Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle.

AMP-activated protein kinase (AMPK) is activated during muscle contraction in response to the increase in AMP and decrease in phosphocreatine (PCr). Once activated, AMPK has been proposed to phosphorylate a number of targets, resulting in increases in glucose transport, fatty acid oxidation, and gene transcription. Although it has been possible to directly observe phosphorylation of one of these targets, acetyl-CoA carboxylase (ACC) in vitro, it has been more difficult to obtain direct evidence of ACC phosphorylation in contracting skeletal muscle. In these experiments using a phosphoserine antibody to ACC and a phosphothreonine antibody to AMPK, evidence was obtained for phosphorylation and activation of ACC in vitro, in gastrocnemius muscle electrically stimulated at different frequencies, and in muscle from rats running on the treadmill. Significant negative linear correlations between phospho-ACC and ACC activity were observed in all models (P < 0.01). The decline in ACC activity was related to the decrease in PCr and the rise in AMP. A relationship between phospho-AMPK (threonine 172) and activity of AMPK immunoprecipitated with anti-alpha(2) subunit antibody preparation was also observed. These data provide the first evidence of a direct link between extent of phosphorylation of these proteins at sites recognized by the antibodies and activity of the enzymes in electrically stimulated muscle and in muscle of rats running on the treadmill.

Immunohistochemical detection of ornithine-decarboxylase in primary and metastatic human breast cancer specimens.

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and > 90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.