Obesity and hyperleptinemia in metallothionein (-I and -II) null mice

J Beattie

A Wood

A Newman

I Bremner

^{Trace Element and Gene Expression Group,}^{Molecular Physiology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, United Kingdom; and}^{Murdoch Institute for Research into Birth Defects, Royal Children’s Hospital, Flemington Road, Parkville, 3052, Australia}

^{To whom reprint requests should be addressed: e-mail:}ku.ca.iras.irr@eittaeB.J.

Communicated by John Waterlow, University of London, London, United Kingdom

Received 1997 Aug 13; Accepted 1997 Nov 17.

Abstract

Metallothionein (MT) has several putative roles in metal detoxification, in Zn and Cu homeostasis, in scavenging free radicals, and in the acute phase response. Mice of mixed 129/Ola and C57BL/6J background with targeted disruption of MT-I and MT-II genes are more sensitive to toxic metals and oxidative stress. We noted that these animals were larger than most strains of mice, and we systematically studied aspects of their physiology and biochemistry relating to energy metabolism. During the first 2 weeks after weaning, the growth rates of MT-null and C57BL/6J mice were similar, but the transgenic mice became significantly heavier at age 5–6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with C57BL/6J mice. Most 22- to 39-week-old male MT-null mice were obese, as shown by increased fat accretion, elevated obese (ob) gene expression, and high plasma leptin levels, similar to those recorded in Zucker fatty (fa/fa) rats. Seven-week-old MT-null mice also had significantly higher levels of plasma leptin and elevated expression of ob, lipoprotein lipase, and CCAAT enhancer binding protein α genes as compared with age-matched C57BL/6J mice. These observations indicate that abnormal accretion of body fat and adipocyte maturation is initiated at 5–7 weeks of age, possibly coincident with sexual maturation. Targeted disruption of MT-I and MT-II genes seems to induce moderate obesity, providing a new obese animal model. A link between MT and the regulation of energy balance is implied.

Abstract

The generation of transgenic mice with targeted gene disruption is very valuable in the study of the physiological function of specific proteins. Nevertheless, a surprising number of such animals show no apparent phenotypic abnormalities and procreate normally. For example, glutathione peroxidase is an important antioxidant, yet cellular glutathione peroxidase-null mice are not apparently disadvantaged in terms of their capacity to resist hyperoxia (1). Metallothioneins comprise a family of highly conserved low M_r metal-binding proteins that are reported to function in the detoxification of heavy metals, in Zn and Cu homeostasis, in the scavenging of free radicals, and in the acute phase response (2–4). However, mice with disrupted MT-I and -II genes are apparently phenotypically normal with no adverse effect on their ability to reproduce and rear offspring (5, 6). Nevertheless, they are more sensitive to metal and oxidant toxicity (5–9), and cells from such animals are less viable than equivalent cells from animals expressing MT-I and -II, when cultured in the presence of various toxic agents. (10, 11).

However, we have noted that mature animals from our colony of MT-null mice with a mixed 129/Ola–C57BL/6J background (5) generally attain higher body mass with larger reserves of fat than C57BL/6J mice. Abnormal energy balance was indicated, and a key link in the feedback control of appetite and energy expenditure is the protein leptin, a product of obese (ob) gene expression in white adipose tissue (12–14). Secreted into the plasma, leptin exerts its biological activity by binding to receptors, particularly in the hypothalamus. Defects in leptin expression or activity can result in obesity, for example in mice with a double recessive mutation of the ob gene (ob/ob mice), which do not produce functional leptin (15), or in Zucker fatty (fa/fa) rats, which have a defective leptin receptor protein (14). In both cases, ob gene expression is elevated, and the animals are hyperphagic, but only Zucker (fa/fa) rats have high levels of circulating leptin (14). Injection of ob/ob mice with recombinant murine leptin reduces food intake, increases energy expenditure, and decreases body weight (16–18).

To evaluate the prevalence of obesity in our MT-null mice, we have systematically studied their growth, food intake, lipid accumulation, the expression of ob gene in white adipose tissue, plasma leptin levels, and other indicators of energy metabolism.

