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Genetic Studies of Brown Adipocyte Induction¹

Pennington Biomedical Research Center, Baton Rouge, LA 70808

	ABSTRACT

TOP ABSTRACT Regulation of brown adipocytes REFERENCES

 
We seek to discover an effective method for utilizing thermogenesis to reduce the caloric load in obese individuals. Experimental evidence indicates that nonshivering thermogenesis is the most effective cellular and biochemical mechanism known for reducing excessive adiposity. In this presentation, we describe our experiments aimed at understanding how nonshivering thermogenesis can be induced. In addition, these experiments have led to a genetic approach for the identification of variant genes that coordinate the expression of pathways of gene transcription that are associated with brown adipocyte induction.

KEY WORDS: • brown adipocyte • thermogenesis • quantitative trait locus • obesity • uncoupling protein • transcription factors

	Regulation of brown adipocytes

TOP ABSTRACT Regulation of brown adipocytes REFERENCES

 
Induction of brown adipocytes in traditional white fat depots occurs after adrenergic stimulation by cold or injection of ß3 adrenergic receptor agonists (Champigny et al. 1991 , Collins et al. 1997 , Guerra et al. 1998 , Himms-Hagen et al. 1994 , Loncar et al. 1988 ). Reductions in excess adiposity caused by a high fat diet or mutant genes are substantial in these animals with elevated brown fat expression. Exactly how much of the reduction in adiposity is caused by the thermogenesis of de novo–induced brown adipocytes is unknown. It has been argued that stimulation of thermogenesis as a mechanism to reduce adiposity is ineffective in obese humans because they lack significant numbers of brown adipocytes. The discovery of genetic variability in the induction of brown adipocytes in white fat tissues of inbred strains of mice has permitted a genetic analysis to determine the number and location of genes that are involved in the induction of brown adipocytes. It is possible that such genetic variability also exists among humans (Guerra et al. 1998 ). It is our expectation that understanding the pathways that regulate the induction of brown adipocytes will provide new steps in the differentiation pathways by which drugs can modulate expression (Kozak and Harper 2000 ).

Four quantitative trait loci (QTL)² have been identified in the mouse that control the level of uncoupling protein 1 (Ucp1) mRNA in the retroperitoneal fat depot. These genes are located on chromosomes 2, 3, 8 and 19. Exactly how they function to determine such large differences in the induction of brown adipocytes among strains of mice remains to be determined. Although the pattern of induction of brown adipocytes among the various white fat depots is different, it is unclear whether different genes will be involved in each tissue. It is clear, however, that the mechanisms controlling induction of brown adipocytes in white fat are quite different from those that control expression in interscapular brown fat. No differences can be detected in the induction of Ucp1 mRNA in the brown fat of A/J and C57BL/6J mice.

Although many QTL associated with the obesity phenotype have been mapped in mice, rats and humans, none have yet been identified or positionally cloned. Accordingly, strategies for the successful identification of these important genes must still be developed. We think that the induction of brown adipocytes is a model system upon which strategies for cloning QTL can be developed. A major reason is that the simplicity of the phenotype, i.e., induction of a mRNA is the genetic endpoint of a complex inductive pathway. In addition, much is known about this pathway for inducing Ucp1 expression in brown adipocytes. The Ucp1 gene has been cloned and several motifs that are involved in the expression of the gene have been identified in the 5' flanking region of the gene. The Ucp1 gene is regulated by adrenergic activity through adenyl cyclase and the protein kinase A signal transduction pathway. This pathway appears to induce the expression of the Pgc1 gene that encodes a coactivator that interacts with peroxisome proliferator–activated receptor (PPAR) to regulate transcription of Ucp1. Evidence has also been presented showing that PGC1 also interacts with the nuclear respiratory factors (NRF)1 and NRF2 to regulate mitochondrial biogenesis, the second major phenotype of the brown adipocyte. Thus, pathways for the regulation of two of the major subphenoytpes of the brown adipocyte, Ucp1 expression and mitochondrial biogenesis, have been described.

The logical first question to ask is whether any of these transcription factors are encoded by the QTL controlling Ucp1 expression. The available linkage information in the Jackson laboratory home page http://www.informatics.jax.org does not place any of the signaling molecules or transcription factors associated with gene expression in brown fat in the chromosomal region of our QTL. Because the linkage of Pgc1 had not been determined, Wolfgang Hofmann in our laboratory identified a single strand conformational polymorphism and mapped Pgc1 to Chromosome 5 at 23 cM. Consequently, none of the QTL encode known regulatory molecules. Although these regulatory molecules have not been mapped to any of the QTL, the Ucp1 structural gene on chromosome 8 is located close to the Iba3 QTL. We are evaluating whether the QTL on chromosome 8 is the Ucp1 gene.

