Regulation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) catalyses the formation of malonyl-CoA, an essential substrate for fatty acid synthesis in lipogenic tissues and a key regulatory molecule in muscle, brain and other tissues. ACC contributes importantly to the overall control of energy metabolism and has provided an important model to explore mechanisms of enzyme control and hormone action. Mammalian ACCs are multifunctional dimeric proteins (530-560 kDa) with the potential to further polymerize and engage in multiprotein complexes. The enzymatic properties of ACC are complex, especially considering the two active sites, essential catalytic biotin, the three-substrate reaction and effects of allosteric ligands. The expression of the two major isoforms and splice variants of mammalian ACC is tissue-specific and responsive to hormones and nutritional status. Key regulatory elements and cognate transcription factors are still being defined. ACC specific activity is also rapidly modulated, being increased in response to insulin and decreased following exposure of cells to catabolic hormones or environmental stress. The acute control of ACC activity is the product of integrated changes in substrate supply, allosteric ligands, the phosphorylation of multiple serine residues and interactions with other proteins. This review traces the path and implications of studies initiated with Dick Denton in Bristol in the late 1970s, through to current proteomic and other approaches that have been consistently challenging and immensely rewarding.

Glucocorticoid-induced glucose release is abolished in trout hepatocytes with elevated hsp70 content.

The metabolic potential of cells with elevated heat shock protein 70 (hsp70) content was examined by measuring unstimulated and glucocorticoid-stimulated glucose release in trout hepatocytes maintained in primary culture. Exposure of hepatocytes to either heat shock (HS;+15 degrees C) or sodium arsenite (50 microM) did not affect cell viability, but resulted in significantly higher hsp70 levels over a 24 h recovery period. Hsp70 accumulation had no significant impact on unstimulated glucose release, but completely abolished cortisol-induced glucose release in trout hepatocytes. This lack of glucocorticoid responsiveness corresponded with lower glucocorticoid receptor protein levels. Together, our results suggest that stressor-induced hsp70 accumulation, while important for maintaining cellular homeostasis, may impair metabolic adjustments to subsequent stressors in animals, especially those that are glucocorticoid-dependent.

Bimodal activation of acetyl-CoA carboxylase by glutamate.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.

Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes: evidence that cAMP-dependent protein kinase mediates effects of beta-adrenergic stimulation.

Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-alpha and ACC-beta, have been described and the sequences of the isoforms deduced. ACC-beta is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid beta-oxidation. The regulatory properties of ACC-beta are incompletely defined but it is known that some cellular stresses lead to inhibition in parallel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-beta within intact rat cardiac ventricular myocytes. Treatment of myocytes with the beta-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-beta phosphorylation that was maximal within 2 min and with 50 nM agonist. Effects of isoprenaline were revealed by the incorporation of 32P into ACC in cells incubated with [32P]Pi and also by a marked decrease (approx. 80%) in subsequent phosphorylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of tryptic phosphopeptides revealed that ACC-beta was phosphorylated at multiple sites by incubation in vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-beta that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. beta-Adrenergic stimulation led to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenization and partial purification of proteins. The results suggest that: (1) ACC-beta is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after beta-adrenergic stimulation, (2) ACC-beta is phosphorylated in vitro by PKA and AMP-PK at multiple sites, including at least one site accessible to each kinase, as well as kinase-selective sites, and (3) PKA is a physiologically significant ACC-beta kinase.