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Chromium(III) propionate complex supplementation improves carbohydrate metabolism in insulin-resistance rat model
Food and Chemical Toxicology 48 (2010) 2791–2796
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Chromium(III) propionate complex supplementation improves carbohydrate metabolism in insulin-resistance rat model Ewelina Król, Zbigniew Krejpcio * Department of Human Nutrition and Hygiene, University of Life Sciences, 31 Wojska Polskiego, 60-624 Poznan, Poland
a r t i c l e
i n f o
Article history: Received 11 May 2010 Accepted 7 July 2010
Keywords: Chromium(III) propionate complex Insulin resistance Rat
a b s t r a c t The purpose of this study was to evaluate the antidiabetic potential and safety of the chromium(III) propionate complex (CrProp) in insulin resistance induced by a high-fructose diet in rats. The experiment was carried out on 32 nine-week old male Wistar rats divided into 4 groups of 8 rats each. Animals were fed at libitum: the control diet (AIN-93M), and high-fructose diets (HF) containing various levels of Cr(III) given as CrProp (1 mg Cr kg1 diet (HF) and supplemented with 10 mg Cr kg1 diet (HFCr10), or 50 mg Cr kg1 diet (HFCr50), equal to approx. 0.1, 1 and 5 mg kg1 body mass per day) for 8 weeks. It was found that supplemental CrProp improved carbohydrate metabolism indices (decreasing serum insulin levels and insulin resistance indices HOMA-IR and HOMA-B, while increasing insulin sensitivity index QUICKI). Supplemental CrProp did not affect overall nutritional indices, blood morphology, most of the toxicity indices, blood glucose and lipids levels, while it increased kidney Cr level (HFCr50), normalized decreased liver Cu concentrations, and decreased kidney Fe and Cu levels (HFCr50). Supplemental CrProp administered at 10- and 50-fold doses of the basal dietary Cr level has a significant antidiabetic effect in insulin resistant rats. However, a prolonged treatment with this compound can affect Fe status. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Insulin resistance is the first phase in type 2 diabetes progression that often results in hyperinsulinemia and disruption of glucose and/or lipid metabolism. Prolonged glucose intolerance and hyperglycemia lead to diabetes and its various complications. The prevalence of diabetes morbidity increases dramatically all over the world, therefore implementation of efficient preventive and treatment approaches is advisable. There are several elaborated treatment practices and efficient therapeutic drugs applied in handling diabetes and its complications. Apart from those standard medical interventions, there is a great deal of information that some nutraceuticals given in the form of dietary supplements can improve diabetes treatment (Bartlett and Eperjesi, 2008). One of such agents is trivalent Cr, considered as an essential element. Over 30 years ago Jeejeebhoy et al. (1977) reported that Cr(III) deficiency may contribute to insulin resistance and type 2 diabetes. Some authors (Davies et al., 1997; Morris et al., 1999; Rajpathak et al., 2004) observed that there is a correlation between type 2 diabetes and low serum, hair and toenail Cr levels. As a result some researchers (Martin et al., 2006; Pei et al., 2006; Racek et al., 2006)
* Corresponding author. Address: Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, 31 Wojska Polskiego, 60-624 Poznan, Poland. Fax: +48 618487332. E-mail address: [email protected] (Z. Krejpcio). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.07.008
performed clinical studies showing a significant improvement of glucose tolerance after Cr(III) supplementation in type 2 diabetics. However, there are also other trials that did not confirm a positive effect of Cr(III) supplementation in diabetes (Gunton et al., 2005; Kleefstra et al., 2006). The inconsistence in clinical results still does not allow to draw definite conclusions, thus the American Diabetes Association (ADA) as well as the European Association for the Study of Diabetes (EASD) do not recommend Cr(III) supplementation as a standard treatment of diabetes (American Diabetes Association, 2008; Mann et al., 2004). On the other hand, the pharmaceutical and dietary supplement market is growing fast all over the world, offering various chemical forms of Cr(III) compounds, such as Cr picolinate (CrPic), Cr histidinate (CrHis), Cr nicotinate (CrNic), the Cr D-phenylalanine complex (Cr(D-phe), etc. The most popular Cr(III) compound in supplements is Cr(III) tris-picolinate (CrPic). The beneficial effects of CrPic on glucose and lipid metabolism and its helpful role in the treatment of diabetes have been extensively studied in the last decade, but the mechanisms of its action are not completely understood. Besides, the safety of CrPic and other forms has been a matter of considerable debate over the last 20 years (Speetjens et al., 1999a; Bailey et al., 2006; Hininger et al., 2007; Staniek et al., 2010a). Apart from the widely used CrPic, the Cr(III) propionate cation, [Cr3O(O2CCH2CH3)6(H2O)3]+, known as CrProp or Cr3 (Harton et al., 1997; Vincent, 2000; Sun et al., 2002; Clodfelder et al., 2001, 2004a, 2005; Clodfelder and Vincent, 2005) is of particular interest.
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Clodfelder et al. (2005) reported that Cr3O(O2CCH2CH3)6(H2O)3]+ is absorbed with a very high efficiency of 40–60%, while popular Cr supplements such as CrCl3, Cr(III) nicotinate, or CrPic are absorbed at only 0.5–1.3% of the gavaged dose. The difference in the degree of absorption is readily explained by the stability and solubility of the cation in the physiological milieu. In our last publications (Staniek and Krejpcio, 2009; Staniek et al., 2010a,b) we provided experimental evidence that CrProp has got low genotoxic potential, is not teratogenic, and of low acute toxicity in the rat (the 5th category in the GHS system or the 4th class in the EU classification system). Moreover, CrProp improves carbohydrate and lipid metabolism in healthy Wistar rats (Kuryl et al., 2006). The objective of this study was to evaluate the antidiabetic potential of CrProp given orally at dosages of 10 and 50 mg Cr kg1 diet (equal to 1 and 5 mg Cr kg1 body mass per day for 8 weeks) to the insulin-resistance rat model (rats fed a high-fructose diet). 2. Materials and methods 2.1. Animals and diets The experiment was carried out on nine-weeks old male Wistar rats (n = 32) with an initial body mass of 246 g, purchased in the Licensed Laboratory Animals Breeding Center (Poznan, Poland). Animals were housed under controlled temperature (21 ± 2 °C), humidity (55–60%) and with 12 h/12 h day/night cycle. After 5 days of adaptation period animals were divided into 4 groups of 8 rats each, and kept individually in semi-metabolic cages. Animals were fed ad libitum semi-synthetic standard and high-fructose diets composed, according to the AIN-93M recommendations (Reeves et al., 1993), from casein (20%), sunflower oil (7%), wheat starch (53.2%), sucrose (10%), potato starch (5%), L-cysteine (0.3%), vitamin mix AIN-93M (1%) and mineral mix AIN-93M (3.5%). The highfructose diets were obtained from the basal AIN-93 diet, by replacement of wheat starch with fructose (up to 60%). The source of supplemental Cr(III) was CrProp added to the mineral mix to obtain: 1, 10 and 50 mg Cr kg1 diet. Food intake was monitored daily and body mass gain weekly. The chemical composition of experimental diets are presented in Table 1. 2.2. Test chemicals Cr(III) propionate cation (CrProp) in the form of nitrate salt (chemical formula [Cr3O(O2CCH2CH3)6(H2O)3]+(NO3) was synthesized in the laboratory of Department of Product Ecology, Poznan University of Economics, according to the method described previously by Earnshaw et al. (1966). The contents of elemental Cr was determined by the AAS method (spectrometer AAS-3 with BC correction, Zeiss, Germany). The authenticity was established using the physicochemical characteristics of CrProp as described previously (Wieloch et al., 2007). 2.3. Data collection At the end of experiment, after 16 h fasting, rats were anaesthesized with intraperitoneal thiopental injection and dissected to collect blood from aorta and remove inner organs (liver, kidneys, heart, spleen, pancreas, testes) for appropriate biochemical tests. Organs were washed in saline, weighed and stored at 20 °C until analyzed. All the procedures used in this study were accepted by The Animal Bioethics Committee of Poznan, Poland (Approval # 37/2007).
Table 1 Chemical composition of diets used in experiment (mean ± SD). Ingredient
Energy (MJ 100 g1) Protein (%) Fat (%) Carbohydrates (%) Dry mass (%) Ash (%) Cr (lg g1)
Control (AIN-93M)
High-fructose diets HF
HFCr10
HFCr50
1.87 ± 0.01
1.86 ± 0.01
1.85 ± 0.01
1.87 ± 0.01
17.11 ± 0.33 7.66 ± 0.08 63.68
17.12 ± 0.45 6.87 ± 0.37 70.69
17.31 ± 0.23 6.31 ± 0.01 71.26
17.03 ± 0.26 7.21 ± 0.07 69.92
91.36 ± 0.03 2.91 ± 0.15 1.52 ± 0.17
97.45 ± 0.05 2.77 ± 0.17 1.33 ± 0.82
97.50 ± 0.53 2.62 ± 0.21 11.5 ± 0.19
96.93 ± 0.08 2.77 ± 0.04 54.32 ± 3.21
Abbreviations: C – control group, HF – high-fructose fed group.
2.4. Laboratory analyses 2.4.1. Blood morphology Blood hemoglobin (Hb) level was determined by the Drabkin’s cyanohemoglobin method. Red blood count (RBC) and other blood morphology parameters (hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), platelets (PLT), platelet distribution width (PDW), mean platelet volume (MPV), platelet large-cell ratio (p-LCR), red cell distribution width based on standard deviation (RDW-SD)) was obtained by analytical hematology system CELLDYN-1700 (Abbot Laboratories, 1995). 2.4.2. Blood biochemistry Blood serum indices were determined by the following methods: glucose concentration by the hexokinase method (Sacks et al., 2002), total, LDL, HDL cholesterols and triacylglycerols concentrations by the colorimetric methods (Miki, 1999; Riesen,1998; Shephard and Whiting, 1990) using Olympus AU 560 equipment. Plasma insulin concentration was measured by RIA method using kit specific for rats (Linco Reaserch , St. Charles, MO, USA). Activity of ALT and AST enzymes was measured by the kinetic methods (Schumann and Klauke, 2003), while urea concentration by the kinetic method using urease and glutamine dehydrogenase (Newmann and Price, 1999). The total protein concentration was determined by the colorimetric method using Cu+2 ions (Thomas, 1998), and creatinine concentration was measured by the Jaffe’s kinetic method with picric acid (Newmann and Price, 1999). The efficacy of glucose utilization, insulin resistance and b-cell function were characterized by the homeostasis model assessment (HOMA) indices (Matthews et al., 1985). 3
HOMA IR ¼ ðFasting Glucose½mmol dm 3
Fasting Insulin½mIU dm Þ=22:5HOMA B 3
3
¼ ð20 Fasting Insulin½mIU dm Þ=ðFasting Glucose½mmo dm -3:5Þ: The insulin sensitivity was assessed by the quantitative insulin-sensitivity check index (QUICKI) calculated according to the formula: QUICKI = (1/(log (Fasting Glucose [mmol dm3]) + log (Fasting Insulin [mIU dm3]) (Katz et al., 2000). 2.4.3. Microelements determination Prior to analysis, the rats’ tissues were digested in 65% (w/w) spectra pure HNO3 (Merck) in the Microwave Digestion System (MARS 5, CEM). Thereafter, the concentrations of Fe, Zn and Cu in the mineral solutions were measured by the flame-AAS method (spectrometer AAS-, Zeiss, with BC, Germany), while the content of Cr was determined by the graphite furnace AAS method (spectrometer AA EA 5, with BC, Jenoptic, Germany). The accuracy of quantitative determinations of Zn, Cu, Fe and Cr was assured by simultaneous analysis of the certified reference material (Pig Kidney BCRÒ No. 186, Brussels, fortified with Cr standard). 2.5. Statistical analysis All results are presented as means ± standard deviation. Significance of differences of means were calculated using the one-way ANOVA and Tukey’s test. Means were considered statistically different at p < 0.05. All calculations were made using the STATISTICA (ver. 7.0) program (StatSoft, Inc., Tulsa).
