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Effects of various forms of dietary chromium on growth and body composition in the rat
Nutrition Research. Vol. 17. No. 2. pp. 283-294.1997 Copyright 8 1997 Elsevier Science Inc. Rimed in the USA. AlI rights reserved 0271-5317/97 $17.00+ .a0
ELSEVIER
PII SO271-5317(96)00258-S
EFFECTS OF VARIOUS FORMS OF DIETARY CHROMIUM ON GROWTH AND BODY COMPOSITION IN THE RAT Deborah L. Hasten, Ph.D.‘, Maren Hegsted, Ph.D.‘, Michael J. Keenan, Ph.D.‘, and G. Stephen Morris, Ph.D.” Washington University School of Medicine, St. Louis, MO 63110; Departments of Human Ecology* and Kinesiology”, Louisiana State University, Baton Rouge, LA 70803
ABSTRACT
Several human and animal studies of chromium (0) supplementation have reported increases in lean body mass, often with concurrent decreases in body fat. Since the majority of studies have tested Cr in the form of Cr picolinate (CrPic), this 12-week study contrasted the effects of three available forms of dietary Cr (300 ppb Cr as CrPic, Cr nicotinate (CrNic), or Cr chloride (CrCI)}, as well as no added dietary Cr, upon growth and body composition in the rat. Sixty male Harlan Sprague-Dawley weanling rats arrived in three groups of 20 animals (referred to as Blocks 1, 2, and 3). Five animals from each block were assigned to each treatment, resulting in a 4 X 3 (treatment X block) Randomized Block Design. Body composition was assessed at Weeks 5 and 10 via dual energy X-ray absorptiometry (DEXA). Significant treatment X block interactions were found for fat weight and percent body fat at both time points (P I 0.05). These interactions reflected decreases in body fat with all forms of Cr supplementation for the Block 1 animals. No treatment interactions or effects were seen for growth rate, lean body mass, or tissue weights, but most block effects were highly significant (P I 0.011, reflecting possible genetic differences between blocks. Since feed intake did not vary between dietary treatments, the reduced body fat levels for the Cr-supplemented animals of Block 1 may have been due to an enhancement of insulin-induced Copyright 8 1997 Elsevier Science Inc. thermogenesis. Key Words:
Rat, Chromium,
Growth,
Body
Composition
‘Address Correspondence to: Dr. Deborah L. Hasten, Washington of Medicine, 660 S. Euclid, Box 8127, St. Louis, MO 631 IO
283
University
School
284
D.L. HASTEN et al.
INTRODUCTION
Various forms of trivalent chromium (0) have been shown to potentiate the cellular actions of insulin (I-5). Insulin has been shown to increase glucose and amino acid uptake into muscle cells (61, activate ribosomal translational activity (61, and enhance the actions of growth hormone (7). Additionally, insulin inhibits enzymes that catabolize amino acids and protein (8). Chromium may potentiate the cellular and subcellular actions of insulin, leading to changes in muscle mass. In support of this suggestion, both human and animal studies have indicated that Cr supplementation enhances the development of lean body mass (9-l 5). Most Cr supplementation studies that have reported increases in lean body mass have used Cr in the form of Cr picolinate (CrPic) (9-l 3,151. Two human studies that used Cr as Cr nicotinate (CrNic) failed to observe any changes in lean body mass (8,161. However, oral ingestion of CrNic has been shown to increase the body weight of rats as compared to the Cr-deficient state (17). Although several studies of Cr as Cr chloride (CrCI) have observed increased growth rates (3,4,14,18), only one (14) observed a concurrent increase in lean body mass. Chromium supplementation has also been found to reduce body fat levels in humans (9.13) and animals (15,191. It has been hypothesized that Cr may achieve this by promoting hypothalamic insulin-mediated activation of the sympathetic nervous system, subsequently increasing dietary-induced thermogenesis 120,21 I. In addition to promoting thermogenesis, centrally administered insulin has been found to suppress food intake in the rat (22). Hence, Cr supplementation may decrease body fat levels by increasing energy expenditure or by decreasing caloric intake. It appears that Cr supplementation is capable of altering body composition, but the ability of different forms of Cr to do this may vary. Since most of the body composition studies of Cr have utilized the mineral in the form of CrPic, and only a limited number of studies of CrNic and of CrCl have been reported, further work needs to be conducted before any final conclusions can be made. The current study examined the effects of Cr in the forms of CrPic, CrNic, and CrCl on growth and body composition in the rat. Since previous studies of Cr supplementation have reported body composition changes after both 6 and 12 weeks (g-151, we assessed body composition at two time intervals (5 and 10 weeks) before the completion of this study at 12 weeks.
