Publications
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Amrutkar, Manoj; Li, Yuchuan; Vefferstad, Anette; Verbeke, Caroline Sophie & Gladhaug, Ivar Prydz
(2025).
Proteomic Characterization of Primary Human Pancreatic Cancer Cell Lines Following Long-Term Exposure to Gemcitabine.
Proteomes.
13(4),
p. 1–22.
doi:
10.3390/proteomes13040048.
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Background: Gemcitabine (GEM) remains a cornerstone in the treatment of pancreatic cancer. Upon exposure to GEM, pancreatic cancer cells (PCCs) tend to adapt quickly to outcompete drug-induced cytotoxicity, thereby contributing to treatment failure. Thus, understanding GEM-induced molecular changes in PCCs is important.
Methods: Three primary PCC lines (PCC-1, PCC-2, PCC-7) and Mia PaCa-2 cultured for 40 passages (p) in the absence (control) or presence of GEM (GemR) were assessed for phenotypic changes. Proteome profiles for all PCCs at p10, p20, p25, p30, p35, and p40 were obtained using mass spectrometry (MS). Protein expression was determined using immunoblotting. Differentially abundant proteins (DAPs) were evaluated for enrichment of functional and biological attributes and protein–protein interactions.
Results: GEM sensitivity and growth were both reduced in GemR versus paired controls for all four PCC lines. MS mapped > 7000 proteins in each PCC line, and the abundance of 70–83% of these was found to be significantly altered when comparing all sample groups. Proteomic changes in GemR versus paired controls differed remarkably among the PCCs and were affected by passaging and treatment duration. DAPs at p40 were mostly related to metabolic pathways, including nucleotide metabolism and diverse cell growth processes. Several closely related DAPs and multiple hub proteins in each PCC line were identified.
Conclusions: Overall, this study revealed cell-line-specific, heterogeneous changes in proteome profiles of PCCs following their long-term exposure to GEM, and these were likely affected by treatment duration, dosage, and passaging.
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Li, Yuchuan; Amrutkar, Manoj; Finstadsveen, Anette V.; Dalen, Knut Tomas; Verbeke, Caroline Sophie & Gladhaug, Ivar Prydz
(2023).
Fatty acids abrogate the growth-suppressive effects induced by inhibition of cholesterol flux in pancreatic cancer cells.
Cancer Cell International.
23.
doi:
10.1186/s12935-023-03138-8.
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Background Despite therapeutic advances, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. Metabolic reprogramming is increasingly recognized as a key contributor to tumor progression and therapy resistance in PDAC. One of the main metabolic changes essential for tumor growth is altered cholesterol flux. Targeting cholesterol flux appears an attractive therapeutic approach, however, the complex regulation of cholesterol balance in PDAC cells remains poorly understood. Methods The lipid content in human pancreatic duct epithelial (HPDE) cells and human PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) was determined. Cells exposed to eight different inhibitors targeting different regulators of lipid flux, in the presence or absence of oleic acid (OA) stimulation were assessed for changes in viability, proliferation, migration, and invasion. Intracellular content and distribution of cholesterol was assessed. Lastly, proteome profiling of PANC-1 exposed to the sterol O-acyltransferase 1 (SOAT1) inhibitor avasimibe, in presence or absence of OA, was performed. Results PDAC cells contain more free cholesterol but less cholesteryl esters and lipid droplets than HPDE cells. Exposure to different lipid flux inhibitors increased cell death and suppressed proliferation, with different efficiency in the tested PDAC cell lines. Avasimibe had the strongest ability to suppress proliferation across the three PDAC cell lines. All inhibitors showing cell suppressive effect disturbed intracellular cholesterol flux and increased cholesterol aggregation. OA improved overall cholesterol balance, reduced free cholesterol aggregation, and reversed cell death induced by the inhibitors. Treatment with avasimibe changed the cellular proteome substantially, mainly for proteins related to biosynthesis and metabolism of lipids and fatty acids, apoptosis, and cell adhesion. Most of these changes were restored by OA. Conclusions The study reveals that disturbing the cholesterol flux by inhibiting the actions of its key regulators can yield growth suppressive effects on PDAC cells. The presence of fatty acids restores intracellular cholesterol balance and abrogates the alternations induced by cholesterol flux inhibitors. Taken together, targeting cholesterol flux might be an attractive strategy to develop new therapeutics against PDAC. However, the impact of fatty acids in the tumor microenvironment must be taken into consideration.