3. Results The effects of feeding high-fructose diets and supplemental CrProp on overall growth indices are presented in Table 2. Neither high-fructose diet nor supplemental CrProp affected most of the growth indices in rats. However, high-fructose diet fed rats had significantly lower (by 12%) relative testes mass, in comparison to the control group. Table 3 shows the effects of high-fructose diet and supplemental CrProp on blood serum carbohydrates and lipid indices. Although experimental factors did not affect serum glucose concentrations in rats, they influenced other biomarkers of glucose utilization, such as insulin concentration, insulin resistance (HOMA-IR), insulin sensitivity (QUICKI), and the b-cell function (HOMA-B) indices. In particular, high-fructose diet significantly increased blood insulin level (by 188% of the control), and increased insulin resistance markers (HOMA-IR by 239%, and HOMA-B by 138%), while decreased b-cell function index (QUICKI by 20%). Feeding rats high-fructose diet was able to induce insulin resis-
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36%). These effects suggest a significant antidiabetic potential of CrProp in rat. Since prolonged supplementation with Cr(III) compounds may cause adverse or toxic effects, in this study several morphological and biochemical blood indices were measured and presented in Tables 4 and 5. Neither high-fructose fed diets nor supplemental CrProp influenced blood morphology and hematology profiles, as well as most of toxicity indices, with the exception of serum ALT that was significantly elevated in the HF group by 26% (but still contained in the in the physiological range for rat). Both high-fructose diet and supplemental CrProp have the potential to affect mineral metabolism, therefore the concentrations of Zn, Fe, Cu and Cr were determined in liver, kidneys and spleen of rats and presented in Table 6. High-fructose fed diet slightly decreased the kidney Fe level, whereas given together with CrProp (10 and 50 mg Cr kg1 diet) significantly decreased its concentration by 17%, in comparison with the control group. High-fructose diet significantly decreased the liver Cu level by 13%, while increased the spleen Cu levels. Supplemental CrProp (50 mg Cr kg1 diet), on the contrary, broad up the liver Cu level to normal, while decreased the kidney and spleen Cu concentrations. The tissular Zn concentrations remained intact in all the experimental groups. High-fructose diet decreased the liver Cr concentrations by 32%, while supplemental CrProp (10 mg Cr kg1 diet) only slightly increased hepatic Cr concentration, while at higher dosages (50 mg kg1 diet), markedly increased its level up to the normal value. Also supplemental CrProp (50 mg Cr kg1 diet) increased the kidney Cr concentration by 188%. Analysis of the tissular Cr concentrations in the critical organs of rats treated with CrProp provides further evidence that CrProp has got low accumulation potential, which is of great importance in case of prolonged application.
Table 2 Effect of high-fructose diet and supplemental CrProp on overall growth indices in rats (mean ± SD). Index
Experimental group
Body mass gain (g) Relative body mass gain (%) Body mass/body length ratio (g cm1) Liver (% b.m.) Kidneys (% b.m.) Spleen (% b.m.) Heart (% b.m.) Testes (% b.m.) Pancreas (% b.m.)
C
HF
HFCr10
HFCr50
132.2 ± 1.6
137.1 ± 18.7
145.3 ± 21.1
145.3 ± 16.2
68.42 ± 6.13
59.31 ± 5.55
67.94 ± 8.81
63.15 ± 6.78
14.39 ± 0.43
14.71 ± 0.89
15.44 ± 0.25
15.13 ± 1.20
3.02 ± 0.26 0.68 ± 0.06 0.18 ± 0.02 0.29 ± 0.02 1.11 ± 0.07b 0.33 ± 0.04
3.30 ± 0.26 0.69 ± 0.04 0.18 ± 0.01 0.29 ± 0.02 0.98 ± 0.08a 0.33 ± 0.05
3.21 ± 0.27 0.72 ± 0.06 0.18 ± 0.02 0.28 ± 0.02 0.93 ± 0.05a 0.32 ± 0.03
3.20 ± 0.22 0.67 ± 0.03 0.18 ± 0.01 0.28 ± 0.02 0.99 ± 0.08ab 0.35 ± 0.07
Abbreviations: C – control group, HF – high-fructose fed group; means in a row with different letters differ significantly (p < 0.05).
tance characteristic for pre-diabetic state, therefore this method may be applied in experimental models of diabetes in animals. Cr(III) ions are postulated as insulin sensitizers. Supplemental CrProp given to insulin resistant rats at dosages of 10 and 50 mg Cr kg1 diet (equals to 1 and 5 mg Cr kg1 body mass/day, for 8 weeks) was able to modulate insulin secretion (decrease by 40% and 46%, respectively), decreased the HOMA-IR index (by 44% and 41%, respectively), as well as increased the QUICKI index by 10% and 18%, respectively. Supplemental CrProp did not affect serum total, HDL and LDL cholesterol levels. High-fructose diet caused hyperactivity of b-cells, while supplemental CrProp normalized their function (decreased the HOMA-B index by 30% and
Table 3 Effect of high-fructose diet and supplemental CrProp on blood serum carbohydrates and lipids indices in rats (mean ± SD). Parameter
Experimental group
Glucose concentration (mmol dm3) Insulin concentration (mIU dm3) HOMA-IR index HOMA-B index (%) QUICKI index Total cholesterol concentration (mmol dm3) HDL cholesterol concentration (mmol dm3) LDL cholesterol concentration (mmol dm3) Triacylglycerols concentration (mmol dm3)
C
HF
HFCr10
HFCr50
6.45 ± 0.61 18.41 ± 6.51a 5.28 ± 1.82a 111.1 ± 35.1a 0.49 ± 0.04c 2.14 ± 0.21 1.42 ± 0.14 0.47 ± 0.05 0.35 ± 0.09a
7.23 ± 0.83 53.06 ± 16.91b 17.91 ± 5.26b 264.3 ± 130.5b 0.39 ± 0.04a 2.48 ± 0.31 1.63 ± 0.21 0.57 ± 0.14 0.57 ± 0.14ab
6.95 ± 0.89 32.03 ± 9.51a 10.13 ± 3.64a 184.7 ± 60.1ab 0.43 ± 0.03ab 2.25 ± 0.21 1.58 ± 0.21 0.52 ± 0.13 0.50 ± 0.15ab
6.34 ± 1.56 28.91 ± 11.33a 10.61 ± 4.83a 167.7 ± 46.4a 0.46 ± 0.05bc 2.43 ± 0.36 1.52 ± 0.26 0.44 ± 0.13 0.67 ± 0.18b
Abbreviations the same as in Table 2.
Table 4 Effect of high-fructose diet and supplemental CrProp on blood morphology and hematology indices in rats (mean ± SD). Blood index
12
Experimental group
3
RBC (10 dm ) HGB (mmol dm3) Hematocrit (%) MCV (1015 dm3) MCH (1015 kg) MCHC (102 kg dm3) WBC (109 dm3) PLT (1012 dm3) PDW (%) MPV (1015 dm3) p-LCR (%) RDW-SD (102 kg dm3) Abbreviations the same as in Table 2.
C
HF
HFCr10
HFCr50
8.43 ± 0.26 14.30 ± 0.42 43.04 ± 1.64 51.49 ± 2.21 17.09 ± 0.79 33.10 ± 0.91 2.95 ± 0.27 928.0 ± 53.4 8.97 ± 0.34 7.92 ± 0.20 10.21 ± 1.64 18.21 ± 1.10
8.70 ± 0.16 14.59 ± 0.31 44.69 ± 1.02 51.63 ± 1.61 16.85 ± 0.51 32.61 ± 0.52 2.98 ± 0.78 887.4 ± 69.5 9.04 ± 0.48 7.92 ± 0.20 10.29 ± 1.30 18.05 ± 0.92
8.85 ± 0.28 14.90 ± 0.55 45.08 ± 1.42 50.93 ± 1.61 16.76 ± 0.62 32.86 ± 0.52 2.92 ± 0.69 962.2 ± 77.0 8.71 ± 0.52 7.74 ± 0.27 9.55 ± 1.81 18.63 ± 1.38
8.89 ± 0.92 14.43 ± 1.47 46.33 ± 1.93 51.59 ± 1.62 16.65 ± 0.75 32.05 ± 1.13 2.73 ± 0.79 939.4 ± 69.3 8.44 ± 0.36 7.55 ± 0.23 8.12 ± 1.37 18.40 ± 1.36
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Table 5 Effect of high-fructose diet and supplemental CrProp on blood toxicity markers in rats (mean ± SD). Blood index
Experimental group
ALT (U dm3) AST (U dm3) Total Protein (102 kg dm3) Creatinine (lmol dm3) Urea (mmol dm3)
C
HF
HFCr10
HFCr50
22.67 ± 3.73a 107.8 ± 13.4 5.85 ± 0.12 35.41 ± 3.54 5.30 ± 0.52
28.71 ± 3.20b 113.3 ± 23.1 5.97 ± 0.22 37.12 ± 3.54 5.09 ± 1.10
21.88 ± 2.75a 106.3 ± 10.4 6.03 ± 0.18 33.64 ± 3.54 5.39 ± 1.04
26.67 ± 3.25ab 123.4 ± 32.2 5.80 ± 0.16 35.42 ± 3.54 5.76 ± 0.62
Abbreviations the same as in Table 2.
Table 6 Effect of high-fructose diet and supplemental CrProp on the tissular zinc, iron, copper, and chromium levels in rats (mean ± SD). Index
Experimental group C
HF
HFCr10
HFCr50
Zn (lg g1 Liver Kidney Spleen
dry mass) 100.4 ± 9.3 81.22 ± 7.32 41.37 ± 4.26
102.9 ± 10.0 82.64 ± 8.75 41.45 ± 7.24
101.3 ± 8.1 82.24 ± 6.86 46.04 ± 3.02
95.6 ± 7.1 76.43 ± 9.90 48.05 ± 4.08
Fe (lg g1 Liver Kidney Spleen
dry mass) 316.1 ± 37.2 367.6 ± 55.6b 2571 ± 638
351.4 ± 50.2 337.2 ± 59.1ab 2398 ± 310
263.6 ± 50.0 305.0 ± 18.2a 2412 ± 356
306.0 ± 53.1 301.4 ± 41.1a 2657 ± 470
Cu (lg g1 Liver Kidney Spleen
dry mass) 22.86 ± 2.51b 39.35 ± 6.84b 8.71 ± 1.81ab
19.87 ± 2.72a 35.46 ± 6.21ab 11.12 ± 2.09b
21.74 ± 1.84ab 38.84 ± 8.12ab 6.49 ± 1.11a
23.34 ± 1.51b 29.51 ± 8.41a 6.20 ± 0.95a
0.61 ± 0.16ab 0.84 ± 0.08a 4.60 ± 1.35
0.83 ± 0.34b 1.90 ± 0.30b 4.80 ± 1.43
Cr (lg g1 dry mass) Liver 0.71 ± 0.18ab Kidney 0.57 ± 0.23a Spleen 5.30 ± 1.22
0.48 ± 0.06a 0.66 ± 0.36a 4.90 ± 1.41
Abbreviations the same as in Table 2.
