QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schuler, A. M. Articles by Wood, P. A. Search for Related Content PubMed PubMed Citation Articles by Schuler, A. M. Articles by Wood, P. A.

---

Biochemical and Molecular Actions of Nutrients
Research Communication

Dietary Phytoestrogens Increase Metabolic Resistance (Cold Tolerance) in Long-Chain Acyl-CoA Dehydrogenase–Deficient Mice¹

A. Michele Schuler^*, Stephen Barnes, Barbara A. Gower^** and Philip A. Wood^*,**,2

Departments of ^* Genetics, Pharmacology and Toxicology, and ^** Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL

²To whom correspondence should be addressed. E-mail: paw{at}uab.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We evaluated the role of dietary phytoestrogens (PE) in the disease phenotype of cold intolerance that characterizes long-chain acyl-CoA dehydrogenase–deficient (LCAD–/–) mice, a model of inborn errors of mitochondrial fatty acid ß-oxidation. Male LCAD–/– mice were fed a standard diet containing endogenous PE, a PE-free diet, or a PE-free diet that was supplemented with genistein (250 µg/g diet). The standard diet did not restore complete cold tolerance, but it provided more resistance (P = 0.004) to cold challenge than the PE-free diet. There was a nonsignificant difference (P < 0.07) between LCAD–/– mice fed the genistein-supplemented diet and those fed the PE-free diet. There were no differences in end-point serum glucose concentrations among the 3 groups. Serum FFA were decreased in LCAD–/– mice fed the standard diet compared with those fed the PE-free diet (P = 0.005) and the diet supplemented with genistein (P < 0.001). Serum triglyceride concentrations were greater (P < 0.05) only in LCAD–/– mice fed the genistein-supplemented diet than those fed the standard diet. These results demonstrate the beneficial effects of dietary PE on metabolic tolerance in LCAD–/– mice. Furthermore, they suggest changes that could improve pediatric formula constituents, especially with regard to management of children with inborn errors of fatty acid oxidation.

KEY WORDS: • phytoestrogens • genistein • long-chain acyl-CoA dehydrogenase • cold tolerance

Inherited enzyme deficiencies of mitochondrial fatty acid ß-oxidation represent a collection of diseases that affect neonates and young children. These diseases present as life-threatening episodes resulting from an inability to oxidize fat stores to produce energy during fasting or other catabolic situations. The hallmark of an inborn error of mitochondrial fatty acid ß-oxidation is the development of hypoketotic-hypoglycemia, hyperammonemia, and fatty change in liver. This clinical presentation is often termed, "Reye-like" syndrome. The most common of these conditions is medium-chain acyl-CoA dehydrogenase deficiency followed by very long-chain acyl-CoA dehydrogenase deficiency (1).

Mitochondrial ß-oxidation of fatty acids occurs through a series of spiraling steps within the mitochondria. The acyl-CoA dehydrogenase (ACAD)³ enzymes catalyze essential steps that result in the shortening of the fatty acyl-CoA and the production of acetyl-CoA (2). When there is a deficiency in one of these enzymes, this cascade of events will stall, and fatty acid oxidation will not proceed at a rate necessary for homeostasis of energy production.

To facilitate the study of these human diseases, we developed 4 mouse models of ACAD deficiency (2). Long-chain acyl-CoA dehydrogenase (LCAD)-deficient (LCAD–/– mice) are the most severely affected. The LCAD–/– mouse model is characterized by hepatic and cardiac steatosis, food deprivation and cold intolerance, hypoketotic-hypoglycemia, and sudden death (3–6). Cold tolerance requires fully intact mitochondrial fatty acid oxidation for nonshivering thermogenesis to maintain adequate thermoregulation. ACAD-deficient mice uniformly become hypothermic during this type of metabolic challenge (6).