METHODS
AND MATERIALS
Animals and Dietarv Treatments: Sixty male Harlan Sprague-Dawley outbred weanling rats (21 days of age) were used in this study. The rats were received in three groups of 20, arriving at two-week intervals. These three groups of animals were referred
DIETARY CHROMIUM AND BODY COMPOSITION
285
to as Blocks 1, 2, and 3. The animals were divided into four treatment groups of 15 (five rats each from Blocks 1, 2, and 3) at 25 days of age. Although the initial body weights (BW) of the animals did not differ by treatment, there were significant block differences for initial BW (p = 0.011, with Block 2 having a greater mean weight than Blocks 1 and 3. A follow-up call to representatives at Harlan Sprague-Dawley, Inc. revealed that the Block 1 animals had originated from a breeding colony in Houston, TX (Building 21 I), whereas Blocks 2 and 3 had come from a facility in Prattville, AL (Building 218). The animals were kept in stainless steel housing with wire mesh flooring. Diets were placed in glass containers, and the animals had free access to food and ultrapure water. The study was carried out for 12 weeks, and the animals were 112 to 116 days of age at sacrifice. The basal diet consumed by all animals in this study was the AIN-76A semi-purified diet without the addition of Cr. Animals received one of the following diets: I) basal; or basal with the addition of 300 fig Cr/kg diet (hereafter referred to as parts per billion, ppb) in the forms of 2) CrPic; 3) CrNic; and 4) CrCI. Basal diet samples were analyzed for Cr content by Dr. Richard A. Anderson of the U.S.D.A. Beltsville Human Nutrition Resource Center (Beltsville, MD). The Cr content of eight random samples taken from different basal diet batches was found to be 180 ( f IO) ppb. Blood Glucose, Insulin. and Glvcated Hemoalobin: At Week 11, after an overnight fast, blood samples were collected from the tail vein and analyzed for glucose and insulin levels. Glucose was analyzed by the Trinder enzymatic procedure (Sigma Kit #315). Insulin was assayed by means of the DPC Coat-a-Count Radioimmunoassay. Blood samples taken at sacrifice (Week 12) were analyzed for glycated hemoglobin. For these analyses, whole blood was collected in EDTA, and samples were assayed on the same day by means of the cis-diol affinity resin column procedure (Sigma Kit #442). Bodv Comoosition: Body composition was determined after 5 weeks and again after 10 weeks of dietary treatment by means of dual energy X-ray absorptiometry (DEXA) on the Hologic QDR-2000 system. For this procedure, four rats from Block 1, three rats from Block 2, and three rats from Block 3 were chosen. Therefore, a total of ten rats from each treatment group underwent DEXA analyses. Before the procedure, the animals were anaesthetized with a combination of ketamine (75 mg/kg BW) and xylazine (25 mglkg BW). The rat whole body was then scanned and was used to determine BW, lean body mass (LBM), body fat weight (FAT), and percent body fat (%BF). Growth Rate. Dailv Feed Intake. and Feed Efficiencv: Body weight and feed intake were recorded 3 times/week over the 12-week period. Average daily gain (ADG) (g/day), daily feed intake (DFI) (g/day), and feed efficiency (FE) (g BW/lOO g feed) were calculated from these records for Weeks 2-l 1. Tissue Weiahts: At the end of the 12-week treatment period, the rats were anaesthetized (ketamine, 75 mg/kg BW, and xylazine, 25 mg/kg BW) and weighed for final BW. The soleus, plantaris, and extensor digitorum longus (EDL) muscles were
286
D.L. HASTEN et al.
excised, connective tissue was carefully trimmed away, and the wet muscle was determined. The tissues were frozen in liquid nitrogen 70°C for later analyses. After removal of the hindlimb muscles, sacrificed by exsanguination. The abdominal cavity was opened, drawn directly from the abdominal aorta. As described earlier, this for glycated hemoglobin analyses.
weight of each and stored at the rats were and blood was blood was used
Statistical Analvses: The data from the three blocks of animals were to be pooled if no significant differences were found to exist between blocks. However, the block effect was highly significant for several variables, and it was determined that a Randomized Block Design (RBD) was most appropriate for the data. A 4 X 3 (treatment X block) RBD was used to analyze each of the glucose tolerance, body composition, tissue weight, and feed characteristic variables. The level of significant differences was set at P 4 0.05. Duncan’s multiple range test was conducted for any significant main effects. As before, the level of significance for post hoc analyses was set at P I 0.05.
RESULTS
Blood Glucose. Insulin. and Glvcated Hemoalobin: There were no treatment X block interactions, nor treatment or block effects for fasting blood glucose (P = 0.501, insulin (P = 0.121, or glycated hemoglobin (P = 0.46) (Data not shown). Bodv Comoosition: There was a significant block effect for BW at both Weeks 5 and 10 (P = 0.01); Block 2 was the heaviest, followed by Block 3, and followed by Block 1. As with BW, there was a significant block effect for LBM at both time points (P = 0.01). At Week 5, Block 2 had a greater LBM than Blocks 1 and 3, and at Week 10, LBM was greatest for Block 2, followed by Block 3, and followed by Block 1. There were significant treatment X block interactions for FAT at Week 5 (P = 0.01) and at Week 10 (P = 0.05). At both Weeks 5 and 10, the main cause of the interaction was a decrease in FAT with all forms of Cr supplementation for Block 1. This same effect was not seen for Blocks 2 and 3. There were also significant treatment X block interactions for %BF at Weeks 5 and 10 (P = 0.01 and P = 0.02, respectively). The interactions at both time periods were due to a decrease in %BF with all forms of Cr supplementation for Block 1. This same decrease in %BF with Cr supplementation was not seen in Blocks 2 and 3. The anthropometric data for Week 10 are given by treatment in Table 1 and by block in Table 2 (Data for Week 5 not shown). The significant treatment X block interactions for FAT and %BF at Week 10 are shown in Figures 1 and 2, respectively. Growth Rate, Dailv Feed Intake. and Feed Efficiencv: For ADG, there were no interactions nor treatment effects, but the block effect was highly significant (P = 0.01). Average daily gain was greatest for Block 2, followed by Block 3, and followed by Block 1. There was also a highly significant block effect for DFI (P = 0.011, with
DIETARY CHROMIUM AND BODY COMPOSITION
287
the greatest mean intake for Block 2, followed by Block 1, followed by Block 3. For FE, there was a treatment effect (P = 0.02). and a trend towards a block effect (P = 0.06). For treatment, the CrCl diet produced a greater FE than the CrPic and basal diets. Furthermore, Blocks 2 and 3 tended to have greater values for FE than Block 1. These data are shown by treatment and by block in Tables 3 and 4, respectively.