4. Discussion Insulin resistance is a major factor in pathogenesis of type 2 diabetes. Insulin sensitivity is frequently linked to obesity, however individuals with correct body mass can also suffer from insulin resistance (Banerji et al., 1999). Independently on the body mass, insulin-resistant individuals have greater insulin responses and lower hepatic insulin utilization than insulin-sensitive individuals. In healthy individuals, there is a feedback loop between the insulin-sensitive tissues and the b-cells, with b-cells increasing insulin supply in response to demand by the liver, muscles and adipose tissue (Kahn et al., 2006). In order to maintain normoglycemia, circulating insulin levels have to increase. Failure of this mechanism changes blood glucose level and is the first step in the progression of type 2 diabetes. In humans, type 2 diabetes mellitus develops slowly over a long period of time, and is preceded by insulin resistance. In experimental studies on animals, it is impossible to mimic exactly the same physiological conditions leading to insulin resistance and hyperglycemia, that occur in humans, however it has been proved that feeding rats high-fructose diet can cause hyperinsulinemia followed by hyperglycemia (Yadav et al., 2009). The mechanism responsible for this effect is not fully known. Probably, high levels of circulating triacylglycerols and free fatty acids increase hepatic endogenous glucose production (EGP) caused by increased gluconeogenesis (Rajasekar and Anuradha, 2007), as insulin mediated suppression of hepatic EGP is impaired. In this study, feeding rats high-fructose diet (up to 60%) was able to cause hyperinsulinemia and a slight increase of blood glucose and triacylglyceroles levels, characteristic of the pre-diabetes state. Cr(III) compounds are postulated to improve insulin sensitivity. In this study the antidiabetic potential of CrProp was evaluated
using insulin-resistance rat model (high-fructose diet). It was found that this compound given at dosages of 10 and 50 mg Cr kg1 diet (equals to 1 and 5 mg Cr kg1 body mass/day for 8 weeks) is able to restore insulin sensitivity as normalize the b-cell function, almost to the level of the healthy rats. In literature, there are only few studies where researchers applied similar model for insulin resistance induction. In the experiment performed by Dong et al. (2008) mice fed high-sucrose diet and supplemented with chromium (D-phenylalanine)3 at daily dosages of 45 lg Cr kg1 body mass for 9 weeks, had decreased serum insulin and improved glucose disposal rate in intraperitoneal glucose tolerance test. Similarly, Xu and Guo (2009) found that mice fed high-sucrose diet with Cr(III) enriched Grifola frondosa given in dosages of 4 and 5 mg Cr kg1 body mass per day for 9 weeks had improved glucose tolerance and decreased serum insulin levels, without changes in the overall growth parameters. In the mice genetic model of obesity and insulin resistance, Chen et al. (2009) found that 7 week supplementation with Cr(III)-containing milk powder reduced serum glucose, insulin and triglycerides levels, as well as improved glucose and insulin tolerance. Additionally, such intervention decreased levels of proinflammatory cytokines (CRP, IL-6, TNF-a) in blood and muscles. It is generally accepted that the chemical form of Cr(III) compound is decisive for its biological activity (including bioavailability and stability in the physiological milieu). Most of studies on Cr(III) have been performed using CrPic, a widely available form of Cr(III) used in dietary supplements. Sahin et al. (2007) reported that high-fat fed streptozotocin injected rats supplementated with 80 lg CrPic kg1 body mass per day, had deceased blood glucose, total cholesterol, triacylglycerols levels, FFA as well as increased serum insulin and the composite insulin sensitivity index (CISI). Similar results were obtained by Shinde and Goyal (2003) and Inceli et al. (2007). Many clinical trials evaluated the antidiabetic potential of Cr(III) supplementation (mostly CrPic) in diabetic type 2 patients (insulin resistance or metabolic syndrome), however the results obtained are inconclusive, which suggests that there are still some not fully understood factors determining the efficacy of Cr(III) action. Iqbal et al. (2009) found that CrPic ingested at dosages of 1000 lg per day for 4 months by 63 adults with metabolic syndrome did not affect neither plasma glucose concentration, lipids, body mass nor indices of inflammation process. Cefalu et al. (2010) reported that Cr(III) supplementation was efficient in people who were insulin-resistant and had elevated plasma glucose and HbA1c levels. Wang et al. (2007) suggested that the subject’s phenotype may be responsible for the response to Cr(III) in diabetes states. Concerning the novel and promising form of Cr(III) as CrProp, almost all reports come from the Vincent’s laboratory. Sun et al. (2002) studied the effects of Cr3 (CrProp) on healthy, type 1 diabetic and type 2 diabetic rats. The authors found that 24 week intravenous administration of 20 lg Cr kg1 body mass, significantly lowered fasting plasma total, HDL and LDL, cholesterol, tria-
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cylglyceroles, and insulin concentrations, as well as 2-h plasma insulin levels in Zucker obese rats. Similar results were obtained when CrProp was given to healthy rats, however in case of type 1 diabetes, this compound had little effect on carbohydrates and lipid metabolism. These results were further confirmed by Clodfelder et al. (2005) who used the same complex, but at higher dosages (up to 1000 lg Cr kg1 body mass per day) in healthy and type 2 diabetic rats. Also in this laboratory’s previous study (Kuryl et al., 2006), healthy Wistar rats fed diet supplemented with CrProp (5 mg Cr kg1 diet, 10 weeks) had markedly decreased serum insulin levels (15%), whereas the levels of red blood cell (RBC) glucose transmembrane transport and beta-oxidation of fatty acids in white blood cells (WBCs) were elevated by 9% and 77%, respectively. These effects were accompanied by a slight decrease of the insulin-resistance index. It is known that feeding rats high-fructose diet brings about negative physiological effects in the organism, such as disturbance in carbohydrate, lipid and mineral metabolism, and induce oxidative stress. In this study feeding rats high-fructose diet significantly decreased the hepatic Cu levels, probably due to reduction of Cu(II) to Cu(I), which is poorly available. It is believed that hyperglycemia and diabetes decreases Cr status due to increased urinary Cr excretion. In diabetic rats and mice, greater urinary Cr losses, and increased movement of Cr from blood to the tissues and alternation of its distribution in blood plasma was reported (Clodfelder et al., 2004b; Mita et al., 2005, 2008). Seaborn and Stocker (1989) reported that feeding mice high-fructose diet (50%) for 26 days resulted in the decreased blood Cr level in internal organs (liver, spleen, testis and femur). In this study, feeding rats high-fructose diet also decreased the hepatic Cr, while supplemental CrProp (10 and 50 mg Cr kg1 diet) was able to restore its reserves, and improve insulin sensitivity indices. All these changes provide further evidence that CrProp has got a significant antidiabetic potential in the insulin-resistance rat model. Cr(III) compounds used for dietary supplements, no matter what chemical form is given, always rise safety concerns. CrProp is generally considered as a non-toxic compound (Speetjens et al., 1999b). In our recent study (Staniek et al., 2010b), it was demonstrated that the toxicity potential of CrProp is low, the LD50 index for this compound, when administrated orally to rat, is greater than 2000 mg kg body mass, which places CrProp in the fifth category in the GHS system or the fourth class (‘‘unclassified”) in the EU classification system. Treatment of rats with the CrProp, in contrast to Cr(VI), did not affect significantly the Comet assay results in lymphocytes, which shows that the compound is not genotoxic for rat (Staniek et al., 2010a). Also dietary exposure to CrProp at repeated dosages of 100 mg Cr kg1 diet (7.2 mg Cr kg1 body mass per day) did not produce deleterious effects on pregnancy outcome, maternal and fetal organism but affected maternal and foetal tissue Zn and Cu levels of rat. It is known that Cr(III) and Fe have the same transport protein, thus may compete for binding sites in transferrin. For this reason, the question of Cr–Fe interactions should be addressed, whenever Cr(III) is to be administrated orally. In this study, supplemental CrProp given at dosages of 10 and 50 mg Cr kg1 diet (the higher Cr level is comparable with the dietary Fe level) did not decrease the liver and spleen Fe contents, but the higher Cr dosages decrease the kidney Fe concentrations. Sun et al. (2002) reported that CrProp given at dosage of 20 lg Cr kg1 body mass per day for 22 weeks increased the liver Fe content, while decreased the kidney Fe content of Zucker lean rats. However, such changes were not found in healthy and animal models of type 1 and obese type 2 diabetes. In the other study performed in that laboratory (Clodfelder et al., 2005) supplemental
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CrProp (1000 lg Cr kg1 body mass per day, 24-weeks) also decreased kidney Fe content in a rat model of early stage of type 2 diabetes. Dogukan et al. (2009) observed that high-fat fed streptozotocin injected rats supplemented with Cr(III) histidinate (8.2% elemental Cr) in dosages of 110 lg CrHis kg1 body mass per day (for 10 weeks) had decreased liver and kidney Cu content with parallel increasing Zn levels in both organs. Similar results were previously noticed in other work of this team with regard to young and pregnant rabbits (Sahin et al., 1999). Scibor and Zaporowska (2007) reported that healthy rats given by gavage aqueous solution of CrCl3 in dosages of 0.42 mg Cr kg1 body mass per day for 12 weeks, had increased the kidney Zn contents, without changes in the liver and kidney Fe and Cu contents. In conclusion, supplemental CrProp given orally at dosages of 10 and 50 mg Cr kg1 kg diet (equals to 1 and 5 mg Cr kg1 body mass per day for 8 weeks) to high-fructose fed Wistar rats is able to ameliorate insulin resistance symptoms, without giving toxic effects. However prolonged treatment with this compound can affect Fe status.
Conflict of Interest The authors declare that there are no conflicts of interest.
References American Diabetes Association, 2008. A position statement of the American Diabetes Association. Nutrition recommendations and interventions for diabetes. Diabetes Care 31, 61–78. Banerji, M.A., Faridi, N., Atluri, R., Chaiken, R.L., Lebovitz, H.E., 1999. Body composition, visceral fat, leptin, and insulin resistance in Asian Indian men. Journal of Clinical Endocrinology and Metabolism 84, 137–144. Bartlett, H.E., Eperjesi, F., 2008. Nutritional supplementation for type 2 diabetes: a systematic review. Ophthalmic and Physiological Optic 28, 503–523. Bailey, M., Boohaker, J., Sawyer, R., Behling, J., Rasco, J., Jernigan, J., Hood, R., Vincent, J.B., 2006. Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring. Birth Defects Research 77, 244–249. Cefalu, W.T., Rood, J., Pinsonat, P., Qin, J., Sereda, O., Levitan, L., Anderson, R.A., Zhang, X.H., Martin, J.M., Martin, C.K., Wang, Z.Q., Newcomer, B., 2010. Characterization of the metabolic and physiologic response to chromium supplementation in subjects with type 2 diabetes mellitus. Metabolism 59, 755–762. CELL-DYN 1700 System, 1995. Operations Manual. Abbott Laboratories, USA. Chen, W.Y., Chen, C.J., Liu, C.H., Mao, F.C., 2009. Chromium supplementation enhances insulin signalling in skeletal muscle of obese KK/HlJ diabetic mice. Diabetes Obesity Metabolism 11, 293–303. Clodfelder, B.J., Chang, C., Vincent, J.B., 2004a. Absorption of the biomimetic chromium cation triaqua-l-oxo-hexapropionatotrichromium(III) in rats. Biological Trace Element Research 98, 159–169. Clodfelder, B.J., Emamaullee, J., Hepburn, D.D., Chakov, N.E., Nettles, H.S., Vincent, J.B., 2001. The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin and chromodulin. Journal of Biological Inorganic Chemistry 6, 608–617. Clodfelder, B.J., Gullick, B.M., Lukaski, H.C., Neggers, Y., Vincent, J.B., 2005. Oral administration of the biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic Chemistry 10, 119–130. Clodfelder, B.J., Upchurch, R.G., Vincent, J.B., 2004b. A comparison of the insulinsensitive transport of chromium in healthy and model diabetic rats. Journal of Inorganic Biochemistry 98, 522–533. Clodfelder, B.J., Vincent, J.B., 2005. The time-dependent transport of chromium in adult rats from the bloodstream to the urine. Journal of Biological Inorganic Chemistry 10, 383–393. Davies, S., Howard, J.M., Hinnisett, A., Howard, M., 1997. Age-related decreases in chromium levels in 51, 665 hair, sweat, and serum samples from 40, 872 patients: Implications for the prevention of cardiovascular disease and type II diabetes mellitus. Metabolism 46, 469–473. Dogukan, A., Sahin, N., Tuzcu, M., Juturu, V., Orhan, C., Onderci, M., Komorowski, J., Sahin, K., 2009. The effects of chromium histidinate on mineral status of serum and tissue in fat-fed and streptozotocin-treated type II diabetic rats. Biological Trace Element Research 131, 124–132. Dong, F., Kandadi, M.R., Ren, J., Sreejayan, N., 2008. Chromium (D-phenylalanine)3 supplementation alters glucose disposal, insulin signaling, and glucose transporter-4 membrane translocation in insulin-resistant mice. Journal of Nutrition 138, 1846–1851.
2796
E. Król, Z. Krejpcio / Food and Chemical Toxicology 48 (2010) 2791–2796
Earnshaw, A., Figgis, B.N., Lewis, J., 1966. Chemistry of polynuclear compounds. Part VI. Magnetic properties of trimeric chromium and iron carboxylates. Journal of Chemical Society A, 1656–1663. Gunton, J.E., Cheung, N.W., Hitchman, R., Hams, G., O‘Sullivan, C., Foster-Powell, K., O‘Sullivan, C., McElduff, A., 2005. Chromium supplementation does not improve glucose tolerance, insulin sensitivity, or lipid profile. Diabetes Care 28, 712–714. Harton, A., Terrell, K., Huffman, J.C., MacDonald, C., Beatty, A., Li, S., O’Connor, C.J., Vincent, J.B., 1997. Synthesis and characterization of novel oxo-bridged dinuclear and hydroxo-bridged trinuclear chromium(III) assemblies. Inorganic Chemistry 36, 4875–4882. Hininger, I., Benaraba, R., Osman, M., Faure, H., Roussel, A.-M., Anderson, R.A., 2007. Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes. Free Radical Biology and Medicine 15, 1759–1765. Inceli, M.S., Bolkent, S., Doger, M.M., Yanardag, R., 2007. The effects of combined treatment with niacin and chromium on the renal tissues of hyperlipidemic rats. Molecular and Cellular Biochemistry 294, 37–44. Iqbal, N., Cardillo, S., Volger, S., Bloedon, L.T., Anderson, R.A., Boston, R., Szapary, P.O., 2009. Chromium picolinate does not improve key features of metabolic syndrome in obese nondiabetic adults. Metabolic Syndrome and Related Disorders 7, 143–150. Jeejeebhoy, K.N., Chu, R.C., Marliss, E.B., Greenberg, G.R., Bruce-Robertson, A., 1977. Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation in a patient receiving long-term total parenteral nutrition. American Journal of Clinical Nutrition 30, 531–538. Kahn, S., Hull, R., Utzschneider, K.M., 2006. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444, 840–846. Katz, A., Nambi, S.S., Mather, K., Baron, A.D., Follmann, D.A., Sullivan, G., Quon, M.J., 2000. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. Journal of Clinical Endocrinology and Metabolism 85, 2402–2410. Kleefstra, N., Houweling, S., Jansman, F.G.A., Groenier, K.H., Gans, R.O.B., Mayboomde Jong, B., Bakker, S.J.L., Bilo, H.J.G., 2006. Chromium treatment has no effect in patients with poorly controlled, insulin-treated type 2 diabetes in an obese Western population. Diabetes Care 29, 321–325. Kuryl, T., Krejpcio, Z., Wójciak, R.W., Lipko, M., Debski, B., Staniek, H., 2006. Chromium(III) propionate and dietary fructans supplementation stimulate erythrocyte glucose uptake and beta-oxidation in lymphocytes of rats. Biological Trace Element Research 114, 237–248. Mann, J.I., De Leeuw, I., Hermansen, K., Karamanos, B., Karlström, B., Katsilambros, N., Riccardi, G., Rivellese, A.A., Rizkalla, S., Slama, G., Toeller, M., Uusitupa, M., Vessby, B., 2004. Diabetes and Nutrition Study Group (DNSG) of the European Association for the Study of Diabetes (EASD), Evidence-based nutritional approaches to the treatment and prevention of diabetes mellitus. Nutrition Metabolism and Cardiovascular Diseases 14, 373–394. Martin, J., Zhang, X.H., Zhong, Q., Wang, X.H., Wachtel, D., Volaufova, J., Matthews, D.E., Cefalu, W.T., 2006. Chromium picolinate supplementation attenuates body weight gain and increases insulin sensitivity in subjects with type 2 diabetes. Diabetes Care 29, 1826–1832. Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, D.F., Turner, R.C., 1985. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412–419. Miki, Y., 1999. A homogenous assay for the selective measurement of LDLcholesterol in serum. Enzymatic selective protection method. Clinical Laboratory 45, 398–401. Mita, Y., Ishihara, K., Fukuchi, Y., Fukuya, Y., Yasumoto, K., 2005. Supplementation with chromium picolinate recovers renal Cr concentration and improves carbohydrate metabolism and renal function in type 2 diabetic mice. Biological Trace Element Research 105, 229–248. Mita, Y., Ishihara, K., Ishiguru, M., Takeda, M., Hattori, R., Murakami, K., Yamada, A., Yasumoto, K., 2008. Elevated urinary Cr loss induces a reduction in renal Cr concentration and the negative Cr balance in streptozotocin-induced diabetic mice. Journal of Nutritional Science and Vitaminology 54, 303–308. Morris, B.W., MacNeil, S., Hardisty, C.A., Heller, S., Burgin, C., Gray, T.A., 1999. Chromium homeostasis in patients with type II (NIDDM) diabetes. Journal of Trace Elements in Medicine and Biology 13, 57–61. Newmann, D.J., Price, C.P., 1999. Renal function and nitrogen metabolites. In: Burtis, C.A., Ashwood, E.R. (Eds.), Tietz Book of Clinical Chemistry. WB Saunders Company, Philadelphia, pp. 1239–1242. Pei, D., Hsieh, C.H., Hung, Y.J., Li, J.C., Lee, C.H., Kuo, S.W., 2006. The influence of chromium chloride-containing milk to glycemic control of patients with type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Metabolism 55, 923–927. Racek, J., Trefil, L., Rajdl, D., Mudrova, V., Hunter, D., Senft, V., 2006. Influence of chromium-enriched yeast on blood glucose and insulin variables, blood lipids, and markers of oxidative stress in subjects with type 2 diabetes mellitus. Biological Trace Element Research 109, 215–230. Rajasekar, P., Anuradha, C.V., 2007. Fructose induced gluconeogenesis: effect of lcarnitine. Life Sciences 80, 1176–1183.
Rajpathak, S., Rimm, E.B., Li, T., Morris, J.S., Stampfer, M.J., Willet, W.C., Hu, F.B., 2004. Lower toenail chromium in men with diabetes and cardiovascular disease compared with healthy men. Diabetes Care 27, 2211–2216. Reeves, P.G., Nielsen, F.H., Fahey Jr., G.C., 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. Journal of Nutrition 123, 1939–1951. Riesen, W.F., 1998. Lipid metabolism. In: Thomas, L. (Ed.), Clinical Laboratory Diagnostics. Use and Assessment of Clinical Laboratory Results. TH-Books Verlagssesellschaft, Frankfurt Main, pp. 167–169. Sacks, D.B., Bruns, D.E., Goldstein, D.E., Maclaren, N.K., McDonald, J.M., Parrott, M., 2002. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clinical Chemistry 48, 436– 472. Sahin, K., Gülar, T., Sahin, N., Ertas, O.N., 1999. The effect of chromium added into basal diet on serum total protein, urea, triglyceride, cholesterol and serum and tissue chromium, zinc, copper levels in rabbits. Turkish Journal of Veterinary and Animal Sciences 23, 113–119. Sahin, K., Onderci, M., Tuzcu, M., Ustundag, B., Cikim, G., Ozercan, I.H., Sriramoju, V., Juturu, V., Komorowski, J.R., 2007. Effect of chromium on carbohydrate and lipid metabolism in a rat model of type 2 diabetes mellitus: the fat-fed, streptozotocin-treated rat. Metabolism Clinical and Experimental 56, 1233– 1240. Schumann, G., Klauke, R., 2003. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clinica Chimica Acta 327, 69–79. Seaborn, C.D., Stocker, B.J., 1989. Effects of starch, sucrose, fructose and glucose on chromium absorption and tissue concentrations in obese and lean mice. Journal of Nutrition 119, 1444–1451. Shephard, M.D., Whiting, M.J., 1990. Falsely low estimation of triglycerides in lipemic plasma by the enzymatic triglyceride method with modified Trinder’s chromogen. Clinical Chemistry 36, 325–329. Shinde, U.A., Goyal, R.K., 2003. Effects of chromium picolinate on histopatological alternations in STZ and neonatal STZ diabetic rats. Journal of Cellular and Molecular Medicine 7, 322–329. Speetjens, J.K., Collins, R.A., Vincent, J.B., Woski, S.A., 1999a. The nutritional supplement chromium(III) tris(picolinate) cleaves DNA. Chemical Research in Toxicology 12, 483–487. Speetjens, J.K., Parand, A., Crowder, M.W., Vincent, J.B., 1999b. Low-molecularweight chromium-binding substance and biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ do not cleave DNA under physiologically relevant conditions. Polyhedron 18, 2617–2624. Staniek, H., Krejpcio, Z., 2009. The effects of tricentric chromium(III) propionate complex supplementation on pregnancy outcome and maternal and foetal mineral status in rat. Food and Chemical Toxicology 47, 2673–2678. Staniek, H., Kostrzewska-Poczekaj, M., Arndt, M., Szyfter, K., Krejpcio, Z., 2010a. Genotoxicity assessment of chromium(III) propionate complex in the rat model using the comet assay. Food and Chemical Toxicology 48, 89–92. Staniek, H., Krejpcio, Z., Iwanik, K., 2010b. Evaluation of the acute oral toxicity class of tricentric chromium(III) propionate complex in rat. Food and Chemical Toxicology 48, 859–864. Sun, Y., Clodfelder, B.J., Shute, A.A., Irvin, T., Vincent, J.B., 2002. The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglicerides in healthy and type II diabetic rats but not type I diabetic rats. Journal of Biological Inorganic Chemistry 7, 852–862. Scibor, A., Zaporowska, H., 2007. Effects of vanadium(V) and/or chromium(III) on L-ascorbic acid and glutathione as well as iron, zinc, and copper levels in rat liver and kidney. Journal of Toxicology and Environmental Health. Part A 70, 696–704. Thomas, L., 1998. Total protein. In: Thomas, L. (Ed.), Clinical Laboratory Diagnostics. Use and Assessment of Clinical Laboartory Results. TH-Books Verlagsgesellschaft, FrankfuntMain, pp. 644–647. Xu, Q., Guo, J., 2009. Activity and toxicity of Cr(III)-enriched Grifola frondosa in insulin-resistant mice. Biological Trace Element Research 131, 271–277. Yadav, H., Jain, S., Yadav, M., Sinha, P.R., Prasad, G.B., Marotta, F., 2009. Epigenomic derangement of hepatic glucose metabolism by feeding of high-fructose diet and its prevention by Rosiglitazone in rats. Digestive and Liver Disease 41, 500– 508. Vincent, J.B., 2000. Elucidating a biological role for chromium at a molecular level. Accounts of Chemical Research 37, 503–510. Wang, Z.Q., Qin, J., Martin, J., Zhang, X.H., Sereda, O., Anderson, R.A., Pinsonatm, P., Cefalu, W.T., 2007. Phenotype of subjects with type 2 diabetes mellitus may determine clinical response to chromium supplementation. Metabolism Clinical and Experimental 56, 1652–1655. Wieloch, A., Wieczorek, D., Staniek, H., Szymusiak, H., Krejpcio, Z., Zielin´ski, R., 2007. Tricentric complexes of chromium(III) with glycine and serie ligands. Current Trends in Commodity Science II 1080, 1086.
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Chromium(III) propionate complex supplementation improves carbohydrate metabolism in insulin-resistance rat model Ewelina Król, Zbigniew Krejpcio * Department of Human Nutrition and Hygiene, University of Life Sciences, 31 Wojska Polskiego, 60-624 Poznan, Poland
a r t i c l e
i n f o
Article history: Received 11 May 2010 Accepted 7 July 2010
Keywords: Chromium(III) propionate complex Insulin resistance Rat
a b s t r a c t The purpose of this study was to evaluate the antidiabetic potential and safety of the chromium(III) propionate complex (CrProp) in insulin resistance induced by a high-fructose diet in rats. The experiment was carried out on 32 nine-week old male Wistar rats divided into 4 groups of 8 rats each. Animals were fed at libitum: the control diet (AIN-93M), and high-fructose diets (HF) containing various levels of Cr(III) given as CrProp (1 mg Cr kg1 diet (HF) and supplemented with 10 mg Cr kg1 diet (HFCr10), or 50 mg Cr kg1 diet (HFCr50), equal to approx. 0.1, 1 and 5 mg kg1 body mass per day) for 8 weeks. It was found that supplemental CrProp improved carbohydrate metabolism indices (decreasing serum insulin levels and insulin resistance indices HOMA-IR and HOMA-B, while increasing insulin sensitivity index QUICKI). Supplemental CrProp did not affect overall nutritional indices, blood morphology, most of the toxicity indices, blood glucose and lipids levels, while it increased kidney Cr level (HFCr50), normalized decreased liver Cu concentrations, and decreased kidney Fe and Cu levels (HFCr50). Supplemental CrProp administered at 10- and 50-fold doses of the basal dietary Cr level has a significant antidiabetic effect in insulin resistant rats. However, a prolonged treatment with this compound can affect Fe status. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Insulin resistance is the first phase in type 2 diabetes progression that often results in hyperinsulinemia and disruption of glucose and/or lipid metabolism. Prolonged glucose intolerance and hyperglycemia lead to diabetes and its various complications. The prevalence of diabetes morbidity increases dramatically all over the world, therefore implementation of efficient preventive and treatment approaches is advisable. There are several elaborated treatment practices and efficient therapeutic drugs applied in handling diabetes and its complications. Apart from those standard medical interventions, there is a great deal of information that some nutraceuticals given in the form of dietary supplements can improve diabetes treatment (Bartlett and Eperjesi, 2008). One of such agents is trivalent Cr, considered as an essential element. Over 30 years ago Jeejeebhoy et al. (1977) reported that Cr(III) deficiency may contribute to insulin resistance and type 2 diabetes. Some authors (Davies et al., 1997; Morris et al., 1999; Rajpathak et al., 2004) observed that there is a correlation between type 2 diabetes and low serum, hair and toenail Cr levels. As a result some researchers (Martin et al., 2006; Pei et al., 2006; Racek et al., 2006)
* Corresponding author. Address: Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, 31 Wojska Polskiego, 60-624 Poznan, Poland. Fax: +48 618487332. E-mail address: [email protected] (Z. Krejpcio). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.07.008
performed clinical studies showing a significant improvement of glucose tolerance after Cr(III) supplementation in type 2 diabetics. However, there are also other trials that did not confirm a positive effect of Cr(III) supplementation in diabetes (Gunton et al., 2005; Kleefstra et al., 2006). The inconsistence in clinical results still does not allow to draw definite conclusions, thus the American Diabetes Association (ADA) as well as the European Association for the Study of Diabetes (EASD) do not recommend Cr(III) supplementation as a standard treatment of diabetes (American Diabetes Association, 2008; Mann et al., 2004). On the other hand, the pharmaceutical and dietary supplement market is growing fast all over the world, offering various chemical forms of Cr(III) compounds, such as Cr picolinate (CrPic), Cr histidinate (CrHis), Cr nicotinate (CrNic), the Cr D-phenylalanine complex (Cr(D-phe), etc. The most popular Cr(III) compound in supplements is Cr(III) tris-picolinate (CrPic). The beneficial effects of CrPic on glucose and lipid metabolism and its helpful role in the treatment of diabetes have been extensively studied in the last decade, but the mechanisms of its action are not completely understood. Besides, the safety of CrPic and other forms has been a matter of considerable debate over the last 20 years (Speetjens et al., 1999a; Bailey et al., 2006; Hininger et al., 2007; Staniek et al., 2010a). Apart from the widely used CrPic, the Cr(III) propionate cation, [Cr3O(O2CCH2CH3)6(H2O)3]+, known as CrProp or Cr3 (Harton et al., 1997; Vincent, 2000; Sun et al., 2002; Clodfelder et al., 2001, 2004a, 2005; Clodfelder and Vincent, 2005) is of particular interest.
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Clodfelder et al. (2005) reported that Cr3O(O2CCH2CH3)6(H2O)3]+ is absorbed with a very high efficiency of 40–60%, while popular Cr supplements such as CrCl3, Cr(III) nicotinate, or CrPic are absorbed at only 0.5–1.3% of the gavaged dose. The difference in the degree of absorption is readily explained by the stability and solubility of the cation in the physiological milieu. In our last publications (Staniek and Krejpcio, 2009; Staniek et al., 2010a,b) we provided experimental evidence that CrProp has got low genotoxic potential, is not teratogenic, and of low acute toxicity in the rat (the 5th category in the GHS system or the 4th class in the EU classification system). Moreover, CrProp improves carbohydrate and lipid metabolism in healthy Wistar rats (Kuryl et al., 2006). The objective of this study was to evaluate the antidiabetic potential of CrProp given orally at dosages of 10 and 50 mg Cr kg1 diet (equal to 1 and 5 mg Cr kg1 body mass per day for 8 weeks) to the insulin-resistance rat model (rats fed a high-fructose diet). 2. Materials and methods 2.1. Animals and diets The experiment was carried out on nine-weeks old male Wistar rats (n = 32) with an initial body mass of 246 g, purchased in the Licensed Laboratory Animals Breeding Center (Poznan, Poland). Animals were housed under controlled temperature (21 ± 2 °C), humidity (55–60%) and with 12 h/12 h day/night cycle. After 5 days of adaptation period animals were divided into 4 groups of 8 rats each, and kept individually in semi-metabolic cages. Animals were fed ad libitum semi-synthetic standard and high-fructose diets composed, according to the AIN-93M recommendations (Reeves et al., 1993), from casein (20%), sunflower oil (7%), wheat starch (53.2%), sucrose (10%), potato starch (5%), L-cysteine (0.3%), vitamin mix AIN-93M (1%) and mineral mix AIN-93M (3.5%). The highfructose diets were obtained from the basal AIN-93 diet, by replacement of wheat starch with fructose (up to 60%). The source of supplemental Cr(III) was CrProp added to the mineral mix to obtain: 1, 10 and 50 mg Cr kg1 diet. Food intake was monitored daily and body mass gain weekly. The chemical composition of experimental diets are presented in Table 1. 2.2. Test chemicals Cr(III) propionate cation (CrProp) in the form of nitrate salt (chemical formula [Cr3O(O2CCH2CH3)6(H2O)3]+(NO3) was synthesized in the laboratory of Department of Product Ecology, Poznan University of Economics, according to the method described previously by Earnshaw et al. (1966). The contents of elemental Cr was determined by the AAS method (spectrometer AAS-3 with BC correction, Zeiss, Germany). The authenticity was established using the physicochemical characteristics of CrProp as described previously (Wieloch et al., 2007). 2.3. Data collection At the end of experiment, after 16 h fasting, rats were anaesthesized with intraperitoneal thiopental injection and dissected to collect blood from aorta and remove inner organs (liver, kidneys, heart, spleen, pancreas, testes) for appropriate biochemical tests. Organs were washed in saline, weighed and stored at 20 °C until analyzed. All the procedures used in this study were accepted by The Animal Bioethics Committee of Poznan, Poland (Approval # 37/2007).
Table 1 Chemical composition of diets used in experiment (mean ± SD). Ingredient
Energy (MJ 100 g1) Protein (%) Fat (%) Carbohydrates (%) Dry mass (%) Ash (%) Cr (lg g1)
Control (AIN-93M)
High-fructose diets HF
HFCr10
HFCr50
1.87 ± 0.01
1.86 ± 0.01
1.85 ± 0.01
1.87 ± 0.01
17.11 ± 0.33 7.66 ± 0.08 63.68
17.12 ± 0.45 6.87 ± 0.37 70.69
17.31 ± 0.23 6.31 ± 0.01 71.26
17.03 ± 0.26 7.21 ± 0.07 69.92
91.36 ± 0.03 2.91 ± 0.15 1.52 ± 0.17
97.45 ± 0.05 2.77 ± 0.17 1.33 ± 0.82
97.50 ± 0.53 2.62 ± 0.21 11.5 ± 0.19
96.93 ± 0.08 2.77 ± 0.04 54.32 ± 3.21
Abbreviations: C – control group, HF – high-fructose fed group.
2.4. Laboratory analyses 2.4.1. Blood morphology Blood hemoglobin (Hb) level was determined by the Drabkin’s cyanohemoglobin method. Red blood count (RBC) and other blood morphology parameters (hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), platelets (PLT), platelet distribution width (PDW), mean platelet volume (MPV), platelet large-cell ratio (p-LCR), red cell distribution width based on standard deviation (RDW-SD)) was obtained by analytical hematology system CELLDYN-1700 (Abbot Laboratories, 1995). 2.4.2. Blood biochemistry Blood serum indices were determined by the following methods: glucose concentration by the hexokinase method (Sacks et al., 2002), total, LDL, HDL cholesterols and triacylglycerols concentrations by the colorimetric methods (Miki, 1999; Riesen,1998; Shephard and Whiting, 1990) using Olympus AU 560 equipment. Plasma insulin concentration was measured by RIA method using kit specific for rats (Linco Reaserch , St. Charles, MO, USA). Activity of ALT and AST enzymes was measured by the kinetic methods (Schumann and Klauke, 2003), while urea concentration by the kinetic method using urease and glutamine dehydrogenase (Newmann and Price, 1999). The total protein concentration was determined by the colorimetric method using Cu+2 ions (Thomas, 1998), and creatinine concentration was measured by the Jaffe’s kinetic method with picric acid (Newmann and Price, 1999). The efficacy of glucose utilization, insulin resistance and b-cell function were characterized by the homeostasis model assessment (HOMA) indices (Matthews et al., 1985). 3
HOMA IR ¼ ðFasting Glucose½mmol dm 3
Fasting Insulin½mIU dm Þ=22:5HOMA B 3
3
¼ ð20 Fasting Insulin½mIU dm Þ=ðFasting Glucose½mmo dm -3:5Þ: The insulin sensitivity was assessed by the quantitative insulin-sensitivity check index (QUICKI) calculated according to the formula: QUICKI = (1/(log (Fasting Glucose [mmol dm3]) + log (Fasting Insulin [mIU dm3]) (Katz et al., 2000). 2.4.3. Microelements determination Prior to analysis, the rats’ tissues were digested in 65% (w/w) spectra pure HNO3 (Merck) in the Microwave Digestion System (MARS 5, CEM). Thereafter, the concentrations of Fe, Zn and Cu in the mineral solutions were measured by the flame-AAS method (spectrometer AAS-, Zeiss, with BC, Germany), while the content of Cr was determined by the graphite furnace AAS method (spectrometer AA EA 5, with BC, Jenoptic, Germany). The accuracy of quantitative determinations of Zn, Cu, Fe and Cr was assured by simultaneous analysis of the certified reference material (Pig Kidney BCRÒ No. 186, Brussels, fortified with Cr standard). 2.5. Statistical analysis All results are presented as means ± standard deviation. Significance of differences of means were calculated using the one-way ANOVA and Tukey’s test. Means were considered statistically different at p < 0.05. All calculations were made using the STATISTICA (ver. 7.0) program (StatSoft, Inc., Tulsa).
