Interferon-gamma-induced regulation of peroxisome proliferator-activated receptor gamma and STATs in adipocytes.

Interferon-gamma (IFN-gamma) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-gamma on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in 3T3-L1 adipocytes. Although IFN-gamma regulated the expression of several adipocyte transcription factors, IFN-gamma treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-gamma also resulted in a decrease of both CCAAT/enhancer-binding alpha and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARgamma mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARgamma in the presence of IFN-gamma. Our studies clearly demonstrated that the IFN-gamma-induced loss of PPARgamma protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-gamma also inhibited the transcription of PPARgamma, which was accompanied by a decrease in PPARgamma mRNA accumulation. In addition, exposure to IFN-gamma resulted in a substantial increase in STAT 1 expression and a small increase in STAT 3 expression. IFN-gamma treatment of 3T3-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-gamma treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARgamma.

Erratum to Mutation spectrum of 2-chloroethyl methanesulfonate in Drosophila melanogaster premeiotic germ cells' [Mutation Res. 331 (1995) 213-224].

The 2-chloroethyl methanesulfonate (2CIEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC --> AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2CIEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2CIEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2CIEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2CIEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2CIEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2CIEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2CIEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.

Mutation spectrum of 2-chloroethyl methanesulfonate in Drosophila melanogaster premeiotic germ cells.

The 2-chloroethyl methanesulfonate (2ClEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC-->AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2ClEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2ClEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2ClEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2ClEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2ClEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2ClEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2ClEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.

The influence of large deletions on the mutation frequency induced by tritiated water and X-radiation in male Drosophila melanogaster post-meiotic germ cells.

Tritium beta radiation (3H beta-radiation) in the form of tritiated water was used to induce mutations at the alcohol dehydrogenase (Adh) locus in male Drosophila melanogaster post-meiotic germ cells. All 23 Adh null mutations were large deletions (> 20 kb), determined by genetic complementation and Southern blot analyses. 27 Adh null mutations have been induced by 100-kVp X-rays (Aaron, 1979) and have been genetically and molecularly characterized (Ashburner et al., 1982; Chia et al., 1985; LoMonaco et al., 1987; Mahmoud et al., 1991). In contrast to 3H beta-radiation, 100-kVp X-rays induced a bimodal distribution of Adh null mutations, intragenic mutations, < or = 250 bp, and large deletions, > 100 kb. A statistically significant difference was observed between the frequency of large deletions (23/23 or 1.0) induced by 3H beta-radiation and the frequency of large deletions (19/27 or 0.7) induced by 100-kVp X-rays. However, a statistical difference was not observed between the size distribution of the large deletions induced by 3H beta-radiation and X-rays. The relative deletion frequency (RDF) induced by 3H beta-radiation and 100-kVp X-rays was (1.0/0.7 = 1.4). The relative biological effectiveness (RBE) of these two radiation sources was 1.4, determined from the ratio of the regression coefficients of the respective 3H beta-radiation and X-ray sex-linked recessive lethal (SLRL) dose-response data. The large difference in size between the two classes of X-ray-induced Adh null mutations and the increase in mutation frequency and deletion frequency for 3H beta-radiation with respect to X-rays may indicate that the relative deletion frequency (RDF) is the molecular biological basis for the increase in the RBE for radiation sources with a mean LET value < or = 10 keV/microns.

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster.

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. Only one of the X-ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X-ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and "point mutations." Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the break-point with one of the repeats deleted.

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster.

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)