bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒08‒30
twenty papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Metabolites. 2020 Aug 25. pii: E345. [Epub ahead of print]10(9):
      Fatty acids are closely involved in lipid synthesis and metabolism in cancer. Their amount and composition are dependent on dietary supply and tumor microenviroment. Research in this subject highlighted the crucial event of membrane formation, which is regulated by the fatty acids' molecular properties. The growing understanding of the pathways that create the fatty acid pool needed for cell replication is the result of lipidomics studies, also envisaging novel fatty acid biosynthesis and fatty acid-mediated signaling. Fatty acid-driven mechanisms and biological effects in cancer onset, growth and metastasis have been elucidated, recognizing the importance of polyunsaturated molecules and the balance between omega-6 and omega-3 families. Saturated and monounsaturated fatty acids are biomarkers in several types of cancer, and their characterization in cell membranes and exosomes is under development for diagnostic purposes. Desaturase enzymatic activity with unprecedented de novo polyunsaturated fatty acid (PUFA) synthesis is considered the recent breakthrough in this scenario. Together with the link between obesity and cancer, fatty acids open interesting perspectives for biomarker discovery and nutritional strategies to control cancer, also in combination with therapies. All these subjects are described using an integrated approach taking into account biochemical, biological and analytical aspects, delineating innovations in cancer prevention, diagnostics and treatments.
    Keywords:  cancer cell membranes; desaturase enzymes; essential fatty acids; fatty acid biomarker; fatty acid biosynthesis; fatty acid signaling; inflammation; molecular nutrition; sapienic acid; sebaleic acid
  2. J Lipid Res. 2020 Aug 26. pii: jlr.RA120000991. [Epub ahead of print]
      Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols.We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent labs assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e. 7 quality control plasmas), standard calibration series and analytical methods. The quantitative analysis is based on a standard calibration series with isotopically labelled internal standards.Using the standardized protocol the technical variance was within ±15% for 73% of oxylipins, however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the labs was examined using consensus value estimates and unsupervised /supervised multivariate analysis (i.e. PCA and PLS-DA). Inter-lab variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas.In summary, we show that using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible and comparable oxylipin concentrations in independent laboratories allowing the generation of biologically meaningful oxylipin patterns.
    Keywords:  Eicosanoids; Leukotrienes; Lipidomics; Mass spectrometry; Oxidized fatty acids; harmonization; liquid chromatography; quantitation
  3. Nat Commun. 2020 Aug 27. 11(1): 4279
      Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.
  4. Mol Cell Proteomics. 2020 Aug 26. pii: mcp.R120.002234. [Epub ahead of print]
      MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.
    Keywords:  Cell biology*; Cell sorting; FAIMS; Label-free quantification; Mass Spectrometry; Tandem Mass Spectrometry; Tissues*; nanoLC; nanoPOTS; single-cell; ultrasensitive
  5. Metabolites. 2020 Aug 25. pii: E342. [Epub ahead of print]10(9):
      In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.
    Keywords:  CSF; LC-MS; LDA; XCMS; metabolomics; plasma
  6. Trends Analyt Chem. 2019 Sep;118 158-169
      Mass spectrometry (MS) plays an important role in seeking biomarkers for disease detection. High-quality quantitative data is needed for accurate analysis of metabolic perturbations in patients. This article describes recent developments in MS-based non-targeted metabolomics research with applications to the detection of several major common human diseases, focusing on study cohorts, MS platforms utilized, statistical analyses and discriminant metabolite identification. Potential disease biomarkers recently discovered for type 2 diabetes, cardiovascular disease, hepatocellular carcinoma, breast cancer and prostate cancer through metabolomics are summarized, and limitations are discussed.
    Keywords:  Disease biomarkers; data analysis; mass spectrometry; non-targeted metabolomics
  7. Metabolomics. 2020 Aug 26. 16(9): 91
      INTRODUCTION: Repurposing of cationic amphiphilic drugs (CADs) emerges as an attractive therapeutic solution against various cancers, including leukemia. CADs target lysosomal lipid metabolism and preferentially kill cancer cells via induction of lysosomal membrane permeabilization, but the exact effects of CADs on the lysosomal lipid metabolism remain poorly illuminated.OBJECTIVES: We aimed to systematically monitor CAD-induced alterations in the quantitative lipid profiles of leukemia cell lines in order to chart effects of CADs on the metabolism of various lipid classes present in these cells.
    METHODS: We conducted this study on eight cultured cell lines representing two leukemia types, acute lymphoblastic leukemia and acute myeloid leukemia. Mass spectrometry-based quantitative shotgun lipidomics was employed to quantify the levels of around 400 lipid species of 26 lipid classes in the leukemia cell lines treated or untreated with a CAD, siramesine.