3. Results The effects of feeding high-fructose diets and supplemental CrProp on overall growth indices are presented in Table 2. Neither high-fructose diet nor supplemental CrProp affected most of the growth indices in rats. However, high-fructose diet fed rats had significantly lower (by 12%) relative testes mass, in comparison to the control group. Table 3 shows the effects of high-fructose diet and supplemental CrProp on blood serum carbohydrates and lipid indices. Although experimental factors did not affect serum glucose concentrations in rats, they influenced other biomarkers of glucose utilization, such as insulin concentration, insulin resistance (HOMA-IR), insulin sensitivity (QUICKI), and the b-cell function (HOMA-B) indices. In particular, high-fructose diet significantly increased blood insulin level (by 188% of the control), and increased insulin resistance markers (HOMA-IR by 239%, and HOMA-B by 138%), while decreased b-cell function index (QUICKI by 20%). Feeding rats high-fructose diet was able to induce insulin resis-
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36%). These effects suggest a significant antidiabetic potential of CrProp in rat. Since prolonged supplementation with Cr(III) compounds may cause adverse or toxic effects, in this study several morphological and biochemical blood indices were measured and presented in Tables 4 and 5. Neither high-fructose fed diets nor supplemental CrProp influenced blood morphology and hematology profiles, as well as most of toxicity indices, with the exception of serum ALT that was significantly elevated in the HF group by 26% (but still contained in the in the physiological range for rat). Both high-fructose diet and supplemental CrProp have the potential to affect mineral metabolism, therefore the concentrations of Zn, Fe, Cu and Cr were determined in liver, kidneys and spleen of rats and presented in Table 6. High-fructose fed diet slightly decreased the kidney Fe level, whereas given together with CrProp (10 and 50 mg Cr kg1 diet) significantly decreased its concentration by 17%, in comparison with the control group. High-fructose diet significantly decreased the liver Cu level by 13%, while increased the spleen Cu levels. Supplemental CrProp (50 mg Cr kg1 diet), on the contrary, broad up the liver Cu level to normal, while decreased the kidney and spleen Cu concentrations. The tissular Zn concentrations remained intact in all the experimental groups. High-fructose diet decreased the liver Cr concentrations by 32%, while supplemental CrProp (10 mg Cr kg1 diet) only slightly increased hepatic Cr concentration, while at higher dosages (50 mg kg1 diet), markedly increased its level up to the normal value. Also supplemental CrProp (50 mg Cr kg1 diet) increased the kidney Cr concentration by 188%. Analysis of the tissular Cr concentrations in the critical organs of rats treated with CrProp provides further evidence that CrProp has got low accumulation potential, which is of great importance in case of prolonged application.
Table 2 Effect of high-fructose diet and supplemental CrProp on overall growth indices in rats (mean ± SD). Index
Experimental group
Body mass gain (g) Relative body mass gain (%) Body mass/body length ratio (g cm1) Liver (% b.m.) Kidneys (% b.m.) Spleen (% b.m.) Heart (% b.m.) Testes (% b.m.) Pancreas (% b.m.)
C
HF
HFCr10
HFCr50
132.2 ± 1.6
137.1 ± 18.7
145.3 ± 21.1
145.3 ± 16.2
68.42 ± 6.13
59.31 ± 5.55
67.94 ± 8.81
63.15 ± 6.78
14.39 ± 0.43
14.71 ± 0.89
15.44 ± 0.25
15.13 ± 1.20
3.02 ± 0.26 0.68 ± 0.06 0.18 ± 0.02 0.29 ± 0.02 1.11 ± 0.07b 0.33 ± 0.04
3.30 ± 0.26 0.69 ± 0.04 0.18 ± 0.01 0.29 ± 0.02 0.98 ± 0.08a 0.33 ± 0.05
3.21 ± 0.27 0.72 ± 0.06 0.18 ± 0.02 0.28 ± 0.02 0.93 ± 0.05a 0.32 ± 0.03
3.20 ± 0.22 0.67 ± 0.03 0.18 ± 0.01 0.28 ± 0.02 0.99 ± 0.08ab 0.35 ± 0.07
Abbreviations: C – control group, HF – high-fructose fed group; means in a row with different letters differ significantly (p < 0.05).
tance characteristic for pre-diabetic state, therefore this method may be applied in experimental models of diabetes in animals. Cr(III) ions are postulated as insulin sensitizers. Supplemental CrProp given to insulin resistant rats at dosages of 10 and 50 mg Cr kg1 diet (equals to 1 and 5 mg Cr kg1 body mass/day, for 8 weeks) was able to modulate insulin secretion (decrease by 40% and 46%, respectively), decreased the HOMA-IR index (by 44% and 41%, respectively), as well as increased the QUICKI index by 10% and 18%, respectively. Supplemental CrProp did not affect serum total, HDL and LDL cholesterol levels. High-fructose diet caused hyperactivity of b-cells, while supplemental CrProp normalized their function (decreased the HOMA-B index by 30% and
Table 3 Effect of high-fructose diet and supplemental CrProp on blood serum carbohydrates and lipids indices in rats (mean ± SD). Parameter
Experimental group
Glucose concentration (mmol dm3) Insulin concentration (mIU dm3) HOMA-IR index HOMA-B index (%) QUICKI index Total cholesterol concentration (mmol dm3) HDL cholesterol concentration (mmol dm3) LDL cholesterol concentration (mmol dm3) Triacylglycerols concentration (mmol dm3)
C
HF
HFCr10
HFCr50
6.45 ± 0.61 18.41 ± 6.51a 5.28 ± 1.82a 111.1 ± 35.1a 0.49 ± 0.04c 2.14 ± 0.21 1.42 ± 0.14 0.47 ± 0.05 0.35 ± 0.09a
7.23 ± 0.83 53.06 ± 16.91b 17.91 ± 5.26b 264.3 ± 130.5b 0.39 ± 0.04a 2.48 ± 0.31 1.63 ± 0.21 0.57 ± 0.14 0.57 ± 0.14ab
6.95 ± 0.89 32.03 ± 9.51a 10.13 ± 3.64a 184.7 ± 60.1ab 0.43 ± 0.03ab 2.25 ± 0.21 1.58 ± 0.21 0.52 ± 0.13 0.50 ± 0.15ab
6.34 ± 1.56 28.91 ± 11.33a 10.61 ± 4.83a 167.7 ± 46.4a 0.46 ± 0.05bc 2.43 ± 0.36 1.52 ± 0.26 0.44 ± 0.13 0.67 ± 0.18b
Abbreviations the same as in Table 2.
Table 4 Effect of high-fructose diet and supplemental CrProp on blood morphology and hematology indices in rats (mean ± SD). Blood index
12
Experimental group
3
RBC (10 dm ) HGB (mmol dm3) Hematocrit (%) MCV (1015 dm3) MCH (1015 kg) MCHC (102 kg dm3) WBC (109 dm3) PLT (1012 dm3) PDW (%) MPV (1015 dm3) p-LCR (%) RDW-SD (102 kg dm3) Abbreviations the same as in Table 2.
C
HF
HFCr10
HFCr50
8.43 ± 0.26 14.30 ± 0.42 43.04 ± 1.64 51.49 ± 2.21 17.09 ± 0.79 33.10 ± 0.91 2.95 ± 0.27 928.0 ± 53.4 8.97 ± 0.34 7.92 ± 0.20 10.21 ± 1.64 18.21 ± 1.10
8.70 ± 0.16 14.59 ± 0.31 44.69 ± 1.02 51.63 ± 1.61 16.85 ± 0.51 32.61 ± 0.52 2.98 ± 0.78 887.4 ± 69.5 9.04 ± 0.48 7.92 ± 0.20 10.29 ± 1.30 18.05 ± 0.92
8.85 ± 0.28 14.90 ± 0.55 45.08 ± 1.42 50.93 ± 1.61 16.76 ± 0.62 32.86 ± 0.52 2.92 ± 0.69 962.2 ± 77.0 8.71 ± 0.52 7.74 ± 0.27 9.55 ± 1.81 18.63 ± 1.38
8.89 ± 0.92 14.43 ± 1.47 46.33 ± 1.93 51.59 ± 1.62 16.65 ± 0.75 32.05 ± 1.13 2.73 ± 0.79 939.4 ± 69.3 8.44 ± 0.36 7.55 ± 0.23 8.12 ± 1.37 18.40 ± 1.36
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Table 5 Effect of high-fructose diet and supplemental CrProp on blood toxicity markers in rats (mean ± SD). Blood index
Experimental group
ALT (U dm3) AST (U dm3) Total Protein (102 kg dm3) Creatinine (lmol dm3) Urea (mmol dm3)
C
HF
HFCr10
HFCr50
22.67 ± 3.73a 107.8 ± 13.4 5.85 ± 0.12 35.41 ± 3.54 5.30 ± 0.52
28.71 ± 3.20b 113.3 ± 23.1 5.97 ± 0.22 37.12 ± 3.54 5.09 ± 1.10
21.88 ± 2.75a 106.3 ± 10.4 6.03 ± 0.18 33.64 ± 3.54 5.39 ± 1.04
26.67 ± 3.25ab 123.4 ± 32.2 5.80 ± 0.16 35.42 ± 3.54 5.76 ± 0.62
Abbreviations the same as in Table 2.
Table 6 Effect of high-fructose diet and supplemental CrProp on the tissular zinc, iron, copper, and chromium levels in rats (mean ± SD). Index
Experimental group C
HF
HFCr10
HFCr50
Zn (lg g1 Liver Kidney Spleen
dry mass) 100.4 ± 9.3 81.22 ± 7.32 41.37 ± 4.26
102.9 ± 10.0 82.64 ± 8.75 41.45 ± 7.24
101.3 ± 8.1 82.24 ± 6.86 46.04 ± 3.02
95.6 ± 7.1 76.43 ± 9.90 48.05 ± 4.08
Fe (lg g1 Liver Kidney Spleen
dry mass) 316.1 ± 37.2 367.6 ± 55.6b 2571 ± 638
351.4 ± 50.2 337.2 ± 59.1ab 2398 ± 310
263.6 ± 50.0 305.0 ± 18.2a 2412 ± 356
306.0 ± 53.1 301.4 ± 41.1a 2657 ± 470
Cu (lg g1 Liver Kidney Spleen
dry mass) 22.86 ± 2.51b 39.35 ± 6.84b 8.71 ± 1.81ab
19.87 ± 2.72a 35.46 ± 6.21ab 11.12 ± 2.09b
21.74 ± 1.84ab 38.84 ± 8.12ab 6.49 ± 1.11a
23.34 ± 1.51b 29.51 ± 8.41a 6.20 ± 0.95a
0.61 ± 0.16ab 0.84 ± 0.08a 4.60 ± 1.35
0.83 ± 0.34b 1.90 ± 0.30b 4.80 ± 1.43
Cr (lg g1 dry mass) Liver 0.71 ± 0.18ab Kidney 0.57 ± 0.23a Spleen 5.30 ± 1.22
0.48 ± 0.06a 0.66 ± 0.36a 4.90 ± 1.41
Abbreviations the same as in Table 2.