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Doncheva, Atanaska Ivanova; Li, Yuchuan; Khanal, Prabhat; Hjorth, Marit; Kolset, Svein Olav & Norheim, Frode Amador
[Show all 8 contributors for this article]
(2023).
Altered hepatic lipid droplet morphology and lipid metabolism in fasted Plin2-null mice.
Journal of Lipid Research.
ISSN 0022-2275.
64(12).
doi:
10.1016/j.jlr.2023.100461.
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Perilipin 2 (Plin2) binds to the surface of hepatic lipid droplets (LDs) with expression levels that correlate with triacylglyceride (TAG) content. We investigated if Plin2 is important for hepatic LD storage in fasted or high-fat diet-induced obese Plin2+/+ and Plin2−/− mice. Plin2−/− mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels compared with Plin2+/+ mice, regardless of the dietary regime. Both fasted and high-fat fed Plin2−/− mice stored reduced levels of hepatic TAG compared with Plin2+/+ mice. Fasted Plin2−/− mice stored fewer but larger hepatic LDs compared with Plin2+/+ mice. Detailed hepatic lipid analysis showed substantial reductions in accumulated TAG species in fasted Plin2−/− mice compared with Plin2+/+ mice, whereas cholesteryl esters and phosphatidylcholines were increased. RNA-Seq revealed minor differences in hepatic gene expression between fed Plin2+/+ and Plin2−/− mice, in contrast to marked differences in gene expression between fasted Plin2+/+ and Plin2−/− mice. Our findings demonstrate that Plin2 is required to regulate hepatic LD size and storage of neutral lipid species in the fasted state, while its role in obesity-induced steatosis is less clear.
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Li, Yuchuan; Khanal, Prabhat; Norheim, Frode; Hjorth, Marit; Bjellaas, Thomas & Drevon, Christian A
[Show all 9 contributors for this article]
(2021).
Plin2 deletion increases cholesteryl ester lipid droplet content and disturbs cholesterol balance in adrenal cortex.
Journal of Lipid Research.
ISSN 0022-2275.
62.
doi:
10.1016/J.JLR.2021.100048.
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Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2−/− mice. Adrenal glands of Plin2−/− mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2+/+ and Plin2−/− mice. Plin2−/− adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2−/− mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.
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Li, Yuchuan; Torp, May-Kristin; Norheim, Frode; Khanal, Prabhat; Kimmel, Alan R. & Stensløkken, Kåre-Olav
[Show all 8 contributors for this article]
(2020).
Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids.
ISSN 1388-1981.
1866(4).
doi:
10.1016/j.bbalip.2020.158873.
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Plin5 is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia. Cardiomyocytes isolated from Plin5-/- mice cultured with oleic acid stored less LDs than Plin5+/+, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia compared to Plin5+/+. In isolated, perfused hearts, deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight, and apart from modest elevated expression of mRNAs for beta myosin heavy chain Myh7 and the fatty acid transporter Cd36, other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content of isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in mouse models during myocardial ischemia.
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Mariero, Lars Henrik; Torp, May-Kristin; Heiestad, Christina Mathisen; Baysa, Anton; Li, Yuchuan & Valen, Guro
[Show all 8 contributors for this article]
(2019).
Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation.
British Journal of Pharmacology.
ISSN 0007-1188.
176(22),
p. 4360–4372.
doi:
10.1111/bph.14830.
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Background and purpose Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage‐associated‐molecular‐pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll‐like receptor 9 (TLR9). Experimental Approach We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia‐reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA. Key results Expression of the pro‐inflammatory cytokines IL‐1β and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia‐reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL‐1β/TNFα and the nucleolin inhibitor AS1411 reduced interleukin‐6 release in adult mouse cardiomyocytes. mtDNA bound 10‐fold stronger than nDNA to nucleolin. In HEK293‐NF‐κB reporter cells, mtDNA induced NF‐κB activity in normoxia, while CpG‐DNA and hypoxia‐reoxygenation, synergistically induced TLR9‐dependent NF‐κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG‐DNA. Inhibition of endocytosis did not reduce CpG‐DNA uptake in cardiomyocytes. Conclusion and implications mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia‐reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation.