    RESULTS: The two leukemia types displayed high, but variable sensitivities to CADs and distinct profiles of cellular lipids. Treatment with siramesine rapidly altered the levels of diverse lipid classes in both leukemia types. These included sphingolipid classes previously reported to play key roles in CAD-induced cell death, but also lipids of other categories. We demonstrated that the treatment with siramesine additionally elevated the levels of numerous cytolytic lysoglycerophospholipids in positive correlation with the sensitivity of individual leukemia cell lines to siramesine.
    CONCLUSIONS: Our study shows that CAD treatment alters balance in the metabolism of glycerophospholipids, and proposes elevation in the levels of lysoglycerophospholipids as part of the mechanism leading to CAD-induced cell death of leukemia cells.
    Keywords:  Cancer; Lipidomics; Lysoglycerophospholipids; Lysosomes; Sphingolipids; Systems biology
  8. Metabolites. 2020 Aug 25. pii: E343. [Epub ahead of print]10(9):
      Untargeted metabolomics is expected to lead to a better mechanistic understanding of diseases and thus applications of precision medicine and personalized intervention. To further increase metabolite coverage and achieve high accuracy of metabolite quantification, the present proof-of-principle study was to explore the applicability of integration of two-dimensional gas and liquid chromatography-mass spectrometry (GC × GC-MS and 2DLC-MS) platforms to characterizing circulating polar metabolome extracted from plasma collected from 29 individuals with colorectal cancer in comparison with 29 who remained cancer-free. After adjustment of multiple comparisons, 20 metabolites were found to be up-regulated and 8 metabolites were found to be down-regulated, which pointed to the dysregulation in energy metabolism and protein synthesis. While integrating the GC × GC-MS and 2DLC-MS data can dramatically increase the metabolite coverage, this study had a limitation in analyzing the non-polar metabolites. Given the small sample size, these results need to be validated with a larger sample size and with samples collected prior to diagnostic and treatment. Nevertheless, this proof-of-principle study demonstrates the potential applicability of integration of these advanced analytical platforms to improve discrimination between colorectal cancer cases and controls based on metabolite profiles in future studies.
    Keywords:  2DLC-MS; GC × GC-MS; case-control study; colorectal cancer; untargeted metabolomics
  9. Anal Chem. 2020 Aug 25.
      In silico spectral library prediction of all possible peptides from whole organisms has a great potential for improving proteome profiling by data-independent acquisition (DIA) and extending its scope of application. In combination with other recent improvements in the field of mass spectrometry (MS)-based proteomics, including sample preparation, peptide separation, and data analysis, we aimed to uncover the full potential of such an advanced DIA strategy by optimization of the data acquisition. The results demonstrate that the combination of high-quality in silico libraries, reproducible and high-resolution peptide separation using micropillar array columns, as well as neural network supported data analysis enables the use of long MS scan cycles without impairing the quantification performance. The study demonstrates that mean coefficient of variations of 4% were obtained even at only 1.5 data points per peak (full width at half-maximum) across different gradient lengths, which in turn improved proteome coverage up to more than 8000 proteins from HeLa cells using empirically corrected libraries and more than 7000 proteins using a whole human in silico predicted library. These data were obtained using a Q Exactive orbitrap mass spectrometer with moderate scanning speed (12 Hz) and perform very well in comparison to recent studies using more advanced MS instruments, which underline the high potential of this optimization strategy for various applications in clinical proteomics, microbiology, and molecular biology.
  10. J Biomol Tech. 2020 Aug;31(Suppl): S21
      There is increasing need for throughput as the metabolomics studies are getting larger. Throughput can be achieved on the analytical side by using rapid methods or speeding up the data analysis and metabolite identification steps. Series of rapid UPLC-MS/MS methods have been developed on a single platform with identical analysis workflow for high throughput measurement of derivatized amino acids, acylcarnitines, bile acids, free fatty acids, tryptophan metabolites in human serum to support metabolomics research. The separation of isomers (amino acids and bile acids) are achieved in analytical runtimes of <4mins making these methods powerful and are well suited for a Core laboratory. Here we discuss these methods and demonstrate their usability for the analysis of metabolites in patient derived serum samples during targeted multi-omics analysis. Sample preparation involved protein precipitation with methanol (1:4 serum:methanol) for the extraction of acylcarnitines, bile acids, and free fatty acids. For amino acid analysis, serum samples were prepared using the Waters™ AccQTag Kit following the Kit protocol. Tryptophan metabolites sample preparation was achieved using Oasis HLB PRiME µElution Plate. UPLC separation was performed on an ACQUITY UPLC I-Class System (fixed loop), equipped with a CORTECS T3 2.7 µm (2.1 x 30 mm) analytical column. A 2 µ Lextract was injected at a flow rate of 1.3 mL/min. Mobile phase A was 0.01% formic acid (aq) and Mobile phase B was 50% isopropanol in acetonitrile containing 0.01% formic acid. The LC gradient and column equilibration times were optimized for each class of metabolites. The analytical column temperature was maintained at 60°C. Multiple Reaction Monitoring (MRM) analyses were performed using a Xevo TQ-S micro mass spectrometer. All experiments were performed in electrospray ionization mode. Data processing was done using in TargetLynx and Skyline.
