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1.
Hepatol Commun ; 8(6)2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38780316

ABSTRACT

BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.


Subject(s)
Ethanol , Hepatocytes , Lysosomes , rab5 GTP-Binding Proteins , rab5 GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/genetics , Lysosomes/metabolism , Lysosomes/drug effects , Ethanol/pharmacology , Hepatocytes/metabolism , Hepatocytes/drug effects , Humans , Lipid Droplets/metabolism , Autophagy/drug effects , Animals , Endosomes/metabolism
2.
Gastroenterology ; 164(2): 256-271.e10, 2023 02.
Article in English | MEDLINE | ID: mdl-36272457

ABSTRACT

BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.


Subject(s)
CD4-Positive T-Lymphocytes , Colitis , Mice , Humans , Animals , Lipid Metabolism , T-Lymphocytes, Regulatory/metabolism , Colitis/chemically induced , Colitis/drug therapy , Colitis/genetics , Chromatin , Inflammation , Cholesterol , Lipids , Forkhead Transcription Factors/metabolism
3.
J Cell Sci ; 135(5)2022 03 01.
Article in English | MEDLINE | ID: mdl-35260889

ABSTRACT

Lipophagy is a central cellular process for providing the cell with a readily utilized, high energy source of neutral lipids. Since its discovery over a decade ago, we are just starting to understand the molecular components that drive lipophagy, how it is activated in response to nutrient availability, and its potential as a therapeutic target in disease. In this Cell Science at a Glance article and the accompanying poster, we first provide a brief overview of the different structural and enzymatic proteins that comprise the lipid droplet (LD) proteome and reside within the limiting phospholipid monolayer of this complex organelle. We then highlight key players in the catabolic breakdown of LDs during the functionally linked lipolysis and lipophagy processes. Finally, we discuss what is currently known about macro- and micro-lipophagy based on findings in yeast, mammalian and other model systems, and how impairment of these important functions can lead to disease states.


Subject(s)
Lipid Droplets , Lipolysis , Animals , Autophagy/physiology , Lipid Droplets/metabolism , Lipid Metabolism/physiology , Mammals/metabolism , Phospholipids/metabolism , Proteins/metabolism , Saccharomyces cerevisiae/metabolism
4.
Cancer Res ; 80(22): 4932-4945, 2020 11 15.
Article in English | MEDLINE | ID: mdl-32816911

ABSTRACT

Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone-sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS-HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis. SIGNIFICANCE: KRAS-dependent regulation of HSL biases cells towards lipid storage for subsequent utilization during invasion of pancreatic cancer cells, representing a potential target for therapeutic intervention.See related commentary by Man et al., p. 4886.


Subject(s)
Pancreatic Neoplasms , Sterol Esterase , Humans , Lipase/genetics , Lipid Metabolism/genetics , Lipids , Male , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Sterol Esterase/genetics , Sterol Esterase/metabolism
5.
Mol Biol Cell ; 31(22): 2463-2474, 2020 10 15.
Article in English | MEDLINE | ID: mdl-32845745

ABSTRACT

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.


Subject(s)
Bile Ducts/metabolism , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , Biological Transport , Cell Line , Cell Movement , Cell Polarity/physiology , Endocytosis , Endosomes/metabolism , Epithelium/metabolism , Exosomes/metabolism , Humans , Signal Transduction
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