The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.z 1999 Federation of European Biochemical Societies.

The pituitary hormone prolactin (PRL) plays an important role in mammary gland development. It was also suggested to contribute to breast cancer progression. In vivo data strongly supported a crucial role of PRL in promoting tumour growth; however, PRL demonstrated only a weak, if any, pro-proliferative effect on cancer cells in vitro. Several recent studies indicated that PRL action in vivo may be influenced by the hormonal milieu, e.g. other growth factors such as 17b-oestradiol (E 2 ). Here, we explored the potential interplay between PRL and E 2 in regulation of gene expression and cell growth. PRL alone induced either a weak or no proliferative response of T47D and BT-483 cells respectively, while it drastically enhanced cell proliferation in E 2 -stimulated cultures. Affymetrix microarray analysis revealed 12 genes to be regulated by E 2 , while 57 genes were regulated by PRL in T47D cells. Most of the PRL-regulated genes (42/57) were not previously described as PRL target genes, e.g. WT1 and IER3. One hundred and five genes were found to be regulated upon PRL/E 2 co-treatment: highest up-regulation was found for EGR3, RUNX2, EGR1, MAFF, GLIPR1, IER3, SOCS3, WT1 and AREG. PRL and E 2 synergised to regulate EGR3, while multiple genes were regulated additively. These data show a novel interplay between PRL and E 2 to modulate gene regulation in breast cancer cells.

analysis of the UCP2 core promoter and relationships of variants with obesity. Obes Res. 2003;11:1420 -1427. Objective: To identify polymorphisms in the human uncoupling protein 2 gene (UCP2) promoter and to investigate whether these were associated with obesity or weight gain. Research Methods and Procedures:The human UCP2 promoter was characterized by reporter gene analysis in cell lines derived from skeletal muscle, white adipose tissue, and embryonic tissue. We analyzed the core promoter for polymorphisms in 60 obese subjects. A prevalent polymorphism, the -866 G/A variant, was investigated for association with obesity in 749 men obese as young adults and 816 men of the same age representing the background population. Genotype-phenotype interaction studies were performed in two other population-based samples: one group of middle-aged-to-elderly Danish subjects (mean age, 53 years; range, 30 to 88 years) and one group of 60-yearold Danish subjects. Results: The region up to Ϫ1202 bp relative to the UCP2 transcription initiation site gave rise to the highest promoter activity. Eight mutations in this region were identified comprising

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor Vil to salivary glands and that the corresponding peptide is secreted into saliva.

Objective: To investigate whether the promotion of breakdown of body fat and the increased energy expenditure associated with growth hormone (GH) affect the voluntary food intake of an obese organism. Design: Wistar rats (15 months old) were first fed either a high-fat (HF) or a low-fat (LF) diet for 10 weeks. In the subsequent treatment period, two saline groups continued with either the HF or the LF diet, and rats of three other groups had their diet shifted from HF to LF and were treated with saline, human GH (hGH) or rat GH (rGH). hGH and rGH were given in a dose of 4 mg/kg per day. After 21 days of treatment and registration of food intake, rats were killed, blood was collected and tissues were excised. Results: The HF diet produced a significant ðP , 0:05Þ increase in weight of fat pads compared with the LF diet: 69^5 g compared with 48^2 g: The switch from HF to LF diet combined with injections of saline alone decreased the intake of metabolizable energy, but fat pad weight did not decrease significantly (69^5 g compared with 63^6 g). The latter value was significantly ðP , 0:05Þ decreased (to 37^3 g) in groups treated with either hGH or rGH. Both GH treatments increased serum IGF-I and muscle weight, whereas the activity of adipose tissue lipoprotein lipase decreased significantly ðP , 0:01Þ: During the first 9 days of treatment, food intake was significantly ðP , 0:01Þ depressed, from 27^1 g=kg per day in control rats to 14^2 and 16^4 g=kg per day in the hGH and rGH groups respectively. Conclusion: This study demonstrates that breakdown of adipose tissue and a transient decrease in voluntary food intake are parallel consequences of GH treatment in old and obese rats, and that the actions of hGH and rGH are very similar.