TABLE 1 Body Weight (BW), Lean Body Mass (LBM), Fat Weight (FAT), and Percent Body Fat (%BF) at Week 10 as Assessed by Dual Energy X-ray Absorptiometry (DEXA); Data are presented by treatment.
Treatment
BW (g) LBM (g) FAT (g) %BF
Basal
CrPic
CrNic
CrCl
393 f 35 326 f 33 67 f 11 17.2 zt 2.8
387 f 37 320 f 26 67 f 16 17.2 f 2.7
375 f 47 311 f 38 64 f 14 16.9 f 2.6
379 f 42 321 f 34 58 f 10 15.2 f 1.6
Data are presented treatment effects.
as mean
f
standard
deviation;
There
were
no significant
TABLE 2 Body Weight (BW), Lean Body Mass (LBM), Fat Weight (FAT), and Percent Body Fat (%BF) at Week 10 as Assessed by Dual Energy X-ray Absorptiometry (DEXA); Data are presented by block.
Block 2
1
BW @I*= LBM (g)” FAT (9) %BF
353 295 58 16.5
f f
29 25 f 12 f 2.8
421 347 74 17.6
f f
33 28 f 11 f 2.1
3
387 326 61 15.8
f f
21 17 f 11 f 2.4
Data are presented as mean f stand deviation; “‘There were highly significant effects for BW and LBM, P = 0.01 (2 > 3 > 1).
block
288
D.L. HASTEN
J”
5 $
I
et al.
I
I
80 70 60 50 40 30
Block1 Basal FIG. 1.
Block2 CrPic
Block3 CrNic
CrCl
cl
Fat Weight (FAT) at week 10. Values are mean f standard deviation. Significant treatment X block interaction,
I
~80.05.
24. T 22
& S
l-
20 18 16 14 12 10 --
Block 1 Basal FIG. 2.
Block 2 CrPic
Block 3 CrNic
Percent Body Fat (%BF) at week 10. Values are mean f standard deviation. Significant treatment X block interaction,
cl
CrCl
~80.02.
I
289
DIETARY CHROMIUM AND BODY COMPOSITION
Tissue Weiahts Reflecting the DEXA analyses of LBM, there were no significant treatment effects nor interactions for the wet weights of any of the skeletal muscles (P > 0.05). As expected, however, the block effect was highly significant for each of the skeletal muscle weights (P = O.Ol), with the highest values for Block 2, followed by Blocks 3 and 1 (Data not shown).
TABLE 3 Average Daily Gain (ADG), Daily Feed Intake (DFI), and Feed Efficiency 2-11; Data are presented by treatment.
(FE) for Weeks
Treatment Basal
ADG (g/day) DFI (g/day) FE’ (g BW/lOO
4.7 f 24.6
CrPic
.5
CrNic
4.5 f
f
3.6
24.0
14.9 f g feed)
2.3
15.3 f
.4 f
3.4 1.8
4.5 f 24.2
CrCl
.6 f
4.4 f
2.7
22.7
16.0 zt 1.4
.5 zt 3.3
17.0 f
1.9
Data are presented as mean f standard deviation; ‘There was a significant treatment effect for FE, P = 0.02 (CrCI, CrNic, CrPic, Basal).
TABLE 4 Average Daily Gain (ADG), Daily Feed Intake (DFI), and Feed Efficiency 2-l 1; Data are presented by block.
1
ADG (g/day)” DFI (g/day)” FE (g BW/lOO g feed)
4.1 f .4 24.2 f 3.6 15.0 f 1.8
Block 2
4.9 f .5 26.0 f 2.6 16.1 f 2.2
(FE) for Weeks
3
4.5 f .4 21.4 f 1.4 16.3 f 1.9
Data are presented as mean f stand deviation; ‘“There were highly significant effects for ADG (2 > 3 > 1) and DFI (2 > 1 > 31, P = 0.01.
block
290
D.L. HASTEN et al.