Diet may play a role in the severity of the disease phenotype in human patients and animal models of the ACAD deficiencies, although the role of diet has not been rigorously studied. Previously, we demonstrated the detrimental effects of feeding a PE-free diet to male LCAD–/– mice on the development of cardiac hypertrophy (7). There is interest in devising ways of using diet to increase metabolic tolerance in patients with inborn errors of mitochondrial ß-oxidation of fatty acids. The LCAD–/– mouse model has the most severe disease phenotype mimicking the phenotypes found in human children with similar mitochondrial inborn errors. The current recommendations for children with these enzyme deficiencies are focused on maintenance of adequate energy intake (8).

Most common rodent diets contain soy meal, a substantial source of dietary phytoestrogens (PE), as a source of protein (9,10). PE are similar in structure and function to mammalian endogenous estrogens with documented effects on lipid metabolism (11). In addition, PE may act on cell signaling pathways independently of typical estrogen-like actions. They have antioxidant properties in vitro (12). Clinically, diets rich in PE have been shown to be beneficial in the management of several common disease conditions including hyperlipidemia (11,13).

We tested the hypothesis that dietary PE provides increased resistance to metabolic stress, e.g., cold challenge, in mice with LCAD deficiency. To test this hypothesis, we fed mice a standard laboratory diet that contained endogenous PE, a PE-free diet, or a PE-free diet supplemented with genistein. Genistein is a soy-derived PE with estrogenic properties (10).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Mice and diets. B6,129-Acadl^–/– (LCAD–/–) male mice were produced in a breeding colony at the University of Alabama at Birmingham. All mice were between 55 and 70 d old at the time of data collection. LCAD+/+ mice do not experience hypothermia when cold challenged and were not used in this study. All diets were commercially available. Breeding mice were fed a standard rodent diet (Harlan Teklad, LM-485). Study mice were fed 1 of the experimental diets starting at weaning (21 d of age). The experimental diets (Table 1) included the following diets: 1) standard laboratory mouse diet (n = 8) (Harlan Teklad, LM-485); 2) PE-free diet (n = 12) (a casein-based diet manufactured by Harlan Teklad, TD96155); and 3) PE-free diet (n = 7) supplemented with the genistein at a dose of 250 mg/kg diet. LM-485 contains soy and alfalfa, both of which are rich in PE (Table 2).

    Cold tolerance. Cold tolerance was accessed by individually housing mice at 4°C. Rectal temperature was measured before exposure to the cold and then hourly using a Barnant Thermocouple thermometer (dual J-D-E-K, listed 5R18). When rectal temperature dropped below 25°C or after 4 h, whichever occurred first, the mice were humanely killed. We found that mice with temperatures < 25°C do not recover, and thus are considered terminal without using death as an end-point.

    Serum biochemistry. Blood was collected by cardiac puncture after induction of anesthesia. Glucose was measured on whole blood using a One Touch Basic glucometer (Lifescan Technical Services, a Johnson & Johnson Company) postcold challenge. Triglyceride (TG) concentrations were measured in 10 µL serum with the Ektachem DT II System (Johnson and Johnson Clinical Diagnostics). FFA were measured by an enzymatic, colorimetric method ("NEFA-C" reagents, Wako Diagnostics). The assay was modified to accommodate a reduced sample volume (10 µL) and the use of a microplate reader for measurement of optical density at 550 nm

