Abstract
5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.
Highlights
Phospholipid in bile and in plasma lipoproteins, and is a precursor for the synthesis of sphingomyelin and phosphatidylserine
Line Kinase in Hepatocytes—Our results far suggest that these results indicate that AICAr inhibits PC biosynthesis inde- AICAr reduces PC biosynthesis by inhibiting choline kinase pendently of AMPK activation
Mechanism of Choline Kinase Inhibition by AICAr—Our results indicate that inhibition of choline kinase is responsible for decreased PC biosynthesis through the Kennedy pathway in the presence of AICAr
Summary
Phospholipid in bile and in plasma lipoproteins, and is a precursor for the synthesis of sphingomyelin and phosphatidylserine. The last enzyme in the pathway, CDP-choline:1,2-diacylglycerol cholinephosphotransferase, is in excess in cells and does not determine the rate of PC biosynthesis. This reaction requires the availability of diacylglycerol, which is synthesized from fatty acids and glycerol. AICAr Inhibits Hepatic PC Biosynthesis depletion, such as hypoxia and glucose deprivation, AMPK activates ATP-generating pathways (i.e. lipolysis and glycolysis) while inhibiting anabolic processes, including fatty acid, cholesterol, and triacylglycerol biosynthesis. Under normal conditions, these metabolic changes protect the cell against ATP deprivation. In contrast to our hypothesis, we found that AICAr does inhibit PEMT and choline kinase flux, it does so independently of AMPK activation
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