4. Discussion Insulin resistance is a major factor in pathogenesis of type 2 diabetes. Insulin sensitivity is frequently linked to obesity, however individuals with correct body mass can also suffer from insulin resistance (Banerji et al., 1999). Independently on the body mass, insulin-resistant individuals have greater insulin responses and lower hepatic insulin utilization than insulin-sensitive individuals. In healthy individuals, there is a feedback loop between the insulin-sensitive tissues and the b-cells, with b-cells increasing insulin supply in response to demand by the liver, muscles and adipose tissue (Kahn et al., 2006). In order to maintain normoglycemia, circulating insulin levels have to increase. Failure of this mechanism changes blood glucose level and is the first step in the progression of type 2 diabetes. In humans, type 2 diabetes mellitus develops slowly over a long period of time, and is preceded by insulin resistance. In experimental studies on animals, it is impossible to mimic exactly the same physiological conditions leading to insulin resistance and hyperglycemia, that occur in humans, however it has been proved that feeding rats high-fructose diet can cause hyperinsulinemia followed by hyperglycemia (Yadav et al., 2009). The mechanism responsible for this effect is not fully known. Probably, high levels of circulating triacylglycerols and free fatty acids increase hepatic endogenous glucose production (EGP) caused by increased gluconeogenesis (Rajasekar and Anuradha, 2007), as insulin mediated suppression of hepatic EGP is impaired. In this study, feeding rats high-fructose diet (up to 60%) was able to cause hyperinsulinemia and a slight increase of blood glucose and triacylglyceroles levels, characteristic of the pre-diabetes state. Cr(III) compounds are postulated to improve insulin sensitivity. In this study the antidiabetic potential of CrProp was evaluated
using insulin-resistance rat model (high-fructose diet). It was found that this compound given at dosages of 10 and 50 mg Cr kg1 diet (equals to 1 and 5 mg Cr kg1 body mass/day for 8 weeks) is able to restore insulin sensitivity as normalize the b-cell function, almost to the level of the healthy rats. In literature, there are only few studies where researchers applied similar model for insulin resistance induction. In the experiment performed by Dong et al. (2008) mice fed high-sucrose diet and supplemented with chromium (D-phenylalanine)3 at daily dosages of 45 lg Cr kg1 body mass for 9 weeks, had decreased serum insulin and improved glucose disposal rate in intraperitoneal glucose tolerance test. Similarly, Xu and Guo (2009) found that mice fed high-sucrose diet with Cr(III) enriched Grifola frondosa given in dosages of 4 and 5 mg Cr kg1 body mass per day for 9 weeks had improved glucose tolerance and decreased serum insulin levels, without changes in the overall growth parameters. In the mice genetic model of obesity and insulin resistance, Chen et al. (2009) found that 7 week supplementation with Cr(III)-containing milk powder reduced serum glucose, insulin and triglycerides levels, as well as improved glucose and insulin tolerance. Additionally, such intervention decreased levels of proinflammatory cytokines (CRP, IL-6, TNF-a) in blood and muscles. It is generally accepted that the chemical form of Cr(III) compound is decisive for its biological activity (including bioavailability and stability in the physiological milieu). Most of studies on Cr(III) have been performed using CrPic, a widely available form of Cr(III) used in dietary supplements. Sahin et al. (2007) reported that high-fat fed streptozotocin injected rats supplementated with 80 lg CrPic kg1 body mass per day, had deceased blood glucose, total cholesterol, triacylglycerols levels, FFA as well as increased serum insulin and the composite insulin sensitivity index (CISI). Similar results were obtained by Shinde and Goyal (2003) and Inceli et al. (2007). Many clinical trials evaluated the antidiabetic potential of Cr(III) supplementation (mostly CrPic) in diabetic type 2 patients (insulin resistance or metabolic syndrome), however the results obtained are inconclusive, which suggests that there are still some not fully understood factors determining the efficacy of Cr(III) action. Iqbal et al. (2009) found that CrPic ingested at dosages of 1000 lg per day for 4 months by 63 adults with metabolic syndrome did not affect neither plasma glucose concentration, lipids, body mass nor indices of inflammation process. Cefalu et al. (2010) reported that Cr(III) supplementation was efficient in people who were insulin-resistant and had elevated plasma glucose and HbA1c levels. Wang et al. (2007) suggested that the subject’s phenotype may be responsible for the response to Cr(III) in diabetes states. Concerning the novel and promising form of Cr(III) as CrProp, almost all reports come from the Vincent’s laboratory. Sun et al. (2002) studied the effects of Cr3 (CrProp) on healthy, type 1 diabetic and type 2 diabetic rats. The authors found that 24 week intravenous administration of 20 lg Cr kg1 body mass, significantly lowered fasting plasma total, HDL and LDL, cholesterol, tria-
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cylglyceroles, and insulin concentrations, as well as 2-h plasma insulin levels in Zucker obese rats. Similar results were obtained when CrProp was given to healthy rats, however in case of type 1 diabetes, this compound had little effect on carbohydrates and lipid metabolism. These results were further confirmed by Clodfelder et al. (2005) who used the same complex, but at higher dosages (up to 1000 lg Cr kg1 body mass per day) in healthy and type 2 diabetic rats. Also in this laboratory’s previous study (Kuryl et al., 2006), healthy Wistar rats fed diet supplemented with CrProp (5 mg Cr kg1 diet, 10 weeks) had markedly decreased serum insulin levels (15%), whereas the levels of red blood cell (RBC) glucose transmembrane transport and beta-oxidation of fatty acids in white blood cells (WBCs) were elevated by 9% and 77%, respectively. These effects were accompanied by a slight decrease of the insulin-resistance index. It is known that feeding rats high-fructose diet brings about negative physiological effects in the organism, such as disturbance in carbohydrate, lipid and mineral metabolism, and induce oxidative stress. In this study feeding rats high-fructose diet significantly decreased the hepatic Cu levels, probably due to reduction of Cu(II) to Cu(I), which is poorly available. It is believed that hyperglycemia and diabetes decreases Cr status due to increased urinary Cr excretion. In diabetic rats and mice, greater urinary Cr losses, and increased movement of Cr from blood to the tissues and alternation of its distribution in blood plasma was reported (Clodfelder et al., 2004b; Mita et al., 2005, 2008). Seaborn and Stocker (1989) reported that feeding mice high-fructose diet (50%) for 26 days resulted in the decreased blood Cr level in internal organs (liver, spleen, testis and femur). In this study, feeding rats high-fructose diet also decreased the hepatic Cr, while supplemental CrProp (10 and 50 mg Cr kg1 diet) was able to restore its reserves, and improve insulin sensitivity indices. All these changes provide further evidence that CrProp has got a significant antidiabetic potential in the insulin-resistance rat model. Cr(III) compounds used for dietary supplements, no matter what chemical form is given, always rise safety concerns. CrProp is generally considered as a non-toxic compound (Speetjens et al., 1999b). In our recent study (Staniek et al., 2010b), it was demonstrated that the toxicity potential of CrProp is low, the LD50 index for this compound, when administrated orally to rat, is greater than 2000 mg kg body mass, which places CrProp in the fifth category in the GHS system or the fourth class (‘‘unclassified”) in the EU classification system. Treatment of rats with the CrProp, in contrast to Cr(VI), did not affect significantly the Comet assay results in lymphocytes, which shows that the compound is not genotoxic for rat (Staniek et al., 2010a). Also dietary exposure to CrProp at repeated dosages of 100 mg Cr kg1 diet (7.2 mg Cr kg1 body mass per day) did not produce deleterious effects on pregnancy outcome, maternal and fetal organism but affected maternal and foetal tissue Zn and Cu levels of rat. It is known that Cr(III) and Fe have the same transport protein, thus may compete for binding sites in transferrin. For this reason, the question of Cr–Fe interactions should be addressed, whenever Cr(III) is to be administrated orally. In this study, supplemental CrProp given at dosages of 10 and 50 mg Cr kg1 diet (the higher Cr level is comparable with the dietary Fe level) did not decrease the liver and spleen Fe contents, but the higher Cr dosages decrease the kidney Fe concentrations. Sun et al. (2002) reported that CrProp given at dosage of 20 lg Cr kg1 body mass per day for 22 weeks increased the liver Fe content, while decreased the kidney Fe content of Zucker lean rats. However, such changes were not found in healthy and animal models of type 1 and obese type 2 diabetes. In the other study performed in that laboratory (Clodfelder et al., 2005) supplemental
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CrProp (1000 lg Cr kg1 body mass per day, 24-weeks) also decreased kidney Fe content in a rat model of early stage of type 2 diabetes. Dogukan et al. (2009) observed that high-fat fed streptozotocin injected rats supplemented with Cr(III) histidinate (8.2% elemental Cr) in dosages of 110 lg CrHis kg1 body mass per day (for 10 weeks) had decreased liver and kidney Cu content with parallel increasing Zn levels in both organs. Similar results were previously noticed in other work of this team with regard to young and pregnant rabbits (Sahin et al., 1999). Scibor and Zaporowska (2007) reported that healthy rats given by gavage aqueous solution of CrCl3 in dosages of 0.42 mg Cr kg1 body mass per day for 12 weeks, had increased the kidney Zn contents, without changes in the liver and kidney Fe and Cu contents. In conclusion, supplemental CrProp given orally at dosages of 10 and 50 mg Cr kg1 kg diet (equals to 1 and 5 mg Cr kg1 body mass per day for 8 weeks) to high-fructose fed Wistar rats is able to ameliorate insulin resistance symptoms, without giving toxic effects. However prolonged treatment with this compound can affect Fe status.
Conflict of Interest The authors declare that there are no conflicts of interest.
References American Diabetes Association, 2008. A position statement of the American Diabetes Association. Nutrition recommendations and interventions for diabetes. Diabetes Care 31, 61–78. Banerji, M.A., Faridi, N., Atluri, R., Chaiken, R.L., Lebovitz, H.E., 1999. Body composition, visceral fat, leptin, and insulin resistance in Asian Indian men. Journal of Clinical Endocrinology and Metabolism 84, 137–144. Bartlett, H.E., Eperjesi, F., 2008. Nutritional supplementation for type 2 diabetes: a systematic review. Ophthalmic and Physiological Optic 28, 503–523. Bailey, M., Boohaker, J., Sawyer, R., Behling, J., Rasco, J., Jernigan, J., Hood, R., Vincent, J.B., 2006. Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring. Birth Defects Research 77, 244–249. Cefalu, W.T., Rood, J., Pinsonat, P., Qin, J., Sereda, O., Levitan, L., Anderson, R.A., Zhang, X.H., Martin, J.M., Martin, C.K., Wang, Z.Q., Newcomer, B., 2010. Characterization of the metabolic and physiologic response to chromium supplementation in subjects with type 2 diabetes mellitus. Metabolism 59, 755–762. CELL-DYN 1700 System, 1995. Operations Manual. Abbott Laboratories, USA. Chen, W.Y., Chen, C.J., Liu, C.H., Mao, F.C., 2009. Chromium supplementation enhances insulin signalling in skeletal muscle of obese KK/HlJ diabetic mice. Diabetes Obesity Metabolism 11, 293–303. Clodfelder, B.J., Chang, C., Vincent, J.B., 2004a. Absorption of the biomimetic chromium cation triaqua-l-oxo-hexapropionatotrichromium(III) in rats. Biological Trace Element Research 98, 159–169. Clodfelder, B.J., Emamaullee, J., Hepburn, D.D., Chakov, N.E., Nettles, H.S., Vincent, J.B., 2001. The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin and chromodulin. Journal of Biological Inorganic Chemistry 6, 608–617. Clodfelder, B.J., Gullick, B.M., Lukaski, H.C., Neggers, Y., Vincent, J.B., 2005. Oral administration of the biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic Chemistry 10, 119–130. Clodfelder, B.J., Upchurch, R.G., Vincent, J.B., 2004b. A comparison of the insulinsensitive transport of chromium in healthy and model diabetic rats. Journal of Inorganic Biochemistry 98, 522–533. Clodfelder, B.J., Vincent, J.B., 2005. The time-dependent transport of chromium in adult rats from the bloodstream to the urine. Journal of Biological Inorganic Chemistry 10, 383–393. Davies, S., Howard, J.M., Hinnisett, A., Howard, M., 1997. Age-related decreases in chromium levels in 51, 665 hair, sweat, and serum samples from 40, 872 patients: Implications for the prevention of cardiovascular disease and type II diabetes mellitus. Metabolism 46, 469–473. Dogukan, A., Sahin, N., Tuzcu, M., Juturu, V., Orhan, C., Onderci, M., Komorowski, J., Sahin, K., 2009. The effects of chromium histidinate on mineral status of serum and tissue in fat-fed and streptozotocin-treated type II diabetic rats. Biological Trace Element Research 131, 124–132. Dong, F., Kandadi, M.R., Ren, J., Sreejayan, N., 2008. Chromium (D-phenylalanine)3 supplementation alters glucose disposal, insulin signaling, and glucose transporter-4 membrane translocation in insulin-resistant mice. Journal of Nutrition 138, 1846–1851.
