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 ␤ (ACC-␤), 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-␤ was first purified by immunoprecipitation with a monoclonal antibody. ACC-␤ was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a K m for acetyl-CoA (120 M) similar to that reported for ACC-␤ purified by other means. After 5 min of contraction, ACC-␤ 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 MgCl 2 or treatment of immunoprecipitated ACC-␤ with purified protein phosphatase 2A abolished the decreases in both ACC-␤ activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-␤ 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 ␣2 but not the ␣1 isoform of 5-AMPactivated 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 ␣2 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.In tissues such as liver (1) and heart (2, 3), malonyl-CoA regulates fatty acid oxidation by inhibiting carnitine palmitoyl transferase I, the enzyme that catalyzes the transfer of cytosolic long chain fatty acyl-CoA into mitochondria. Evidence has been presented that it plays a similar role in skeletal muscle (4 -7), although definitive evidence is still lacking (see "Discussion"). Malonyl-CoA is synthesized from cytosolic acetyl-CoA by a reaction catalyzed by acetyl-CoA carboxylase (ACC).1 In liver, the principal ACC ...

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspasedependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders.degenerations | necroptosis P hotoreceptor death and subsequent visual decline occurs when the photoreceptors are separated from the underlying retinal pigment epithelium. Physical separation of photoreceptors is seen in various retinal disorders, including age-related macular degeneration (1), diabetic retinopathy (2), as well as rhegmatogenous (i.e., caused by a break in the retina) retinal detachment (3, 4). Although surgery is carried out to reattach the retina, only two fifths of patients with rhegmatogenous retinal detachment involving the macula, a region essential for central vision, recover 20/40 or better vision because of photoreceptor death (4, 5). Thus, identification of the mechanisms that underlie photoreceptor death is critical to developing new treatment strategies for these diseases.Apoptosis and necrosis are two major cell death modalities (6). Apoptosis is a highly regulated process involving the caspase family of cysteine proteases. In contrast, necrosis has been considered a passive, unregulated form of cell death; however, recent evidence indicates that some necrosis can be induced by regulated signal transduction pathways such as those mediated by receptor interacting protein (RIP) kinases, especially in conditions in which caspases are inhibited or cannot be activated efficiently (7,8). Stimulation of the Fas and TNFR family of death domain receptors is known to mediate apoptosis in most cell types through the activation of the extrinsic caspase pathway. In addition, in certain cells deficient for caspase-8 or treated with the pan-caspa...