    Statistics. Survival rates were reported as the percentage survival and evaluated by Kaplan-Meier Survival Analysis, i.e., log rank test with multiple comparisons performed by the Holm-Sidak method. For comparison of biochemical measurements at study termination, one-way ANOVA was performed with all pairwise multiple comparison procedures (Holm-Sidak method) for fatty acid concentrations and a Kruskal-Wallis one-way ANOVA on ranks using Dunn’s method of all pairwise multiple comparisons for triglyceride concentrations. Significant difference for all data analysis was accepted at P < 0.05. Data were analyzed with SigmaStat. All results are expressed as mean ± SD.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All LCAD–/– mice fed the PE-free diet were terminally hypothermic before h 2 of the study (Fig. 1). This was in marked contrast to those fed the standard diet or the PE-free diet supplemented with genistein. Of the LCAD–/– mice fed the genistein-supplemented diet, only 1 became terminally hypothermic before h 2. Thus, there was an 86% survival until h 3 in the LCAD–/– mice fed the genistein-supplemented diet. Of the LCAD–/– mice fed the standard diet, 100% survived past 1 h, 75% survived to 2 h, 50% survived to 3 h, and 25% survived to the study’s termination (4 h) (Fig. 1). The standard diet did not restore complete cold tolerance, but it provided more resistance (P = 0.004) to cold challenge than the PE-free diet. There was a nonsignificant difference (P < 0.07) between LCAD–/– mice fed the genistein-supplemented diet and those fed the PE-free diet.

View larger version (15K): [in this window] [in a new window]   FIGURE 1 Survival during cold challenge in LCAD–/– mice fed standard (n = 8), PE-free + genistein (n = 7), or PE-free (n = 12) diets. Mice fed standard diet differed from those fed PE free diet, P = 0.004 (log rank).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In contrast to the potential protective effects, consumption of soy early in life has recently come under criticism. In human infants, if the sole source of nutrition is soy infant formula, the infant receives a dose of up to 11 mg/(kg · d) of dietary PE with presumed estrogenic effects (11,19). In an effort to elucidate the effects of soy consumption on male infants, Sharpe et al. (20) used a primate model to describe the effects of xenoestrogen consumption on the development of secondary sex characteristics. They found a suppression of the normal rise in neonatal serum testosterone and effects on the testis (20). Although the limited investigations in human infants and children have not confirmed these findings (18), the study by Sharpe et al. (20) nonetheless demonstrated the potential of a relationship between biologic effects and consumption of PE. Soy infant formulas contain a high level of dietary PE and could be used if increasing dietary PE was a goal of dietary management in inborn errors of metabolism.

Two common ingredients in laboratory rodent diets are soy meal and alfalfa. Both of these components contain high levels of dietary PE (9,10). These levels may have physiologic effects (21–23), especially in metabolic studies. Brown et al. (24) demonstrated elevated serum PE levels in both rats and mice fed commercial diets compared with rodents fed a low-PE diet.

Exogenous estrogen may have either physiologic or pharmacologic effects, depending on the dose, preparation, and mode of administration. In ovariectomized female rats fed oral estrogen, serum TG concentrations were elevated (25). This is similar to human studies that demonstrated dyslipidemia in postmenopausal women treated with oral estrogen (26). In contrast, dietary PE were shown to decrease dyslipidemia in postmenopausal women, and transdermal estrogen had a minimal effect on lipids (11,13,21). In the present study, increased TG levels in the genistein-supplemented group mimics what we would predict in rodents receiving oral estrogen therapy rather than a PE dietary regimen. This suggests a pharmacologic rather than a dietary effect of genistein.

In the LCAD–/– mice, the standard diet had a greater influence (P < 0.004) on cold tolerance than did the PE-free diet that was supplemented with genistein (P < 0.07). The standard mouse diet contains many different PE other than genistein, including daidzein and glycitein (Table 2), which may also affect cold challenge. Further studies are warranted to elucidate the potential effects of these PE on cold tolerance in LCAD–/– mice.

Our current study demonstrated the protective effects of PE on cold tolerance in mice with inborn errors of mitochondrial ß-oxidation. These results have implications for dietary treatment of children with inborn errors of metabolism. Perhaps a commercial soy-based infant formula (27) should be considered in the dietary management of human infants with any of the ACAD deficiency diseases. The present results also suggest potentially undesirable effects of isolated genistein supplementation in individuals with genetic defects in fatty acid oxidation. LCAD–/– mice fed a genistein-supplemented diet developed hypertriglyceridemia. Similarly, human patients with inborn errors of mitochondrial fatty acid metabolism may have elevated serum TG after exposure to dietary genistein. On the basis of the serum TG levels in our mice fed the genistein-supplemented diet, serum lipids should be monitored carefully in these patients.

