James L. Gamble

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase β (ACC-β), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-β was first purified by immunoprecipitation with a monoclonal antibody. ACC-β was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and aK m for acetyl-CoA (120 μm) similar to that reported for ACC-β purified by other means. After 5 min of contraction, ACC-β activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-β with purified protein phosphatase 2A abolished the decreases in both ACC-β activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-β activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60–90 min) were paralleled temporally by reciprocal changes in the activity of the α2 but not the α1 isoform of 5′-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the α2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

Posttranslational Modifications of the 5′-AMP-activated Protein Kinase β1 Subunit

The AMP-activated protein kinase (AMPK) consists of catalytic α and noncatalytic β and γ subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK α1 and α2 catalytic subunits are associated with β1 and γ1 noncatalytic subunits. We find that the isolated γ1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated β1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable β1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated β1subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylatedin vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that α1 Thr172 is a major, although not exclusive, site of both basal and stimulated α1phosphorylation by an upstream AMPK kinase.

THE FACTORS IN THE DEHYDRATION FOLLOWING PYLORIC OBSTRUCTION

The data presented in this paper were obtained from several dogs following experimental obstruction of the pylorus. The experiments were undertaken with the purpose of learning the chief factors in the causation of the rapid dehydration which follows pyloric obstruction and of explaining the prevention or repair of this change which is obtained by introducing sodium chloride and water into the body. In undertaking to investigate the cause of a reduction of the volume of body water it should at once be admitted that we have as yet only a fragmentary knowledge of the many parts of the regulatory mechanism which under usual circumstances accomplishes the remarkable adjustment of maintaining, in several elastic compartments, a closely stationary total volume of water. Certain gross data are, however, at hand which fairly satisfactorily serve the purposes of this study. It has been shown that in the presence of considerable reductions of the volume of body water the total concentration of dissolved electrolytes tends to remain stationary (1, 2). It is therefore probably permissible to postulate a close dependence of the volume of body water on the total quantity of dissolved electrolytes which the body contains. In this study the premise is used that a withdrawal of the electrolytes of the body fluids will be accompanied by a proportionate reduction of the volume of body water and that this change can only be repaired by replacing both the lost water and the lost electrolytes. The relative structural importance of each of the electrolytes in blood plasma is indicated by the diagram in figure 1, which presents

A STUDY OF THE EFFECTS OF PYLORIC OBSTRUCTION IN RABBITS

In a recent paper Gamble and Ross (1) have demonstrated that, in the presence of pyloric obstruction in the dog, there occurs a loss in vomited gastric secretions of sodium as well as of chloride ion and water. They have shown the relationship of this loss of base to changes in the chemical structure of blood plasma and its significance from the point of view of the reparative action of injections of NaCl solution. In this paper are presented measurements of the amounts of water, fixed base and chloride found in the stomachs of rabbits following experimental obstruction of the pylorus. These measurements were obtained with the purpose of determining quantitatively the extent of the loss into the stomach of each of these important components of the body fluids during the survival period following pyloric obstruction. Rabbits were used for the reason that in these animals the vomiting reflex is absent. Their stomachs will therefore conveniently collect for measurement substances leaving the body in the gastric secretions and contamination of this material by urine or feces is dependably avoided. It was found, however, that the collection period following obstruction could not be begun with an empty stomach. More than a week of fasting is required to completely empty a rabbits stomach. The usual content is of a stiff texture and attempts to either wash it out or remove it surgically without considerably traumatizing the stomach were unsuccessful. The plan used in estimating