Reversine, a selective MPS1 inhibitor, induced autophagic cell death via diminished glucose uptake and ATP production in cholangiocarcinoma cells.
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ABSTRACT:
SUBMITTER: Prajumwongs P
PROVIDER: S-EPMC7797171 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects the oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cell from women of advanced maternal age, implying a potential negative correlation between autophagy level and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect the oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. In molecular level, we found ATP citrate lyase (Acly), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for the selective autophagic degradation. In human follicles, autophagy levels in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting Acly during oocyte maturation.
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Project description:Macroautophagy/autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cells from women of advanced maternal age, implying a potential negative correlation between autophagy levels and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. Mechanistically, we found that ACLY (ATP citrate lyase), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for selective autophagic degradation. In human follicles, the autophagy level in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting ACLY during oocyte maturation.Abbreviations: 3-MA: 3-methyladenine; ACLY: ATP citrate lyase; AMA: advanced maternal age; CG: cortical granule; CHX: cycloheximide; CQ: chloroquine; CS: citrate synthase; COCs: cumulus-oocyte-complexes; GCM: granulosa cell monolayer; GV: germinal vesicle; MII: metaphase II stage of meiosis; PB1: first polar body; ROS: reactive oxygen species; shRNA: small hairpin RNA; SQSTM1/p62: sequestosome 1; TCA: tricarboxylic acid; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild-type.
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Project description:Background: Systemic glucose homeostasis is affected by adipose cell function. Still, the cellular events preceding the onset of insulin resistance in adipocytes are not yet resolved. To capture the immediate cellular changes during diet-induced expansion of cell volume and number, we characterized the initial changes in mature adipocytes during a short high fat diet (HFD) intervention. Methods: C57BL6/J male mice were fed chow diet, and switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA seq analysis and cell size distribution analyzed using coulter counter technique. Insulin response in isolated adipocytes was measured by glucose uptake and Western blotting of insulin signaling intermediates. Confocal microscopy was used to assess autophagic flux in cells pre-incubated with rapamycin and chloroquine. Results: Switching to HFD was accompanied by an immediate adipose cell size expansion followed by recruitment of new adipocytes. Despite an increase in both basal and insulin-stimulated glucose uptake in adipocytes, we observed a progressive decrease in insulin-stimulated activation of Protein Kinase B (PKB) Ser473, PKB Thr308 and Akt substrate of 160 (AS160) Tyr642, and onset of systemic insulin-resistance already after two days. Moreover, RNA-seq analysis of adipose tissue revealed marked changes in gene expression at day four, of which the most significant included genes directly involved in autophagy (Trp53Inp2 and Beclin1). Enhanced autophagy was observed in isolated adipocytes from HFD-fed mice, detected as an increased density of LC3-positive puncta by confocal microscopy, and increase of LC3II. Conclusions: HFD rapidly caused impaired insulin signal transduction in adipocytes, while both basal and insulin-stimulated glucose uptake increased, which support the concept of spare signaling. Interestingly, our data suggests autophagy as one of the early cellular events that could contribute to insulin resistance.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2023-03-22 | GSE188535 | GEO