Alternative splicing of the proadrenomedullin gene results in differential expression of gene products.

The adrenomedullin (AM) gene codifies for two bioactive peptides, AM and proAM N-terminal 20 peptide (PAMP). We have found two forms of the AM mRNA. Form A is devoid of introns and results in a prohormone containing both peptides. Form B retains the third intron, which introduces a premature stop codon, producing a shorter prohormone with only PAMP. Tissues with a higher B/A ratio were more immunoreactive for PAMP than for AM. The form B message was found in the cytoplasmic compartment, thus excluding that the longer message was a result of contaminating nuclear mRNA. Form B was found in cells that express PAMP but not AM. mRNA expression in a variety of cell lines was investigated by ribonuclease protection assay and form B was found in significant amounts in two of them. Treatments that modify AM expression, such as exposure to hypoxia, were shown to change the B/A ratio and the relative secretion of AM and PAMP, indicating that the splicing mechanism for AM can be modulated and is physiologically relevant. Analysis of the sequence of the third intron and the fourth exon of the AM gene found motifs compatible with a highly regulated alternative splicing mechanism.

Structural characterization and tissue-specific expression of the mouse glucose-6-phosphate dehydrogenase gene.

Glucose-6-phosphate dehydrogenase (G6PD) activity differs among tissues and, in liver, with the dietary state of the mouse. Tissue-specific differences in G6PD activity in adipose tissue, liver, kidney, and heart were associated with similar differences in the amount of G6PD mRNA. Regulation of mRNA amount by dietary fat was only observed in liver. In mice fed a low-fat diet, the relative amounts of G6PD mRNA were 3:1:1:0.38, respectively, in the four tissues. Further, the amount of precursor mRNA for G6PD in liver, kidney, and heart reflected the amount of mature mRNA in these tissues, suggesting differing transcriptional activity. Our S1 nuclease and primer-extension analyses indicated that the same transcriptional start site is used in liver, kidney, and adipose tissue, resulting in a common 5' end of the mRNA in these tissues. Thus, differential regulation is not attributable to alternate promoter usage. A DNase hypersensitivity analysis of the 5' end of the G6PD gene identified three hypersensitive sites (HS): HS 1 and HS 2 were present in all tissues, whereas HS 3 was liver specific. Thus, regulation of G6PD expression involves both dietary and tissue-specific signals that appear to act via different mechanisms.

Nutritional regulation of the glucose-6-phosphate dehydrogenase gene is mediated by a nuclear posttranscriptional mechanism.

Expression of the glucose-6-phosphate dehydrogenase (G6PD) gene is inhibited by addition of polyunsaturated fat to a high-carbohydrate diet and stimulated by feeding a high-carbohydrate diet to starved mice. The mechanism of this regulation is posttranscriptional. To define the regulated step, we measured the abundance of G6PD mRNA both in the nucleus and in total RNA. Feeding mice a high-fat diet results in a 70% or greater inhibition of nuclear precursor mRNA (pre-mRNA) and mature mRNA abundance. Amounts of both pre-mRNA and mature mRNA for G6PD are stimulated 13-fold or more by refeeding starved mice. Changes in amount of pre-mRNA for G6PD are of a similar magnitude and precede the changes in amount of mature mRNA for G6PD in total RNA. These changes in pre-mRNA abundance occur in the absence of observable changes in the rate of transport of mRNA from the nucleus to the cytoplasm, splicing of the pre-mRNA, or degradation at the 3'-end of the transcript. Despite large changes in pre-mRNA amount in mice fed a low-fat diet relative to mice fed a high-fat diet, the rate of change in the amount of pre-mRNA during the diurnal feeding cycle is not altered. Thus, expression of G6PD is regulated at an early step after transcription of the pre-mRNA. We suggest that pre-mRNA which enters the processing pathway is stable and can be processed and transported to the cytoplasm where it is translated.

Posttranscriptional regulation of glucose-6-phosphate dehydrogenase by dietary polyunsaturated fat.

The activity of glucose-6-phosphate dehydrogenase (G6PD) is inhibited by the addition of polyunsaturated fat (PUFA) to a high carbohydrate diet. To define the regulated step, we measured enzyme activity, accumulation of G6PD mRNA, and transcriptional activity of the gene. At steady-state, G6PD activity and mRNA abundance were inhibited by 80% in the livers of mice fed a high-fat diet (6% safflower oil) compared to mice fed a low-fat diet (1% safflower oil). Inhibition of mRNA accumulation was 20% by 4 h and was maximal by 9 h after beginning the high-fat diet. Changes in mRNA accumulation preceded changes in enzyme activity, indicating pretranslational regulation. The rapid kinetics of G6PD mRNA accumulation depended on prior dietary history of the mice. In meal-trained mice, abundance of G6PD mRNA increased by twofold within 4 h of the onset of a low-fat meal and was maximal by 12 h. In contrast, an increase in G6PD mRNA was not observed until 12 h after refeeding starved mice and the increase was maximal (12-fold) by 27 h. Transcriptional activity of the gene was measured using the nuclear run-on assay. The G6PD probes were rigorously screened for repetitive elements and for transcription of the noncoding strand of the gene. Throughout the time course of changes in G6PD mRNA accumulation due to PUFA or refeeding, transcriptional activity of the gene did not change. Therefore, regulation of G6PD expression by nutritional status occurs at a posttranscriptional step.