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Letters to the Editor

After reading a recent publication by Brodie et al. (1999) in which our work was discussed (Houseknecht et al. 1998 ), we consider it pertinent to clarify the applications of methodologies used so that our data may be interpreted correctly. Most importantly, the clarification of our approaches is important for understanding our hypothesis.

First, with the transactivation assay, we were testing the ability of conjugated linoleic acid (CLA) to induce PPAR activation, not transcription. Indeed, as can be read in the "Research design and methods" section (Houseknecht et al. 1998 ), we transfected CV-1 cells with psG5-PPAR full-length expression vector. Doing so predicts that PPAR is being transcribed and translated in the cell system, thereby "preparing" this transcription factor for activation (i.e., by CLA). Similar to other steroid hormone receptors, PPAR requires activation by a ligand in order to modulate gene expression by interacting with a specific DNA response element (known as PPRE) located upstream of a responsive gene. In our transactivation assay, we cotransfected the cells with pSV-GL2-PPRE-luciferase reporter plasmid and determined the ability of various concentrations of CLA to activate PPAR by measuring activity of the "reporter," luciferase.

Second, we would like to clarify our hypothesis that was incorrectly described by Brodie et al. (1999) when it was stated: "Using a transfection study, they demonstrate that CLA and troglitazone can activate PPAR transcription. They hypothesize from these data that increased PPAR is responsible for increased aP2 mRNA." This statement is completely wrong, as mentioned before, we were not looking at increasing PPAR mRNA in any part of our study. Our hypothesis is that mRNA levels of aP2, a PPAR-responsive gene (see above), were increased as a result of PPAR activation (NOT induction) by CLA.

Third, the authors incorrectly compared our data measuring aP2 mRNA and PPAR-transactivation with work by Okuno et al. (1998) who did not actually measure aP2 mRNA or PPAR activation. In contrast to Brodie's implication that our data do not agree with others, our findings are in close agreement with the dogma that ligands and activators of PPAR result in induction of PPAR-responsive genes such as aP2, in vivo (Pearson et al. 1996 ) and in vitro (Tonotonoz et al. 1994 ).

REFERENCES

1. Brodie A. E., Manning V. A., Ferguson K. R., Jewell D. E., Hu Y. Conjugated linoleic acid inhibits differentiation of pre- and post-confluent 3T3–L1 preadipocytes but inhibits cell proliferation only in preconfluent cells. J. Nutr. 1999;129:602-606[Abstract/Free Full Text]

2. Houseknecht K. L., Vanden Heuvel J. P., Moya-Camarena S. Y., Portocarrero C. P., Peck L. W., Nickel K. P., and Belury M. A. Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat. Biochem. Biophys. Res. Commun. 1998;244:678-682[Medline]

3. Okuno A., Tamemoto H., Tobe K., Ueki K., Mori Y., Iwamoto K., Umesono K., Akanuma Y., Fujiwara T., Horikoshi H., Yazaki Y., and Kadowaki T. Troglitazone increases the number of small adipocytes without change of white adipose tissue mass in obese Zucker rats. J. Clin. Invest. 1998;101:1354-1361[Medline]

4. Pearson S. L., Cawthorne M. A., Clapham J. C., Dunmore S. J., Holmes S. D., Moore G.B.T., Smith S. A., and Tadayyon M. Thiazolidinedione insulin sensitizer BRL 49653 increases the expression of PPAR- and aP2 in adipose tissue of high-fat-fed rats. Biochem. Biophys. Res. Comm. 1996;229:752-757[Medline]

5. Tonotonoz P., Hu E., Graves R. A., Budavari A. I., and Spiegelman B. M. mPPAR2: Tissue-specific regulator of an adipocyte enhancer. Genes & Dev 1994;8:1224-1234[Abstract/Free Full Text]

Response to Drs. Moya-Camarena and Belury

Oregon State University Department of Animal Sciences Corvallis, OR 97331-6702

Dear Dr. Suttie:

We thank Dr. Belury for clarifying their data. The point of their investigation was to show that PPAR is activated by CLA. We did misstate their results to mean that CLA caused more PPAR mRNA. Our confusion came from their discussion where both expression and activation of PPAR were linked with expression of aP2. However, their paper does say "We report that both CLA and TZD significantly increased the steady state level of aP2 mRNA" (p. 681). Even with their interpretation, the aP2 changes which they report do not agree with our data. Therefore, these comments by Drs. Moya-Camarena and Belury do not affect the conclusion of our paper.

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