2796
E. Król, Z. Krejpcio / Food and Chemical Toxicology 48 (2010) 2791–2796
Earnshaw, A., Figgis, B.N., Lewis, J., 1966. Chemistry of polynuclear compounds. Part VI. Magnetic properties of trimeric chromium and iron carboxylates. Journal of Chemical Society A, 1656–1663. Gunton, J.E., Cheung, N.W., Hitchman, R., Hams, G., O‘Sullivan, C., Foster-Powell, K., O‘Sullivan, C., McElduff, A., 2005. Chromium supplementation does not improve glucose tolerance, insulin sensitivity, or lipid profile. Diabetes Care 28, 712–714. Harton, A., Terrell, K., Huffman, J.C., MacDonald, C., Beatty, A., Li, S., O’Connor, C.J., Vincent, J.B., 1997. Synthesis and characterization of novel oxo-bridged dinuclear and hydroxo-bridged trinuclear chromium(III) assemblies. Inorganic Chemistry 36, 4875–4882. Hininger, I., Benaraba, R., Osman, M., Faure, H., Roussel, A.-M., Anderson, R.A., 2007. Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes. Free Radical Biology and Medicine 15, 1759–1765. Inceli, M.S., Bolkent, S., Doger, M.M., Yanardag, R., 2007. The effects of combined treatment with niacin and chromium on the renal tissues of hyperlipidemic rats. Molecular and Cellular Biochemistry 294, 37–44. Iqbal, N., Cardillo, S., Volger, S., Bloedon, L.T., Anderson, R.A., Boston, R., Szapary, P.O., 2009. Chromium picolinate does not improve key features of metabolic syndrome in obese nondiabetic adults. Metabolic Syndrome and Related Disorders 7, 143–150. Jeejeebhoy, K.N., Chu, R.C., Marliss, E.B., Greenberg, G.R., Bruce-Robertson, A., 1977. Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation in a patient receiving long-term total parenteral nutrition. American Journal of Clinical Nutrition 30, 531–538. Kahn, S., Hull, R., Utzschneider, K.M., 2006. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444, 840–846. Katz, A., Nambi, S.S., Mather, K., Baron, A.D., Follmann, D.A., Sullivan, G., Quon, M.J., 2000. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. Journal of Clinical Endocrinology and Metabolism 85, 2402–2410. Kleefstra, N., Houweling, S., Jansman, F.G.A., Groenier, K.H., Gans, R.O.B., Mayboomde Jong, B., Bakker, S.J.L., Bilo, H.J.G., 2006. Chromium treatment has no effect in patients with poorly controlled, insulin-treated type 2 diabetes in an obese Western population. Diabetes Care 29, 321–325. Kuryl, T., Krejpcio, Z., Wójciak, R.W., Lipko, M., Debski, B., Staniek, H., 2006. Chromium(III) propionate and dietary fructans supplementation stimulate erythrocyte glucose uptake and beta-oxidation in lymphocytes of rats. Biological Trace Element Research 114, 237–248. Mann, J.I., De Leeuw, I., Hermansen, K., Karamanos, B., Karlström, B., Katsilambros, N., Riccardi, G., Rivellese, A.A., Rizkalla, S., Slama, G., Toeller, M., Uusitupa, M., Vessby, B., 2004. Diabetes and Nutrition Study Group (DNSG) of the European Association for the Study of Diabetes (EASD), Evidence-based nutritional approaches to the treatment and prevention of diabetes mellitus. Nutrition Metabolism and Cardiovascular Diseases 14, 373–394. Martin, J., Zhang, X.H., Zhong, Q., Wang, X.H., Wachtel, D., Volaufova, J., Matthews, D.E., Cefalu, W.T., 2006. Chromium picolinate supplementation attenuates body weight gain and increases insulin sensitivity in subjects with type 2 diabetes. Diabetes Care 29, 1826–1832. Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, D.F., Turner, R.C., 1985. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412–419. Miki, Y., 1999. A homogenous assay for the selective measurement of LDLcholesterol in serum. Enzymatic selective protection method. Clinical Laboratory 45, 398–401. Mita, Y., Ishihara, K., Fukuchi, Y., Fukuya, Y., Yasumoto, K., 2005. Supplementation with chromium picolinate recovers renal Cr concentration and improves carbohydrate metabolism and renal function in type 2 diabetic mice. Biological Trace Element Research 105, 229–248. Mita, Y., Ishihara, K., Ishiguru, M., Takeda, M., Hattori, R., Murakami, K., Yamada, A., Yasumoto, K., 2008. Elevated urinary Cr loss induces a reduction in renal Cr concentration and the negative Cr balance in streptozotocin-induced diabetic mice. Journal of Nutritional Science and Vitaminology 54, 303–308. Morris, B.W., MacNeil, S., Hardisty, C.A., Heller, S., Burgin, C., Gray, T.A., 1999. Chromium homeostasis in patients with type II (NIDDM) diabetes. Journal of Trace Elements in Medicine and Biology 13, 57–61. Newmann, D.J., Price, C.P., 1999. Renal function and nitrogen metabolites. In: Burtis, C.A., Ashwood, E.R. (Eds.), Tietz Book of Clinical Chemistry. WB Saunders Company, Philadelphia, pp. 1239–1242. Pei, D., Hsieh, C.H., Hung, Y.J., Li, J.C., Lee, C.H., Kuo, S.W., 2006. The influence of chromium chloride-containing milk to glycemic control of patients with type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Metabolism 55, 923–927. Racek, J., Trefil, L., Rajdl, D., Mudrova, V., Hunter, D., Senft, V., 2006. Influence of chromium-enriched yeast on blood glucose and insulin variables, blood lipids, and markers of oxidative stress in subjects with type 2 diabetes mellitus. Biological Trace Element Research 109, 215–230. Rajasekar, P., Anuradha, C.V., 2007. Fructose induced gluconeogenesis: effect of lcarnitine. Life Sciences 80, 1176–1183.
Rajpathak, S., Rimm, E.B., Li, T., Morris, J.S., Stampfer, M.J., Willet, W.C., Hu, F.B., 2004. Lower toenail chromium in men with diabetes and cardiovascular disease compared with healthy men. Diabetes Care 27, 2211–2216. Reeves, P.G., Nielsen, F.H., Fahey Jr., G.C., 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. Journal of Nutrition 123, 1939–1951. Riesen, W.F., 1998. Lipid metabolism. In: Thomas, L. (Ed.), Clinical Laboratory Diagnostics. Use and Assessment of Clinical Laboratory Results. TH-Books Verlagssesellschaft, Frankfurt Main, pp. 167–169. Sacks, D.B., Bruns, D.E., Goldstein, D.E., Maclaren, N.K., McDonald, J.M., Parrott, M., 2002. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clinical Chemistry 48, 436– 472. Sahin, K., Gülar, T., Sahin, N., Ertas, O.N., 1999. The effect of chromium added into basal diet on serum total protein, urea, triglyceride, cholesterol and serum and tissue chromium, zinc, copper levels in rabbits. Turkish Journal of Veterinary and Animal Sciences 23, 113–119. Sahin, K., Onderci, M., Tuzcu, M., Ustundag, B., Cikim, G., Ozercan, I.H., Sriramoju, V., Juturu, V., Komorowski, J.R., 2007. Effect of chromium on carbohydrate and lipid metabolism in a rat model of type 2 diabetes mellitus: the fat-fed, streptozotocin-treated rat. Metabolism Clinical and Experimental 56, 1233– 1240. Schumann, G., Klauke, R., 2003. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clinica Chimica Acta 327, 69–79. Seaborn, C.D., Stocker, B.J., 1989. Effects of starch, sucrose, fructose and glucose on chromium absorption and tissue concentrations in obese and lean mice. Journal of Nutrition 119, 1444–1451. Shephard, M.D., Whiting, M.J., 1990. Falsely low estimation of triglycerides in lipemic plasma by the enzymatic triglyceride method with modified Trinder’s chromogen. Clinical Chemistry 36, 325–329. Shinde, U.A., Goyal, R.K., 2003. Effects of chromium picolinate on histopatological alternations in STZ and neonatal STZ diabetic rats. Journal of Cellular and Molecular Medicine 7, 322–329. Speetjens, J.K., Collins, R.A., Vincent, J.B., Woski, S.A., 1999a. The nutritional supplement chromium(III) tris(picolinate) cleaves DNA. Chemical Research in Toxicology 12, 483–487. Speetjens, J.K., Parand, A., Crowder, M.W., Vincent, J.B., 1999b. Low-molecularweight chromium-binding substance and biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ do not cleave DNA under physiologically relevant conditions. Polyhedron 18, 2617–2624. Staniek, H., Krejpcio, Z., 2009. The effects of tricentric chromium(III) propionate complex supplementation on pregnancy outcome and maternal and foetal mineral status in rat. Food and Chemical Toxicology 47, 2673–2678. Staniek, H., Kostrzewska-Poczekaj, M., Arndt, M., Szyfter, K., Krejpcio, Z., 2010a. Genotoxicity assessment of chromium(III) propionate complex in the rat model using the comet assay. Food and Chemical Toxicology 48, 89–92. Staniek, H., Krejpcio, Z., Iwanik, K., 2010b. Evaluation of the acute oral toxicity class of tricentric chromium(III) propionate complex in rat. Food and Chemical Toxicology 48, 859–864. Sun, Y., Clodfelder, B.J., Shute, A.A., Irvin, T., Vincent, J.B., 2002. The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglicerides in healthy and type II diabetic rats but not type I diabetic rats. Journal of Biological Inorganic Chemistry 7, 852–862. Scibor, A., Zaporowska, H., 2007. Effects of vanadium(V) and/or chromium(III) on L-ascorbic acid and glutathione as well as iron, zinc, and copper levels in rat liver and kidney. Journal of Toxicology and Environmental Health. Part A 70, 696–704. Thomas, L., 1998. Total protein. In: Thomas, L. (Ed.), Clinical Laboratory Diagnostics. Use and Assessment of Clinical Laboartory Results. TH-Books Verlagsgesellschaft, FrankfuntMain, pp. 644–647. Xu, Q., Guo, J., 2009. Activity and toxicity of Cr(III)-enriched Grifola frondosa in insulin-resistant mice. Biological Trace Element Research 131, 271–277. Yadav, H., Jain, S., Yadav, M., Sinha, P.R., Prasad, G.B., Marotta, F., 2009. Epigenomic derangement of hepatic glucose metabolism by feeding of high-fructose diet and its prevention by Rosiglitazone in rats. Digestive and Liver Disease 41, 500– 508. Vincent, J.B., 2000. Elucidating a biological role for chromium at a molecular level. Accounts of Chemical Research 37, 503–510. Wang, Z.Q., Qin, J., Martin, J., Zhang, X.H., Sereda, O., Anderson, R.A., Pinsonatm, P., Cefalu, W.T., 2007. Phenotype of subjects with type 2 diabetes mellitus may determine clinical response to chromium supplementation. Metabolism Clinical and Experimental 56, 1652–1655. Wieloch, A., Wieczorek, D., Staniek, H., Szymusiak, H., Krejpcio, Z., Zielin´ski, R., 2007. Tricentric complexes of chromium(III) with glycine and serie ligands. Current Trends in Commodity Science II 1080, 1086.