DISCUSSION
The purpose of this study was to determine if diets supplemented with different forms of Cr were differentially capable of modifying the body composition of the rat. Surprisingly, all Cr treatments failed to modify LBM as determined by DEXA analyses of whole body density. This finding is in contrast to other studies that have examined the effects of Cr supplementation in the form of CrPic on body composition in both humans (9,10,12,13) and animals (11 ,15,23,24). However, our findings are consistent with those of Evock-Clover et al. (251, who did not find any changes in lean mass of swine after 6 weeks of CrPic supplementation. The failure to observe any increase in LBM may indicate that the Cr status of the animals was adequate without the added dietary Cr. Additional Cr, regardless of its form, would therefore be unable to further modify processes that are sensitive to Cr status. This was further supported by the fact that Cr supplementation had no effect upon any of the variables that reflected glucose tolerance (i.e., fasting blood glucose, insulin, or glycated hemoglobin levels). Alternatively, emerging evidence suggests that Cr supplementation may be most effective when the animal is experiencing some form of stress such as exercise, trauma, or a low-protein diet (14,19,26-29). The animals used in the current study were under no externally applied stress, perhaps making them resistant to the Cr supplementation. Chromium supplementation, however, appeared to have an effect on body fat for the Block 1 animals in this study. The DEXA analysis of FAT and %BF at Weeks 5 and 10 reflected equivalent reductions in body fat levels for all animals who received supplemental dietary Cr. regardless of the form. This is in agreement with some previous human (9,131 and animal (15,23,24) studies that have observed decreases in body fat with CrPic supplementation. Since food intake was equivalent for all dietary treatments, Cr supplementation did not appear to reduce body fat levels in the Block 1 animals through a reduction in caloric intake. Therefore, it is possible that all forms of dietary Cr potentiated insulininduced thermogenesis, thereby enhancing caloric expenditure. The only treatment effect that was significant across all blocks of animals was FE. While the FE for all Cr-supplemented diets tended to be higher than that of the basal diet, only the CrCl diet statistically modified this variable. This indicated that the animals consuming the CrCI-supplemented diet gained more weight per unit of food consumed. The higher FE did not manifest itself in higher BW or changes in body composition, suggesting that the duration of treatment may have been too short to observe any dietary Cr effects upon these variables. Alternatively, it could be proposed that CrCl may have had the greatest absorption across the gut wall. This seems unlikely since CrNic appears to have the greatest absorption and retention of these three Cr-containing compounds (301. Hence, the selective impact of CrCl on feed retention remains difficult to explain.
DIETARY CHROMIUM AND BODY COMPOSITION
291
The central finding of this study is that different blocks of animals responded differently to the imposed dietary intervention. For example, Cr supplementation decreased FAT and %BF in Block 1 animals but not in Blocks 2 and 3. Similarly, ADG and DFI demonstrated significant block effects. These differential responses for ADG and DFI were seen even in animals that were reared in the same breeding facility and presumably from the same breeding colony (i.e., Blocks 2 and 3). Verch et al. (31) reported that the Cr levels of the same organs varied across different shipments of rats, suggesting that the Cr status of animals, even those from the same breeding facility, can vary. Results from the current study support the possibility that the Cr status of animals may differ. Such a variation could account for the inconsistent The block effects observed in the results elicited by dietary Cr supplementation. current study also point to the need to develop a reliable and convenient measure of Cr status. In conclusion, Cr supplementation appeared to decrease body fat levels in only one group of animals in this study. Furthermore, the decreases in body fat were equivalent for all forms of Cr studied, suggesting that the biological activity of these Cr compounds are equivalent under the conditions of this experiment. Since these body fat reductions occurred without concurrent decreases in food consumption, it is possible that Cr supplementation exerted a metabolic effect, perhaps a potentiation of insulin-induced dietary thermogenesis. Unexpectedly, increases in growth rate and in LBM were not seen, regardless of the type of Cr added to the diet. These results suggest that a deficit in Cr status must be created before the effects of supplemental dietary Cr upon these variables can be realized.
ACKNOWLEDGEMENTS
This study
was supported
by a grant
from
Nutrition
21,
REFERENCES
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Schroeder HA. Chromium deficiency in rats: a syndrome mellitus with retarded growth. J Nutr 1966; 88: 439-445.
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Golde DW, Bersch N, Kaplan unresponsiveness to human growth 1980; 303: 1156-I 158.
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Evans GW. in humans.
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Evans GW, Pouchnik DJ. Composition and biological activity of chromiumpyridine carboxylate complexes. J lnorgan Biochem 1993; 49: 177-l 87.
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Hasten DL, Hegsted M, Glickman-Weiss EL. Effects of chromium picolinate and exercise on the body composition of the rat. FASEB J 1993; 7: A77 (abstract).
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Hasten DL, Rome EP, Franks BD, Hegsted M. Effects of chromium picolinate on beginning weight training students. Int J Sport Nutr 1992; 2: 343-350.
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Kaats GR, Fisher JA, supplementation on body 138 (abstract).
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Mertz W, Roginski EE. Effect stress in rats fed low protein
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Page TG, Southern LL, Ward TL, Thompson DL Jr. Effect of chromium picolinate on growth and serum and carcass traits of growing-finishing pigs. J Anim Sci 1993; 71: 656-662.
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Lefavi RG, Anderson RA, Keith RE, Wilson GD, McMillan JL, Stone Efficacy of chromium supplementation in athletes: Emphasis on anabolism. J Sport Nutr 1992; 2: 11 l-l 22.
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Mertz W, Roginski EE. Some biological properties of chromium acid (NA) complexes. Fed Proc 1975; 34: 922 (abstract).
18.
Donaldson DL, Lee DM, Smith CC, Rennert OM. Glucose tolerance and plasma lipid distributions in rats fed a high-sucrose, high-cholesterol, low-chromium diet. Metabolism 1985; 34: 1086-1093.
P.
K, Mertz W. Chromium (III) and the glucose Biophys 1959; 85: 292-295.
Amino
RDG.
Insulin
as a growth
acid metabolism
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Ped Res 1985;
SA, Rimoin DL, Le CH. hormone in Laron dwarfism,
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Ann Rev Biochem
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19: 879-886. Peripheral N Eng J Med
1975;
The effect of chromium picolinate on insulin controlled Int J Biosocial Med Res 1989; 11: 163-I 80.
Blum K. composition
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44:
933-
parameters
The effects of chromium picolinate in different age groups. AGE 1991; 4:
on growth and survival of Cr3+ supplement diets. J Nutr 1969; 97: 531-536.
under
MH. Int
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19.
Hasten DL, Siver F, Fornea S, Savoie chromium picolinate on body composition.
20.