	ACKNOWLEDGMENTS

 
We thank Michael Wyss and Roche Vitamins Ltd., Basel, Switzerland for the kind gift of genistein, Ada Elgavish for assistance with data analysis, and Kenneth Jones for measuring the dietary PE compounds.

	FOOTNOTES

 
¹ Supported by National Institutes of Health grants RO1-RR02599 (P.A.W.), T-32–RR07003 (A.M.S.), P30-DK56336 (P.A.W., B.A.G.) and P50-AT00477 (S.B.).

³ Abbreviations used: ACAD, acyl-CoA dehydrogenases; LCAD, long-chain acyl-CoA dehydrogenase; PE, phytoestrogens; TG, triglycerides.

Manuscript received 29 September 2003. Initial review completed 15 December 2003. Revision accepted 4 February 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Roe, C. & Ding, J. (2001) Mitochondrial fatty acid oxidation disorders. Scriver, C. R. Beaudet, A. L. Sly, W. S. Valle, D. Childs, B. Kinzler, K. W. Vogelstein, B. eds. The Metabolic and Molecular Bases of Inherited Disease 2001:2297-2326 McGraw-Hill New York, NY. .

2. Schuler, A. M. & Wood, P. A. (2002) Mouse models of mitochondrial fatty acid ß-oxidation. Inst. Lab. Anim. Res. J. 43:57-65.

3. Wood, P. A., Amendt, B. A., Rhead, W. J., Millington, D. S., Inoue, F. & Armstrong, D. (1989) Short-chain acyl-coenzyme A dehydrogenase deficiency in mice. Pediatr. Res. 25:38-43.[Medline]

4. Kurtz, D. M., Rinaldo, P., Rhead, W. J., Tian, L., Millington, D. S., Vockley, J., Hamm, D. A., Brix, A. E., Lindsey, J. R., Pinkert, C. A., O’Brien, W. E. & Wood, P. A. (1998) Targeted disruption of a mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation. Proc. Natl. Acad. Sci. U.S.A. 95:15592-15597.[Abstract/Free Full Text]

5. Cox, K. B., Hamm, D. A., Millington, D. S., Matern, D., Vockley, J., Rinaldo, P., Pinkert, C. A., Rhead, W. J., Lindsey, J. R. & Wood, P. A. (2001) Gestational, pathologic, and biochemical differences between very long-chain acyl-CoA dehydrogenase deficiency and long-chain acyl CoA dehydrogenase deficiency in the mouse. Hum. Mol. Genet. 10:2069-2077.[Abstract/Free Full Text]

6. Guerra, C., Koza, R. A., Walsh, K., Kurtz, D. M., Wood, P. A. & Kozak, L. P. (1998) Abnormal nonshivering thermogenesis in mice with inherited defects of fatty acid oxidation. J. Clin. Investig. 102:1724-1731.[Medline]

7. Cox, K. B. & Wood, P. A. (June 14–18, 2000) Factors Influencing Metabolically Induced Cardiac Hypertrophy June 14–18, 2000 International Society for Heart Research Louisville, KY.

8. Winter, S. & Buist, N. (1998) Clinical treatment guide to inborn errors of metabolism. J. Rare Dis. 4:18-46.

9. Thigpen, J. E., Setchell, K.D.R., Goelz, M. F. & Forsyth, D. B. (1999) The phytoestrogen content of rodent diets. Environ. Health Perspect. 107:A182-A183.[Medline]

10. Degen, G. H., Janning, P., Diel, P. & Bolt, H. M. (2002) Estrogenic isoflavones in rodent diets. Toxicol. Lett. 128:145-157.[Medline]

11. Setchell, K.D.R. & Cassidy, A. (1999) Dietary isoflavones: biological effects and relevance to human health. J. Nutr. 129:758S-767S.