  11. Nat Commun. 2020 Aug 28. 11(1): 4334
      The metabolome includes not just known but also unknown metabolites; however, metabolite annotation remains the bottleneck in untargeted metabolomics. Ion mobility - mass spectrometry (IM-MS) has emerged as a promising technology by providing multi-dimensional characterizations of metabolites. Here, we curate an ion mobility CCS atlas, namely AllCCS, and develop an integrated strategy for metabolite annotation using known or unknown chemical structures. The AllCCS atlas covers vast chemical structures with >5000 experimental CCS records and ~12 million calculated CCS values for >1.6 million small molecules. We demonstrate the high accuracy and wide applicability of AllCCS with medium relative errors of 0.5-2% for a broad spectrum of small molecules. AllCCS combined with in silico MS/MS spectra facilitates multi-dimensional match and substantially improves the accuracy and coverage of both known and unknown metabolite annotation from biological samples. Together, AllCCS is a versatile resource that enables confident metabolite annotation, revealing comprehensive chemical and metabolic insights towards biological processes.
  12. Metabolites. 2020 Aug 26. pii: E346. [Epub ahead of print]10(9):
      Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.
    Keywords:  metabolic reprogramming; metabolomics; microglia; stable-isotope tracing; β-hydroxybutyrate
  13. Nat Commun. 2020 Aug 26. 11(1): 4261
      Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.
  14. Proc Natl Acad Sci U S A. 2020 Aug 25. pii: 202011310. [Epub ahead of print]
      While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.
    Keywords:  differentiation; epidermis; keratinocytes; lipidomics; lipids
  15. Int J Biochem Cell Biol. 2020 Aug 19. pii: S1357-2725(20)30151-5. [Epub ahead of print] 105834
      Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.
    Keywords:  AGMO; AGPS; GPER1; ether lipids; sphingolipids
  16. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Aug 22. pii: S1388-1981(20)30198-0. [Epub ahead of print] 158806
      Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H218O, 18O2 gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.
    Keywords:  catalytic activity; eicosanoids; leukotrienes; oxylipins; reaction specificity; specialized pro-resolving mediators
  17. Nat Commun. 2020 Aug 25. 11(1): 4236
      The impact of commensal bacteria on the host arises from complex microbial-diet-host interactions. Mapping metabolic interactions in gut microbial communities is therefore key to understand how the microbiome influences the host. Here we use an interdisciplinary approach including isotope-resolved metabolomics to show that in Drosophila melanogaster, Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp) a syntrophic relationship is established to overcome detrimental host diets and identify Ap as the bacterium altering the host's feeding decisions. Specifically, we show that Ap uses the lactate produced by Lp to supply amino acids that are essential to Lp, allowing it to grow in imbalanced diets. Lactate is also necessary and sufficient for Ap to alter the fly's protein appetite. Our data show that gut bacterial communities use metabolic interactions to become resilient to detrimental host diets. These interactions also ensure the constant flow of metabolites used by the microbiome to alter reproduction and host behaviour.
  18. Front Endocrinol (Lausanne). 2020 ;11 483
      Ceramide synthases (CerS) are central enzymes required for the de-novo synthesis of ceramides and other sphingolipids. They catalyze the addition of different acyl-chains to a sphingoid base, and thus account for much of the rich diversity in the sphingolipid family. Recent studies have demonstrated that the acyl-chain is an important determinant of ceramide function, such that a small subset of ceramides (e.g., those containing the C16 or C18 acyl-chain) alter metabolism by inhibiting insulin signaling or inducing mitochondrial fragmentation. Herein I discuss the therapeutic potential of targeting certain ceramide synthase isoforms for the treatment of obesity, insulin resistance, steatohepatitis, and other metabolic disorders.
    Keywords:  C16 ceramide; NAFLD; NASH and mitochondrial dysfunction; ceramides; insulin resistance; obesity; sphingolipids
  19. Int J Mol Sci. 2020 Aug 25. pii: E6113. [Epub ahead of print]21(17):
      This review discusses how lipophagy and cytosolic lipolysis degrade cellular lipids, as well as how these pathway ys communicate, how they affect lipid metabolism and energy homeostasis in cells and how their dysfunction affects the pathogenesis of lipid storage and lipid metabolism diseases. Answers to these questions will likely uncover novel strategies for the treatment of aforementioned human diseases, but, above all, will avoid destructive effects of high concentrations of lipids-referred to as lipotoxicity-resulting in cellular dysfunction and cell death.
    Keywords:  TFEB; lipid droplets; lipid metabolism; lipid metabolism diseases; lipid storage diseases; lipolysis; lipophagy; mTORC1