Felig P. Insulin is the mediator of feeding-related thermogenesis: insulin resistance and/or deficiency results in a thermogenic defect which contributes to the pathogenesis of obesity. Clin Physiol 1984; 4: 267-273.
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McCarty MF. Hypothesis: Sensitization of insulin-dependent hypothalamic glucoreceptors may account for the fat-reducing effects of chromium picolinate. J Optimal Nutr 1993; 2: 36-53.
22.
Brief DJ, Davis JD. Reduction intraventricular insulin infusion.
23.
Kitchalong L, Fernandez JM, Bunting LD, Chapa AM, Sticker LS, Amoikon Ward TL, Bidner TD, Southern LL. Chromium picolinate supplementation lamb rations: Effects on performance, nitrogen balance, endocrine metabolic parameters. J Anim Sci 1993; 71 (Suppl 1): 291 (abstract).
24.
Lindemann MD, Wood CM, Harper additions to diets of growing/finishing (abstract).
25.
Evock-Clover CM, Polansky supplementation with or hormones and metabolites performance. J Nutr 1993;
26.
Anderson RA, Polansky MM, Bryden NA. Strenuous running: acute effects on chromium, copper, zinc, and selected clinical variables in urine and serum of male runners. Biol Trace Elem Res 1984; 6: 327-336.
27.
Anderson RA, Polansky MM, Bryden NA, Roginski EE, Patterson KY, Veillon C, Glinsmann, W. Urinary chromium excretion of human subjects: Effects of chromium supplementation and glucose loading. Am J Clin Nutr 1982; 36: 1184-I 193.
28.
Bore1 JS, Marjerus TC, Polansky MM, Moser PB, Anderson RA. Chromium intake and urinary chromium excretion of trauma patients. Biol Trace Elem Res 1984; 6: 317-326.
29.
Campbell WW, Anderson trace minerals chromium,
S, Hegsted M. FASEB J 1994;
Dosage effects of 8: Al 95 (abstract).
of food intake and body weight by chronic Brain Res Bull 1984; 12: 571-575.
AF, Kornegay pigs. J Anim
EK, in and
ET. Chromium picolinate Sci 1993; 71 (Suppl 1): 14
MM, Anderson RA, Steele NC. Dietary chromium without somatotropin treatment alters serum in growing pigs without affecting growth 123: 1504-l 512.
RA. Effects of aerobic exercise and training on the zinc and copper. Sports Med 1987; 4: 9-18.
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30.
Olin SK, Sterns DM, Armstrong WH, Keen CL. Comparative retention absorption of chromium-51 (Cr-51) from Cr-51 chloride, nicotinate Cr-51 and Cr-51 picolinate in a rat model. Trace Elem Electrol 1994; 11: 182-l 86.
31.
Verch RL, Chu R, Wallach S, Peabody RA, Jain R, Hannan E. Tissue chromium in the rat. Nutr Rep Int 1983; 27: 531-540. Accepted for publication September 20, 1996
ELSEVIER
PII SO271-5317(96)00258-S
EFFECTS OF VARIOUS FORMS OF DIETARY CHROMIUM ON GROWTH AND BODY COMPOSITION IN THE RAT Deborah L. Hasten, Ph.D.‘, Maren Hegsted, Ph.D.‘, Michael J. Keenan, Ph.D.‘, and G. Stephen Morris, Ph.D.” Washington University School of Medicine, St. Louis, MO 63110; Departments of Human Ecology* and Kinesiology”, Louisiana State University, Baton Rouge, LA 70803
ABSTRACT
Several human and animal studies of chromium (0) supplementation have reported increases in lean body mass, often with concurrent decreases in body fat. Since the majority of studies have tested Cr in the form of Cr picolinate (CrPic), this 12-week study contrasted the effects of three available forms of dietary Cr (300 ppb Cr as CrPic, Cr nicotinate (CrNic), or Cr chloride (CrCI)}, as well as no added dietary Cr, upon growth and body composition in the rat. Sixty male Harlan Sprague-Dawley weanling rats arrived in three groups of 20 animals (referred to as Blocks 1, 2, and 3). Five animals from each block were assigned to each treatment, resulting in a 4 X 3 (treatment X block) Randomized Block Design. Body composition was assessed at Weeks 5 and 10 via dual energy X-ray absorptiometry (DEXA). Significant treatment X block interactions were found for fat weight and percent body fat at both time points (P I 0.05). These interactions reflected decreases in body fat with all forms of Cr supplementation for the Block 1 animals. No treatment interactions or effects were seen for growth rate, lean body mass, or tissue weights, but most block effects were highly significant (P I 0.011, reflecting possible genetic differences between blocks. Since feed intake did not vary between dietary treatments, the reduced body fat levels for the Cr-supplemented animals of Block 1 may have been due to an enhancement of insulin-induced Copyright 8 1997 Elsevier Science Inc. thermogenesis. Key Words:
Rat, Chromium,
Growth,
Body
Composition
‘Address Correspondence to: Dr. Deborah L. Hasten, Washington of Medicine, 660 S. Euclid, Box 8127, St. Louis, MO 631 IO
283
University
School
284
D.L. HASTEN et al.