12. Patel, R. P., Boersma, B., Crawford, J. H., Hogg, N., Kirk, M., Kalyanaraman, B., Parks, D., Barnes, S. & Darley-Usmar, V. M. (2001) Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging. Free Radic. Biol. Med. 31:1570-1581.[Medline]

13. Anderson, J. W., Johnstone, B. M. & Cook-Newell, M. E. (1995) Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333:276-282.[Abstract/Free Full Text]

14. Coward, L., Smith, M., Kirk, M. & Barnes, S. (1998) Chemical modification of isoflavones in soy foods during cooking and processing. Am. J. Clin. Nutr. 68:1486S-1491S.[Abstract]

15. Barnes, S. (1998) Phytoestrogens and breast cancer. Bailliere’s Clin. Endocrinol. 12:559-579.

16. Urban, D., Irwin, W., Kirk, M., Markiewicz, M. A., Myers, R., Smith, M., Weiss, H., Grizzle, W. E. & Barnes, S. (2001) The effect of isolated soy protein on plasma biomarkers in elderly men with elevated serum prostate specific antigen. J. Urol. 165:294-300.[Medline]

17. Day, J. K., Besch-Williford, C., McMann, T. R., Hufford, M. G., Lubahn, D. B. & MacDonald, R. S. (2001) Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice. Nutr. Cancer 39:226-232.[Medline]

18. Mitchell, J. H., Cawood, E., Kinniburgh, D., Provan, A., Collins, A. R. & Irvine, D. S. (2001) Effect of a phytoestrogen food supplement on reproductive health in normal males. Clin. Sci. (Lond.) 100:613-618.[Medline]

19. Barrett, J. R. (2002) Soy and children’s health: a formula for trouble?. Environ. Health Perspect. 110:A294-A295.[Medline]

20. Sharpe, R. M., Martin, B., Morris, K., Greig, I., McKinnell, C., McNeilly, A. S. & Walker, M. (2002) Infant feeding with soy formula milk: effects on the testis and on blood testosterone levels in marmoset monkeys during the period of neonatal testicular activity, Hum. Reprod. 17:1692-1703.

21. Burke, G. L., Vitolins, M. Z. & Bland, D. (2000) Soybean isoflavones as an alternative to traditional hormone replacement therapy: are we there yet?. J. Nutr. 130:664S-665S.[Medline]

22. Kanno, J., Kato, H., Iwata, T. & Inoue, T. (2002) Phytoestrogen-low diet for endocrine disruptor studies, J. Agric. Food Chem. 50:3883-3885.

23. Barnes, S. (1997) The chemopreventive properties of soy isoflavonoids in animal models of breast cancer. Breast Cancer Res. Treat. 46:169-179.[Medline]

24. Brown, N. M. & Setchell, K.D.R. (2001) Animal models impacted by phytoestrogens in commercial chow: implications for pathways influenced by hormones. Lab. Investig. 81:735-747.[Medline]

25. Gower, B. A., Nagy, T. R., Blaylock, M. L., Wang, C. & Nyman, L. (2002) Estradiol may limit lipid oxidation via Cpt 1 expression and hormonal mechanisms. Obes. Res. 10:167-172.[Medline]

26. Walsh, B. W., Schiff, I., Rosner, B., Greenberg, L., Ravnikar, V. & Sacks, F. M. (1991) Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N. Engl. J. Med. 326:1196-1204.

27. Mendez, M. A., Anthony, M. S. & Arab, L. (2002) Soy-based formulae and infant growth and development: a review. J. Nutr. 132:2127-2130.[Abstract/Free Full Text]

28. Reeves, P. G, Nielsen, F. H. & Fahey, G. C., Jr (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. J. Nutr. 123:1939-1951.

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schuler, A. M. Articles by Wood, P. A. Search for Related Content PubMed PubMed Citation Articles by Schuler, A. M. Articles by Wood, P. A.