, insulin-resistant rats may improve insulin action by enhancing intracellular signalling. (Wang et al. 2006). In addition to humans, beneficial effects of supplemented Cr have been observed in rats, mice, squirrel, monkeys, guinea pigs, rabbits, fish, pigs, cattle and horses (Anderson 1993; Mertz 1993). In a cohort of type II diabetes patients, low serum zinc (Zn) level was an independent risk factor for CHD events (Soinio et al. 2007). While it is clear that urinary excretion of Zn is markedly increased in individuals with diabetes, if hyperglycaemia is the primary aetiology, replacement with oral Zn supplementation should provide sufficient treatment. High dose supplementation in Type I diabetes and in normal individuals was evaluated by serum and urine Zn and mononuclear cell Zn concentrations. Zn excretion and mononuclear Zn concentrations were increased by a similar amount in both groups. There did not appear to be any evidence for total body Zn deficiency in the group with diabetes despite the clear hyperzincuria. Somewhat distressing in this study was an increase in haemoglobin Alc in all individuals which was, of course, of greater concern in the subjects with diabetes. The data suggested that there is a possible Zn toxicity for high dose supplementation which has greater ramifications in subjects with diabetes. It also suggests that there is more than hyperzincuria responsible for the abnormalities in Zn metabolism in patients with diabetes (Cunningham et al.1994). The aim of this study was to explore the effects of diets supplemented with certain individual and combined elements such as chromium (Cr), magnesium (Mg), and zinc (Zn), on blood sugar level, lipid profile, liver and kidney functions, T3, T4 and TSH among male Wistar rats. These minerals were studied extensively but the combinations of these minerals were lacking in the literature. Furthermore, in this study the amounts of minerals used in the supplemented diets were different compared to similar studies. The model followed in this study is matching the scenario of obesity that is associated with several complications such as type II diabetes (Environmental Diabetes). MATERIALS AND METHODS Diets and chemicals All diet components such as casein, vitamins, minerals, fibre, were obtained from El-Gomhoreya Company, Cairo, Egypt. Kits used to carry out biochemical parameters in this study were obtained from Egyptian American Company for Laboratory Service. Experimental Animals This study was performed on (n = 66) male Wistar rats at 8 weeks of age, the rats were housed and bred as approved by the Animal Ethics of Nutrition and Food Science Department; Faculty of Home Economics, Helwan University, Egypt. Rats were kept separately in metal cages in a room with controlled temperature (20 to 22°C) and humidity (50 to 55%), and maintained in a cycle of light for 12 h (06:00 to 18:00 h) and dark for 12 h (18:00 to 06:00 h). Rats were allowed to consume their respective diets (Table 2) and water as ad libitum. The animals were kept on rodent chow for a week. After this washout period, rats were divided into 2 groups; group A (n=60) (150-160g) fed on high fat diet (19% hydrogenated fats and 1% corn oil as a source of essential fatty acids) and group B (6 rats) fed on basal diet Ain-93M (Reeves et al. 1993) for 45 days. Using hydrogenated fats in this experiment aimed to induce experimental obesity and increase the risk factor to heart disease as it's a source of trans fatty acids. After this stage, rats (n=66) were divided into 11 sub-groups as explained in Table 1. Sixty of which (n=60) were injected with Alloxan to induce diabetes, (apart from control group n=6), within 48 hours from injection, blood sugar was elevated to above 200 mg/dl. In this stage, rats were (n=60) were fed on normal level of fat according to Ain-93M. Diets were supplemented with either individual or combined minerals; Cr, Mg, and Zn at two levels as presented in Table 1. These supplements were given by tube feeding daily. Lipid profile was measured during the study by taking blood samples from the orbital plexus of all rats, in order to confirm the elevation of lipid profile parameters (Data not shown) compared to control group. Rats were sacrificed after 45 days of feeding on diets supplemented with individual and combined minerals. Body weight was recorded, blood collected