INTRODUCTION
Various forms of trivalent chromium (0) have been shown to potentiate the cellular actions of insulin (I-5). Insulin has been shown to increase glucose and amino acid uptake into muscle cells (61, activate ribosomal translational activity (61, and enhance the actions of growth hormone (7). Additionally, insulin inhibits enzymes that catabolize amino acids and protein (8). Chromium may potentiate the cellular and subcellular actions of insulin, leading to changes in muscle mass. In support of this suggestion, both human and animal studies have indicated that Cr supplementation enhances the development of lean body mass (9-l 5). Most Cr supplementation studies that have reported increases in lean body mass have used Cr in the form of Cr picolinate (CrPic) (9-l 3,151. Two human studies that used Cr as Cr nicotinate (CrNic) failed to observe any changes in lean body mass (8,161. However, oral ingestion of CrNic has been shown to increase the body weight of rats as compared to the Cr-deficient state (17). Although several studies of Cr as Cr chloride (CrCI) have observed increased growth rates (3,4,14,18), only one (14) observed a concurrent increase in lean body mass. Chromium supplementation has also been found to reduce body fat levels in humans (9.13) and animals (15,191. It has been hypothesized that Cr may achieve this by promoting hypothalamic insulin-mediated activation of the sympathetic nervous system, subsequently increasing dietary-induced thermogenesis 120,21 I. In addition to promoting thermogenesis, centrally administered insulin has been found to suppress food intake in the rat (22). Hence, Cr supplementation may decrease body fat levels by increasing energy expenditure or by decreasing caloric intake. It appears that Cr supplementation is capable of altering body composition, but the ability of different forms of Cr to do this may vary. Since most of the body composition studies of Cr have utilized the mineral in the form of CrPic, and only a limited number of studies of CrNic and of CrCl have been reported, further work needs to be conducted before any final conclusions can be made. The current study examined the effects of Cr in the forms of CrPic, CrNic, and CrCl on growth and body composition in the rat. Since previous studies of Cr supplementation have reported body composition changes after both 6 and 12 weeks (g-151, we assessed body composition at two time intervals (5 and 10 weeks) before the completion of this study at 12 weeks.
METHODS
AND MATERIALS
Animals and Dietarv Treatments: Sixty male Harlan Sprague-Dawley outbred weanling rats (21 days of age) were used in this study. The rats were received in three groups of 20, arriving at two-week intervals. These three groups of animals were referred
DIETARY CHROMIUM AND BODY COMPOSITION
285
to as Blocks 1, 2, and 3. The animals were divided into four treatment groups of 15 (five rats each from Blocks 1, 2, and 3) at 25 days of age. Although the initial body weights (BW) of the animals did not differ by treatment, there were significant block differences for initial BW (p = 0.011, with Block 2 having a greater mean weight than Blocks 1 and 3. A follow-up call to representatives at Harlan Sprague-Dawley, Inc. revealed that the Block 1 animals had originated from a breeding colony in Houston, TX (Building 21 I), whereas Blocks 2 and 3 had come from a facility in Prattville, AL (Building 218). The animals were kept in stainless steel housing with wire mesh flooring. Diets were placed in glass containers, and the animals had free access to food and ultrapure water. The study was carried out for 12 weeks, and the animals were 112 to 116 days of age at sacrifice. The basal diet consumed by all animals in this study was the AIN-76A semi-purified diet without the addition of Cr. Animals received one of the following diets: I) basal; or basal with the addition of 300 fig Cr/kg diet (hereafter referred to as parts per billion, ppb) in the forms of 2) CrPic; 3) CrNic; and 4) CrCI. Basal diet samples were analyzed for Cr content by Dr. Richard A. Anderson of the U.S.D.A. Beltsville Human Nutrition Resource Center (Beltsville, MD). The Cr content of eight random samples taken from different basal diet batches was found to be 180 ( f IO) ppb. Blood Glucose, Insulin. and Glvcated Hemoalobin: At Week 11, after an overnight fast, blood samples were collected from the tail vein and analyzed for glucose and insulin levels. Glucose was analyzed by the Trinder enzymatic procedure (Sigma Kit #315). Insulin was assayed by means of the DPC Coat-a-Count Radioimmunoassay. Blood samples taken at sacrifice (Week 12) were analyzed for glycated hemoglobin. For these analyses, whole blood was collected in EDTA, and samples were assayed on the same day by means of the cis-diol affinity resin column procedure (Sigma Kit #442). Bodv Comoosition: Body composition was determined after 5 weeks and again after 10 weeks of dietary treatment by means of dual energy X-ray absorptiometry (DEXA) on the Hologic QDR-2000 system. For this procedure, four rats from Block 1, three rats from Block 2, and three rats from Block 3 were chosen. Therefore, a total of ten rats from each treatment group underwent DEXA analyses. Before the procedure, the animals were anaesthetized with a combination of ketamine (75 mg/kg BW) and xylazine (25 mglkg BW). The rat whole body was then scanned and was used to determine BW, lean body mass (LBM), body fat weight (FAT), and percent body fat (%BF). Growth Rate. Dailv Feed Intake. and Feed Efficiencv: Body weight and feed intake were recorded 3 times/week over the 12-week period. Average daily gain (ADG) (g/day), daily feed intake (DFI) (g/day), and feed efficiency (FE) (g BW/lOO g feed) were calculated from these records for Weeks 2-l 1. Tissue Weiahts: At the end of the 12-week treatment period, the rats were anaesthetized (ketamine, 75 mg/kg BW, and xylazine, 25 mg/kg BW) and weighed for final BW. The soleus, plantaris, and extensor digitorum longus (EDL) muscles were
286
D.L. HASTEN et al.
excised, connective tissue was carefully trimmed away, and the wet muscle was determined. The tissues were frozen in liquid nitrogen 70°C for later analyses. After removal of the hindlimb muscles, sacrificed by exsanguination. The abdominal cavity was opened, drawn directly from the abdominal aorta. As described earlier, this for glycated hemoglobin analyses.
weight of each and stored at the rats were and blood was blood was used
Statistical Analvses: The data from the three blocks of animals were to be pooled if no significant differences were found to exist between blocks. However, the block effect was highly significant for several variables, and it was determined that a Randomized Block Design (RBD) was most appropriate for the data. A 4 X 3 (treatment X block) RBD was used to analyze each of the glucose tolerance, body composition, tissue weight, and feed characteristic variables. The level of significant differences was set at P 4 0.05. Duncan’s multiple range test was conducted for any significant main effects. As before, the level of significance for post hoc analyses was set at P I 0.05.