Having established that transcriptional factors and signaling molecules known to be implicated in Ucp1 and brown adipocyte expression are not encoded by the Iba QTL, we next began experiments to determine whether they could control the expression of these molecules as well as Ucp1. We approached this goal by first determining whether the level of the mRNA for Pgc1, Nrf1 and Pparg varied in the retroperitoneal fat and brown fat of A/J and C57BL/6J mice after exposure to cold for between 3 h and 7 d. No differences were detected for Pparg mRNA; however, the levels of expression for Pgc1 and Nrf1 were approximately twofold higher in A/J mice. It must be noted that unlike Ucp1, which is restricted in expression to the brown adipocyte, Nrf1 and Pgc1 are also expressed in other cell types. The differences in the brown adipocytes may actually be much greater. Encouraged by these data, we began an analysis of Pgc1 and Nrf1 mRNA levels in the retroperitoneal fat of 2-mo-old (A/J x B6)F1 x A/J backcross male mice. The 10% highs and lows from 400 mice were selected for a genome-wide scan to identify chromosomal regions associated with mRNA levels. Pgc1 mRNA levels showed very significant linkage to chromosomes 6 and 3, but not to the other chromosomes associated with Ucp1 expression, i.e., 2, 8 and 19. Surprisingly, Ucp1 also showed significant linkage to chromosome 6 in this new cohort of mice that were generated after a move of our laboratory from the Jackson Laboratory to the Pennington Biomedical Research Center. A similar genome wide scan of Nrf1 showed significant linkage to chromosome 6 only in a region coincident with that associated with Ucp1 and Pgc1 expression.

Our interpretation of the genetic analysis is that chromosomes 3 and 6 are the locations of genes that control the level of Pgc1 and Nrf1 expression. The elevated expression of these transcription factors increases Ucp1 expression, thereby accounting for the positive correlation between genes on chromosomes 3 and 6 with elevated Ucp1 expression. One could also speculate as follows from these data and line of reasoning: because Nrf1 is not influenced by chromosome 3, whereas Pgc1 is, Nrf1 regulates expression of Pgc1 in brown adipocyte induction in vivo. Such a model is at variance with current data emerging from studies of immortalized cell culture lines showing induction of Nrf1 in cells transfected with Pgc1 expression vectors (Wu et al. 1999 ). Additional work is required before these complicated regulatory pathways are understood.

	FOOTNOTES

 
¹ Presented as part of the symposium "Adipocyte Function, Differentiation and Metabolism" given at the Experimental Biology 00 meeting, San Diego, CA on April 16, 2000. This symposium was sponsored by the American Society for Nutritional Sciences and was supported by educational grants from Dupont Pharmaceuticals, Pfizer, Inc. and Zen-Bio, Inc. The proceedings of this symposium are published as a supplement to The Journal of Nutrition. The guest editor for the symposium publication was Naima Moustaid-Moussa, University of Tennessee, Knoxville, TN.

² Abbreviations used: NRF, nuclear respiratory factor; PGC, PPAR gamma coactivator; PPAR, peroxisome proliferator–activated receptor ; QTL, quantitative trait locus; UCP, uncoupling protein.

	REFERENCES

TOP ABSTRACT Regulation of brown adipocytes REFERENCES

 

1. Champigny O., Ricquier D., Blondel O., Mayers R. M., Briscoe M. G., Holloway B. R. Beta 3-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs. Proc. Natl. Acad. Sci. U.S.A. 1991;88:10774-10777[Abstract/Free Full Text]

2. Collins S., Daniel K. W., Petro A. E., Surwit R. S. Strain-specific response to beta 3-adrenergic receptor agonist treatment of diet-induced obesity in mice. Endocrinology 1997;138:405-413[Abstract/Free Full Text]

3. Guerra C., Koza R. A., Yamashita H., Walsh K., Kozak L. P. Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity. J. Clin. Investig. 1998;102:412-420[Medline]

4. Himms-Hagen J., Cui J., Danforth E., Jr, Taatjes D. J., Lang S. S., Waters B. L., Claus T. H. Effect of CL-316,243, a thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats. Am. J. Physiol. 1994;266:R1371-R1382[Abstract/Free Full Text]

5. Kozak L. P., Harper M.-E. Mitochondrial uncoupling proteins in energy expenditure. Annu. Rev. Nutr. 2000;20:339-363[Medline]

6. Loncar D., Afzelius B. A., Cannon B. Epididymal white adipose tissue after cold stress in rats. I. Nonmitochondrial changes. J. Ultrastruct. Mol. Struct. Res. 1988;101:109-122[Medline]

7. Wu Z., Puigserver P., Andersson U., Zhang C., Adelmant G., Mootha V., Troy A., Cinti S., Lowell B., Scarpulla R. C., Spiegelman B. M. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999;98:115-124[Medline]

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