Values are the mean ± SD for six mice and statistical comparisons were made using t tests based on a pooled estimate of error.

* Significantly different from the C57BL/6J control group (P < 0.05).

Acknowledgments

We thank Mr. T. Atkinson for analysis of plasma insulin and also Mrs. D. Bourke and the Rowett Institute animal house staff for helpful advice and assistance with establishment and maintenance of the MT-null mouse colony. Useful discussions with Dr. A. K. West, Biochemistry Department, University of Tasmania, Hobart, Australia, are also appreciated. This work was funded by the Scottish Office Agriculture, Environment, and Fisheries Department, U.K.

Acknowledgments

ABBREVIATIONS

MT	metallothionein
C/EBP α	CCAAT enhancer binding protein
LPL	lipoprotein lipase
Ln	lean
Ave	average
Obs	obese
eWAT	epididymal white adipose tissue

ABBREVIATIONS

References

1. Ho Y S, Magnenat J L, Bronson R T, Cao J, Gargano M, Sugawara M, Funk C D. J Biol Chem. 1997;272:16644–16651.[PubMed]
2. Andrews G K. Prog Food Nutr Sci. 1990;14:193–258.[PubMed]
3. Bremner I, Beattie J H. Annu Rev Nutr. 1990;10:63–83.[PubMed]
4. Beattie J H, Bremner I In: Trace Elements in Man and Animals: 9. Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals. Fischer P W F, L’Abbé M R, Cockell K A, Gibson R S, editors. Ottawa: NRC Research Press; 1997. pp. 367–371. [PubMed][Google Scholar]
5. Michalska A E, Choo K H A. Proc Natl Acad Sci USA. 1993;90:8088–8092.
6. Masters B A, Kelly E J, Quaife C J, Brinster R L, Palmiter R D. Proc Natl Acad Sci USA. 1994;91:584–588.
7. Liu J, Liu Y P, Michalska A E, Choo K H A, Klaassen C D. J Pharmacol Exp Ther. 1996;276:1216–1223.[PubMed]
8. Sato M, Apostolova M D, Hamaya M, Yamaki J, Choo K H A, Michalska A E, Kodama N, Tohyama C. Environ Toxicol Pharmacol. 1996;1:221–225.[PubMed]
9. Kelly E J, Quaife C J, Froelick G J, Palmiter R D. J Nutr. 1996;126:1782–1790.[PubMed]
10. Kondo Y, Woo E S, Michalska A E, Choo K H A, Lazo J S. Cancer Res. 1995;55:2021–2023.[PubMed]
11. Lazo J S, Kondo Y, Dellapiazza D, Michalska A E, Choo K H A, Pitt B R. J Biol Chem. 1995;270:5506–5510.[PubMed]
12. Flier J S. Proc Natl Acad Sci USA. 1997;94:4242–4245.
13. Wolf G. Nutr Rev. 1997;55:85–88.[PubMed]
14. White B D, Martin R J. Proc Soc Exp Biol Med. 1997;214:222–232.[PubMed]
15. Weigle D S, Kuijper J L. BioEssays. 1996;18:867–874.[PubMed]
16. Pelleymounter M A, Cullen M J, Baker M B, Hecht R, Winters D, Boone T, Collins F. Science. 1995;269:540–543.[PubMed]
17. Halaas J L, Gajiwala K S, Maffei M, Cohen S L, Chait B T, Rabinowitz D, Lallone R L, Burley S K, Friedman J M. Science. 1995;269:543–546.[PubMed]
18. Campfield L A, Smith F J, Guisez Y, Devos R, Burn P. Science. 1995;269:546–549.[PubMed]
19. Siu Lo J, Russell C, Taylor A W. J Appl Physiol. 1970;28:234–236.[PubMed]
20. Folch J, Lees M, Sloane Stanley G H. J Biol Chem. 1957;226:497–509.[PubMed]