RESULTS
Blood Glucose. Insulin. and Glvcated Hemoalobin: There were no treatment X block interactions, nor treatment or block effects for fasting blood glucose (P = 0.501, insulin (P = 0.121, or glycated hemoglobin (P = 0.46) (Data not shown). Bodv Comoosition: There was a significant block effect for BW at both Weeks 5 and 10 (P = 0.01); Block 2 was the heaviest, followed by Block 3, and followed by Block 1. As with BW, there was a significant block effect for LBM at both time points (P = 0.01). At Week 5, Block 2 had a greater LBM than Blocks 1 and 3, and at Week 10, LBM was greatest for Block 2, followed by Block 3, and followed by Block 1. There were significant treatment X block interactions for FAT at Week 5 (P = 0.01) and at Week 10 (P = 0.05). At both Weeks 5 and 10, the main cause of the interaction was a decrease in FAT with all forms of Cr supplementation for Block 1. This same effect was not seen for Blocks 2 and 3. There were also significant treatment X block interactions for %BF at Weeks 5 and 10 (P = 0.01 and P = 0.02, respectively). The interactions at both time periods were due to a decrease in %BF with all forms of Cr supplementation for Block 1. This same decrease in %BF with Cr supplementation was not seen in Blocks 2 and 3. The anthropometric data for Week 10 are given by treatment in Table 1 and by block in Table 2 (Data for Week 5 not shown). The significant treatment X block interactions for FAT and %BF at Week 10 are shown in Figures 1 and 2, respectively. Growth Rate, Dailv Feed Intake. and Feed Efficiencv: For ADG, there were no interactions nor treatment effects, but the block effect was highly significant (P = 0.01). Average daily gain was greatest for Block 2, followed by Block 3, and followed by Block 1. There was also a highly significant block effect for DFI (P = 0.011, with
DIETARY CHROMIUM AND BODY COMPOSITION
287
the greatest mean intake for Block 2, followed by Block 1, followed by Block 3. For FE, there was a treatment effect (P = 0.02). and a trend towards a block effect (P = 0.06). For treatment, the CrCl diet produced a greater FE than the CrPic and basal diets. Furthermore, Blocks 2 and 3 tended to have greater values for FE than Block 1. These data are shown by treatment and by block in Tables 3 and 4, respectively.
TABLE 1 Body Weight (BW), Lean Body Mass (LBM), Fat Weight (FAT), and Percent Body Fat (%BF) at Week 10 as Assessed by Dual Energy X-ray Absorptiometry (DEXA); Data are presented by treatment.
Treatment
BW (g) LBM (g) FAT (g) %BF
Basal
CrPic
CrNic
CrCl
393 f 35 326 f 33 67 f 11 17.2 zt 2.8
387 f 37 320 f 26 67 f 16 17.2 f 2.7
375 f 47 311 f 38 64 f 14 16.9 f 2.6
379 f 42 321 f 34 58 f 10 15.2 f 1.6
Data are presented treatment effects.
as mean
f
standard
deviation;
There
were
no significant
TABLE 2 Body Weight (BW), Lean Body Mass (LBM), Fat Weight (FAT), and Percent Body Fat (%BF) at Week 10 as Assessed by Dual Energy X-ray Absorptiometry (DEXA); Data are presented by block.
Block 2
1
BW @I*= LBM (g)” FAT (9) %BF
353 295 58 16.5
f f
29 25 f 12 f 2.8
421 347 74 17.6
f f
33 28 f 11 f 2.1
3
387 326 61 15.8
f f
21 17 f 11 f 2.4
Data are presented as mean f stand deviation; “‘There were highly significant effects for BW and LBM, P = 0.01 (2 > 3 > 1).
block
288
D.L. HASTEN
J”
5 $
I
et al.
I
I
80 70 60 50 40 30
Block1 Basal FIG. 1.
Block2 CrPic
Block3 CrNic
CrCl
cl
Fat Weight (FAT) at week 10. Values are mean f standard deviation. Significant treatment X block interaction,
I
~80.05.
24. T 22
& S
l-
20 18 16 14 12 10 --
Block 1 Basal FIG. 2.
Block 2 CrPic
Block 3 CrNic
Percent Body Fat (%BF) at week 10. Values are mean f standard deviation. Significant treatment X block interaction,
cl
CrCl
~80.02.
I
289
DIETARY CHROMIUM AND BODY COMPOSITION
Tissue Weiahts Reflecting the DEXA analyses of LBM, there were no significant treatment effects nor interactions for the wet weights of any of the skeletal muscles (P > 0.05). As expected, however, the block effect was highly significant for each of the skeletal muscle weights (P = O.Ol), with the highest values for Block 2, followed by Blocks 3 and 1 (Data not shown).
