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AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides.

ABSTRACT: The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-?-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer.

SUBMITTER: Ishibashi Y

PROVIDER: S-EPMC4505067 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides.

Ishibashi Yohei Y Hirabayashi Yoshio Y

The Journal of biological chemistry 20150605 29

The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside a ...[more]

PMID: 26048992

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Project description:Interleukin-1? (IL-1?) and tumor necrosis factor ? (TNF?) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1? and TNF?.Expression of AMPK subunits was examined, and AMPK? activity was ascertained by the phosphorylation status of AMPK? Thr(172) in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL-1? and TNF?, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPK? by small interfering RNA.Normal human knee articular chondrocytes expressed AMPK?1, ?2, ?1, ?2, and ?1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL-1? and TNF?. Knockdown of AMPK? resulted in enhanced catabolic responses to IL-1? and TNF? in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL-1? and TNF? and the capacity of TNF? and CXCL8 (IL-8) to induce type X collagen expression.Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL-1? or TNF?. AMPK activators attenuate dephosphorylation of AMPK? and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation-induced degradation.

Project description:Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase ?1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the ?1 subunit was required for autophagy induction, since in ?1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (?1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the ?1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in ?1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

Dataset Information

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides.

Publications

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides.

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