from orbital plexus for biochemical analyses: a series of biochemical analyses were measured; cholesterol, triacylglycerol, LDL-C, VLDL-C blood glucose, uric acid, urea nitrogen, creatinine, AST, ALT, T3, T4, and TSH. Table 1: Schematic of experimental rat groups Negative control group (NC) Obese diabetes group (PC) Distilled water group (DW) 1 200-mg magnesium sulphate / (kg bw) 5 (Mg 200) n c 300 -mg magnesium sulphate / (kg bw) ° (Mg 300) 10mg zinc chloride / (kg bw) (Zn 10) s o § $ 20 mg zinc chloride / (kg bw) (Zn 20) oo ^ ^ 100 ^g chromium / (kg bw) (Cr 100) 2 | 200 ^g chromium / (kg bw) (Cr 200) ^ 300-mg magnesium sulphate, 20 mg zinc ii a> S5 chloride and 200 ^g chromium / (kg bw) O £ £ (Combined high levels) Table 2: Composition of the basal diets Ingredient g/kg Diet Control High fat Casein 140.000 140.000 Fibre 50.000 50.000 *Corn oil/hydrogenated fats 40.000 200.000 Mineral mix. 35.000 35.000 Vitamin mix. 10.000 10.000 Corn starch 465.692 345.692 Dextrinized corn starch 155.000 155.000 Sucrose 100.000 100.000 L-cytesine 1.800 1.800 Cholin bitartarate 2.500 2.500 Tert-butylhydroquinone 0.008 0.008 *Soybean oil was replaced by corn oil Blood glucose was measured by (Trinder 1959), total thyroxin T4 (Britton et al. 1975), triiodothyronine T3 (Ahmed et al. 1974) and thyroid-stimulating hormones TSH (Wada et al. 1983). Table 3: Effect of diets supplemented with Cr, Mg and Zn on weights of obese rats with diabetes. Parameters Weights BWG% Groups Initial Middle Final Negative control group (NC) 230.8±3.7b 256.6± 5.2e 274.5± 7.6h 18.9± 4.3fg Obese diabetes group (PC) 281.3± 5.9a 331.0± 4.0a 386.6± 8.4a 37.4± 2.3a ■o £ a ^ * 3 tfl <5 .Q is = ° (5 Distilled water group (DW) 200 -mg magnesium sulphate / (kg bw) 300 -mg magnesium sulphate / (kg bw) 10 mg zinc chloride / (kg bw) 20 mg zinc chloride / (kg bw) 100 pg chromium /(kg bw) 200 pg chromium /(kg bw) 200-mg magnesium sulphate, 10 mg zinc chloride and 100 pg chromium / (kg bw) 300-mg magnesium sulphate, 20 mg zinc chloride and 200 pg chromium / (kg bw) 281.0±9.9a 280.8±9.0a 280.8± 9.1a 279.2±6.6a 280.8 ±9.0a 280.0± 3.7a 279.3±5.2 a 279.2 ± 2a 320.5 ± 4.3ab 306.5± 5.7abcd 296.0 ± 6.2bcd 310.5 ±4.2abc 317.6 ± 37.9ab 305.2± 4.2abcd 300.0±39.7 bcd 285.8± 4.8cd 368.2±10.8b 358.5± 5.7c 340.2± 5.9e 362.5± 6.7bc 344.5± 8.4de 349.7 ± 5.6d 329.0± 5.8f 329.7 ± 5.6f 278.7± 5.1a 282.0± 38.5d 309.3 ± 6.3 31.1± 1.6b 27.8± 3.5c 21.2± 2.4ef 29.8 ± 1.6bc 22.7± 1.6de 24.9± 2.0d 17.8± 2.23 18.1±2.3g 11.0± 1.8h Significant differences at P < 0.05; NC= Negative control, (PC) Obese rats with Diabetes Group (High fat diet); Similar letters in the same column indicate non-significant differences; BWG% = Body Weight Gain %. Biostatistics Studies The data analysis was carried out with SPSS Inc. software (version 15.0). One-way ANOVA was used to study a significant difference between means of the groups with a significance level of P<0.05 when ANOVA analyses revealed differences among the rat groups, post-hoc analyses identified where the differences existed (Tukey HSD Test). All data are presented as ± Standard Deviation of Means (STDEV). RESULTS Body weight gain of rats Table 3 Mean weight (g) changes in rats treated with normal fat level diets. Feed intake, body weight gain of rat groups were calculated and displayed in (Table 3). Feed mean intake (g) was significantly (P < 0.05) increased in all rat groups compared with NC. Body weight gain % has significantly (P < 0.05) increased in all rat groups compared to NC. With a closer look at Table 3 rats fed on normal fat level (NC) had significantly (P < 0.05) lower mean of feed intake and body weight gain % compared with both HF and control groups (P < 0.05). PC shows most BWG% increase for rats (37.4). The combined group with Mg, Zn, and Cr at high level showing most decrease in BWG% (11.0). Table 4: Effect of diets supplemented with Cr, Mg and Zn on total cholesterol and triacylglycerol in obese rats with diabetes. mg/dl Parameters Groups Cholesterol Triacylglycerol Negative control group (NC) 80.6 ± 3.1g Obese Diabetes group (PC) 165.3± 4.5a 47.4 ± 1.9fgh 82.2± 5.0a d a) ÜB 2 re w -o tfl tfl <5 n 2 = ° (5 200 ^g chromium /(kg bw) 127.3± 5.4e 70.0 ± 6.0e 32.0± 2.8 efg 200-mg magnesium sulphate, 10 mg zinc chloride and 100 ^g chromium / (kg bw) 121.4± 7.3e 71.6± 3.3e 32.8± 3.2df 300-mg magnesium sulphate, 20 mg zinc chloride