TABLE 3 Average Daily Gain (ADG), Daily Feed Intake (DFI), and Feed Efficiency 2-11; Data are presented by treatment.
(FE) for Weeks
Treatment Basal
ADG (g/day) DFI (g/day) FE’ (g BW/lOO
4.7 f 24.6
CrPic
.5
CrNic
4.5 f
f
3.6
24.0
14.9 f g feed)
2.3
15.3 f
.4 f
3.4 1.8
4.5 f 24.2
CrCl
.6 f
4.4 f
2.7
22.7
16.0 zt 1.4
.5 zt 3.3
17.0 f
1.9
Data are presented as mean f standard deviation; ‘There was a significant treatment effect for FE, P = 0.02 (CrCI, CrNic, CrPic, Basal).
TABLE 4 Average Daily Gain (ADG), Daily Feed Intake (DFI), and Feed Efficiency 2-l 1; Data are presented by block.
1
ADG (g/day)” DFI (g/day)” FE (g BW/lOO g feed)
4.1 f .4 24.2 f 3.6 15.0 f 1.8
Block 2
4.9 f .5 26.0 f 2.6 16.1 f 2.2
(FE) for Weeks
3
4.5 f .4 21.4 f 1.4 16.3 f 1.9
Data are presented as mean f stand deviation; ‘“There were highly significant effects for ADG (2 > 3 > 1) and DFI (2 > 1 > 31, P = 0.01.
block
290
D.L. HASTEN et al.
DISCUSSION
The purpose of this study was to determine if diets supplemented with different forms of Cr were differentially capable of modifying the body composition of the rat. Surprisingly, all Cr treatments failed to modify LBM as determined by DEXA analyses of whole body density. This finding is in contrast to other studies that have examined the effects of Cr supplementation in the form of CrPic on body composition in both humans (9,10,12,13) and animals (11 ,15,23,24). However, our findings are consistent with those of Evock-Clover et al. (251, who did not find any changes in lean mass of swine after 6 weeks of CrPic supplementation. The failure to observe any increase in LBM may indicate that the Cr status of the animals was adequate without the added dietary Cr. Additional Cr, regardless of its form, would therefore be unable to further modify processes that are sensitive to Cr status. This was further supported by the fact that Cr supplementation had no effect upon any of the variables that reflected glucose tolerance (i.e., fasting blood glucose, insulin, or glycated hemoglobin levels). Alternatively, emerging evidence suggests that Cr supplementation may be most effective when the animal is experiencing some form of stress such as exercise, trauma, or a low-protein diet (14,19,26-29). The animals used in the current study were under no externally applied stress, perhaps making them resistant to the Cr supplementation. Chromium supplementation, however, appeared to have an effect on body fat for the Block 1 animals in this study. The DEXA analysis of FAT and %BF at Weeks 5 and 10 reflected equivalent reductions in body fat levels for all animals who received supplemental dietary Cr. regardless of the form. This is in agreement with some previous human (9,131 and animal (15,23,24) studies that have observed decreases in body fat with CrPic supplementation. Since food intake was equivalent for all dietary treatments, Cr supplementation did not appear to reduce body fat levels in the Block 1 animals through a reduction in caloric intake. Therefore, it is possible that all forms of dietary Cr potentiated insulininduced thermogenesis, thereby enhancing caloric expenditure. The only treatment effect that was significant across all blocks of animals was FE. While the FE for all Cr-supplemented diets tended to be higher than that of the basal diet, only the CrCl diet statistically modified this variable. This indicated that the animals consuming the CrCI-supplemented diet gained more weight per unit of food consumed. The higher FE did not manifest itself in higher BW or changes in body composition, suggesting that the duration of treatment may have been too short to observe any dietary Cr effects upon these variables. Alternatively, it could be proposed that CrCl may have had the greatest absorption across the gut wall. This seems unlikely since CrNic appears to have the greatest absorption and retention of these three Cr-containing compounds (301. Hence, the selective impact of CrCl on feed retention remains difficult to explain.
DIETARY CHROMIUM AND BODY COMPOSITION
291
The central finding of this study is that different blocks of animals responded differently to the imposed dietary intervention. For example, Cr supplementation decreased FAT and %BF in Block 1 animals but not in Blocks 2 and 3. Similarly, ADG and DFI demonstrated significant block effects. These differential responses for ADG and DFI were seen even in animals that were reared in the same breeding facility and presumably from the same breeding colony (i.e., Blocks 2 and 3). Verch et al. (31) reported that the Cr levels of the same organs varied across different shipments of rats, suggesting that the Cr status of animals, even those from the same breeding facility, can vary. Results from the current study support the possibility that the Cr status of animals may differ. Such a variation could account for the inconsistent The block effects observed in the results elicited by dietary Cr supplementation. current study also point to the need to develop a reliable and convenient measure of Cr status. In conclusion, Cr supplementation appeared to decrease body fat levels in only one group of animals in this study. Furthermore, the decreases in body fat were equivalent for all forms of Cr studied, suggesting that the biological activity of these Cr compounds are equivalent under the conditions of this experiment. Since these body fat reductions occurred without concurrent decreases in food consumption, it is possible that Cr supplementation exerted a metabolic effect, perhaps a potentiation of insulin-induced dietary thermogenesis. Unexpectedly, increases in growth rate and in LBM were not seen, regardless of the type of Cr added to the diet. These results suggest that a deficit in Cr status must be created before the effects of supplemental dietary Cr upon these variables can be realized.
ACKNOWLEDGEMENTS
This study
was supported
by a grant
from
Nutrition
21,
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