RESUMEN
Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast. Deletion of PLB1, a gene encoding a B-type phospholipase that hydrolyzes PC, in a ypk1Δ mutant curtails the increased PC deacylation. In contrast to previous data, we find that Plb1 resides in the ER and in the medium. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1Δ strain compared with wild type yeast. Furthermore, deletion of PLB1 in a ypk1Δ mutant exacerbates phenotypes associated with loss of YPK1, including slowed growth and sensitivity to cell wall perturbation, suggesting that increased Plb1 activity buffers against the loss of Ypk1. Because Plb1 lacks a consensus phosphorylation site for Ypk1, we probed other processes under the control of Ypk1 that might be linked to PC turnover. Inhibition of sphingolipid biosynthesis by the drug myriocin or through utilization of a lcb1-100 mutant results in increased PLB1 expression. Furthermore, we discovered that the increase in PLB1 expression observed upon inhibition of sphingolipid synthesis or loss of Ypk1 is under the control of the Crz1 transcription factor. Taken together, these results suggest a functional interaction between Ypk1 and Plb1 in which altered sphingolipid metabolism up-regulates PLB1 expression via Crz1.
Asunto(s)
Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Lisofosfolipasa/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Alelos , Colina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos Monoinsaturados/química , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glicerilfosforilcolina/metabolismo , Homeostasis , Hidrólisis , Lípidos/química , Proteínas de la Membrana/metabolismo , Mutación , Fenotipo , Fosforilación , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Saccharomyces cerevisiae produces extracellular glycerophosphoinositol through phospholipase-mediated turnover of phosphatidylinositol and transports glycerophosphoinositol into the cell upon nutrient limitation. A screening identified the RAS GTPase-activating proteins Ira1 and Ira2 as required for utilization of glycerophosphoinositol as the sole phosphate source, but the RAS/cyclic AMP pathway does not appear to be involved in the growth phenotype. Ira1 and Ira2 affect both the production and transport of glycerophosphoinositol.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas Activadoras de GTPasa/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Emerging evidence suggests that impaired regulation of adipocyte lipolysis contributes to the proinflammatory immune cell infiltration of metabolic tissues in obesity, a process that is proposed to contribute to the development and exacerbation of insulin resistance. To test this hypothesis in vivo, we generated mice with adipocyte-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme catalyzing triacylglycerol hydrolysis. In contrast to previous models, adiponectin-driven Cre expression was used for targeted ATGL deletion. The resulting adipocyte-specific ATGL knockout (AAKO) mice were then characterized for metabolic and immune phenotypes. Lean and diet-induced obese AAKO mice had reduced adipocyte lipolysis, serum lipids, systemic lipid oxidation, and expression of peroxisome proliferator-activated receptor alpha target genes in adipose tissue (AT) and liver. These changes did not increase overall body weight or fat mass in AAKO mice by 24 weeks of age, in part due to reduced expression of genes involved in lipid uptake, synthesis, and adipogenesis. Systemic glucose and insulin tolerance were improved in AAKO mice, primarily due to enhanced hepatic insulin signaling, which was accompanied by marked reduction in diet-induced hepatic steatosis as well as hepatic immune cell infiltration and activation. In contrast, although adipocyte ATGL deletion reduced AT immune cell infiltration in response to an acute lipolytic stimulus, it was not sufficient to ameliorate, and may even exacerbate, chronic inflammatory changes that occur in AT in response to diet-induced obesity.
Asunto(s)
Adipocitos/metabolismo , Inflamación/genética , Resistencia a la Insulina/genética , Lipasa/genética , Obesidad/genética , Tejido Adiposo/metabolismo , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Células Dendríticas/metabolismo , Dieta Alta en Grasa/efectos adversos , Expresión Génica , Immunoblotting , Inflamación/sangre , Inflamación/metabolismo , Lipasa/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/sangre , Lipólisis/genética , Hígado/metabolismo , Macrófagos/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A highly sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150 mm × 4.6 mm, 5 µm) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5 mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r² ≥ 0.995) over the range of 0.5-150 nM, with the lower limit of quantitation validated at 0.5 nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between -8.42 to 8.22% and -9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae.