BackgroundObesity research focuses essentially on gene targets associated with the obese phenotype. None of these targets have yet provided a viable drug therapy. Focusing instead on genes that are involved in energy absorption and that are associated with a “human starvation phenotype”, we have identified enteropeptidase (EP), a gene associated with congenital enteropeptidase deficiency, as a novel target for obesity treatment. The advantages of this target are that the gene is expressed exclusively in the brush border of the intestine; it is peripheral and not redundant.Methodology/Principal FindingsPotent and selective EP inhibitors were designed around a boroarginine or borolysine motif. Oral administration of these compounds to mice restricted the bioavailability of dietary energy, and in a long-term treatment it significantly diminished the rate of increase in body weight, despite ad libitum food intake. No adverse reactions of the type seen with lipase inhibitors, such as diarrhea or steatorrhea, were observed. This validates EP as a novel, druggable target for obesity treatment.Conclusions In vivo testing of novel boroarginine or borolysine-based EP inhibitors validates a novel approach to the treatment of obesity.
A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin -avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests doublestranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring MgZf over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgClz or MnCl,. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichiu coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharom yces cerevisia have revealed a potential structural homology between these nucleases.
Deoxyribonuclease I1 has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin -agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease I1 was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme.Deoxyribonuclease I1 cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease I1 is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 pM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichiu coli DNA polymerase I.
In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment.
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