21. Beattie J H, Black D J, Duncan J S, Wood A M, Trayhurn P. Am J Physiol. 1996;270:R971–R977.[PubMed]
22. Burton K. Biochem J. 1956;62:315–323.
23. Trayhurn P, Duncan J S, Rayner D V. Biochem J. 1995;311:729–733.
24. Trayhurn P, Duncan J S, Nestor A, Thomas M E, Rayner D V. Anal Biochem. 1994;222:224–230.[PubMed]
25. Christy R J, Kaestner K H, Geiman D E, Lane M D. Proc Natl Acad Sci USA. 1991;88:2593–2597.
26. Mehra R K, Bremner I. Biochem J. 1983;213:459–465.
27. Hardie L J, Rayner D V, Holmes S, Trayhurn P. Biochem Biophys Res Commun. 1996;223:660–665.[PubMed]
28. Mercer J G, Lawrence C B, Atkinson T. Physiol Behav. 1996;60:121–127.[PubMed]
29. Greenwood M R C, Cleary M, Steingrimsdottir L, Vasselli J R In: Recent Advances in Obesity Research: III. Björntorp P, Cairella M, Howard A N, editors. London: John Libbey; 1981. pp. 75–79. [PubMed][Google Scholar]
30. Umek R M, Friedman A D, McKnight S L. Science. 1991;251:288–292.[PubMed]
31. Philcox J C, Coyle P, Michalska A, Choo K H A, Rofe A M. Biochem J. 1995;308:543–546.
32. Duffy J Y, Baines D, Keen C L, Daston G P. Teratology. 1997;55:54.[PubMed]
33. He Y, Chen H, Quon M J, Reitman M. J Biol Chem. 1995;270:28887–28891.[PubMed]
34. Hwang C S, Mandrup S, MacDougald O A, Geiman D E, Lane M D. Proc Natl Acad Sci USA. 1996;93:873–877.
35. Ronnekleiv O K, Ojeda S R, McCann S M. Biol Reprod. 1978;19:414–424.[PubMed]
36. Cheung C C, Thornton J E, Kuijper J L, Weigle D S, Clifton D K, Steiner R A. Endocrinology. 1997;138:855–858.[PubMed]
37. Chehab F F, Mounzih K, Lu R, Lim M E. Science. 1997;275:88–90.[PubMed]
38. Yu W H, Kimura M, Walczewska A, Karanth S, McCann S M. Proc Natl Acad Sci USA. 1997;94:1023–1028.
39. Chehab F F, Lim M E, Lu R. Nat Genet. 1996;12:318–320.[PubMed]
40. Muller G, Ertl J, Gerl M, Preibisch G. J Biol Chem. 1997;272:10585–10593.[PubMed]
41. Rofe A M, Philcox J C, Coyle P. Biochem J. 1996;314:793–797.
42. Apostolova M D, Choo K H A, Michalska A E, Tohyama C. J Trace Elem Med Biol. 1997;11:1–7.[PubMed]
43. Coyle P, Philcox J C, Rofe A M. Biochem J. 1995;309:25–31.
44. Zheng H, Liu J, Choo K H A, Michalska A E, Klaassen C D. Toxicol Appl Pharmacol. 1996;136:229–235.[PubMed]
45. Liu J, Liu Y, Michalska A E, Choo K H A, Klaassen C D. J Pharmacol Exp Ther. 1996;276:1216–1223.[PubMed]
46. Kelly E J, Palmiter R D. Nat Genet. 1996;13:219–222.[PubMed]
47. Wang Y, Kuropatwinski K K, White D W, Hawley T S, Hawley R G, Tartaglia L A, Baumann H. J Biol Chem. 1997;272:16216–16223.[PubMed]
48. Hoggard N, Mercer J G, Rayner D V, Moar K, Trayhurn P, Williams L M. Biochem Biophys Res Commun. 1997;232:383–387.[PubMed]
49. Serradeil-Le Gal C, Raufaste D, Brossard G, Pouzet B, Marty E, Maffrand J P, Le Fur G. FEBS Lett. 1997;404:185–191.[PubMed]
50. Olson E N, Arnold H-H, Rigby P W J, Wold B J. Cell. 1996;85:1–4.[PubMed]