and 200 ^g chromium / (kg bw) 109.1± 4.4f 69.0± 4.0e 28.8 ± 2.5gh Significant differences at P < 0.05; Similar letters in the same column indicate non-significant differences. more effective than 200 ^g/day. In the current study we used 200 ^g/ Kg.b.w of chromium that reduced blood glucose levels more effectively compared to using 100 ^g Kg.b.w. Similarly, in gestational diabetes 8 ^g/day of chromium was more effective than 4 ^g/day. In contrast, two well-designed studies in the U.S. (Sherman et al 1968; Abraham et al 1992) and two in Finland (Uusitupa 1984; Uusitupa et al 1992) failed to demonstrate any significant benefit of chromium supplementation in patients with diabetes. The latter studies used chromium chloride, which may not be as bioavailable as chromium picolinate. At the present time, benefit from chromium supplementation in rats with diabetes has been conclusively demonstrated. The Institute of Medicine Food and Nutrition Board's DRIs found insufficient evidence to set an estimated average requirement for chromium. An adequate intake was determined based on estimated mean intakes. The adequate intake for adult men >51 years is 30 ^g/day and for women >51 years is 20 ^g/day. However, few serious adverse effects have been associated with excess intake of chromium from food supplements, and therefore a tolerable upper intake level has not been established. A more recent placebo-controlled trial with a formulation of Zn and rabbit prostatic extracts found a significant reduction in HbA1c in subjects randomized to the active treatment arm (Song et al. 1998). However, in that study, those randomized to the active treatment had higher baseline HbA1c levels than those randomized to placebo. From biochemical studies we understand that Zn is crucial for proper thyroid hormone metabolism. A moderate elevation of thyrotropin (TSH) concentrations, which is associated with triiodothyronine (T3) values in or slightly above the upper normal range, is frequently found in obese humans. These alterations seem rather a consequence than a cause of obesity since weight loss leads to a normalization of elevated thyroid hormone levels. In our study, thyroxin hormones were decreased significantly, specifically at higher level of individual supplementation of zinc (20 mg) that was in agreement with Kececi and Keskin (2002) whom supplemented diets of lambs and goats with zinc sulphate adjusted to 250 mg/kg for 12 weeks. They found that the levels of serum total thyroxin (T4) and triiodothyronine (T3) were lower in the lambs and goats of the Zn groups, except in the 4th week, as compared to those in the controls. Conversely, Maxwell and Volpe (2007) reported increase at 4 months, total T3 concentrations in two zinc-deficient female college students (ZD1 and ZD2) that were supplemented with 26.4 mg/day of zinc (as zinc gluconate), while all thyroid hormone concentrations increased in ZD2. The same trend has been obviously reported when minerals are combined at higher levels in rats' diets. Table 8: Effect of diets supplemented with Cr, Mg and Zn on T3, T4 and TSH of obese rats with diabetes Groups Parameters T3 Ng/dl T4 Mg/dl TSH pIu/ml Negative control group (NC) 125.2 ± 3.1g 6.9 ± 0.2h 0.102±0.013' Obese Diabetes Group (PC) 147.8 ± 5.3a 8.9 ± 0.2a 0.169±0.010a Distilled water group (WG) 139.4 ± 1.6b 8.7 ± 0.2ab 0.153±0.007b ° £ T3 ^ CD 4—-- a 200 -mg magnesium sulphate / (kg bw) 134.9 ± 1.6cd 8.4 ± 0.3bc 0.145±0.010bc 300 -mg magnesium sulphate / (kg bw) 133.5± 2.6cde 8.3± 0.1cd 0.140±0.006cd 3 T3 ° oes gas £ ¿= T3 CO TO enr ■iS CO o CD „ ^ .Q CD 10 mg zinc chloride / (kg bw) 135.1 ± 2.8cd 8.1 ± 0.2de 0.127 ± 0.004ef 20 mg zinc chloride / (kg bw) 130.4± 2.8 ef 7.9 ± 0.2ef 0.123± 0.003fg 100 ^g chromium /(kg bw) 137.1 ± 2.5bc 8.3± 0.2cd 0.138± 0.1cd 200 ^g chromium /(kg bw) 131.9 ±3.1def 7.9 ± 0.2ef 0.132 ± 0.1de •SS T3 TO T3 — T3 CD £ W t 200-mg magnesium sulphate, 10 mg zinc chloride and 100 ^g chromium / (kg bw) 132.5 ± 2.9de 7.7± 0.3f 0.115± 0.1gh er On 300-mg magnesium sulphate, 20 mg zinc chloride and 200 ^g chromium / (kg bw) 128.7±1.2f 7.4 ±0.164g 0.111 ±0.005h Significant differences at P < 0.05; Similar letters in the same column indicate non-TSH = thyroid-stimulating hormones 28 ■significant difference; T3 = = Triiodothytronine; T4 = = Thyroxine; REFERENCES 1. 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