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Lund, Jenny; Helle, Siw A; Li, Yuchuan; Løvsletten, Nils Gunnar; Stadheim, Hans Kristian & Jensen, Jørgen
[Show all 9 contributors for this article]
(2018).
Higher lipid turnover and oxidation
in cultured human myotubes from
athletic versus sedentary young
male subjects.
Scientific Reports.
8(1).
doi:
10.1038/s41598-018-35715-7.
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In this study we compared fatty acid (FA) metabolism in myotubes established from athletic and
sedentary young subjects. Six healthy sedentary (maximal oxygen uptake (VO2max)≤ 46 ml/kg/min)
and six healthy athletic (VO2max> 60 ml/kg/min) young men were included. Myoblasts were cultured
and diferentiated to myotubes from satellite cells isolated from biopsy of musculus vastus lateralis. FA
metabolism was studied in myotubes using [14C]oleic acid. Lipid distribution was assessed by thin layer
chromatography, and FA accumulation, lipolysis and re-esterifcation were measured by scintillation
proximity assay.Gene and protein expressions were studied. Myotubes from athletic subjects showed
lower FA accumulation, lower incorporation of FA into total lipids, triacylglycerol (TAG), diacylglycerol
and cholesteryl ester, higherTAG-related lipolysis and re-esterifcation, and higher complete oxidation
and incomplete β-oxidation of FA compared to myotubes from sedentary subjects. mRNA expression
of the mitochondrial electron transport chain complex III gene UQCRB was higher in cells from athletic
compared to sedentary. Myotubes established from athletic subjects have higher lipid turnover and
oxidation compared to myotubes from sedentary subjects.Our fndings suggestthat cultured myotubes
retain some of the phenotypic traits of their donors.
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Feng, Yuan Zeng; Lund, Jenny; Li, Yuchuan; Knabenes, Irlin; Bakke, Siril Skaret & Kase, Eili Tranheim
[Show all 11 contributors for this article]
(2017).
Loss of perilipin 2 in cultured myotubes enhances lipolysis and redirects the metabolic energy balance from glucose oxidation towards fatty acid oxidation.
Journal of Lipid Research.
ISSN 0022-2275.
58(11),
p. 2147–2161.
doi:
10.1194/jlr.M079764.
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Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD coating protein in skeletal muscle, but its importance for muscle function is unclear. We show that myotubes established from Plin2−/− mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) due to elevated LD hydrolysis in comparison with Plin2+/+ myotubes. The reduced ability to store TAG in LDs in Plin2−/− myotubes is accompanied by a shift in energy metabolism. Plin2−/− myotubes are characterized by increased oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation in comparison with Plin2+/+ myotubes, perhaps reflecting competition between FAs and glucose as part of the Randle cycle. In accord with these metabolic changes, Plin2−/− myotubes have elevated expression of Ppara and Ppargc1a, transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. Loss of Plin2 had no impact on insulin-stimulated Akt phosphorylation. Our results suggest that Plin2 is essential for protecting the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of stored TAG and to balance skeletal muscle energy metabolism. This article was originally published in the Journal of Lipid Research. © 2017 American Society for Biochemistry and Molecular Biology
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Li, Yuchuan; Hessvik, Nina Pettersen; Danbolt, Niels Christian & Holen, Torgeir
(2016).
A large-scale quantitative EM study on activation of olfactory glands shows no effect of cholinergic agents.
Journal of Electron Microscopy.
ISSN 0022-0744.
65(5),
p. 438–443.
doi:
10.1093/jmicro/dfw028.
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Little is known about olfactory glands' regulation despite their presumed importance for normal functioning of the cilia of olfactory neurons. The aim of this study was to establish an assay for olfactory gland activation by using large-scale quantitative electron microscopy (EM). In addition we wanted to test the hypothesis that cholinergic drugs activate the olfactory glands, by using our newly established EM assay. In total, over 70 000 secretory gland vesicles were quantified in over 3000 cells. Olfactory gland cell size (40.8 µm2 ± 2.0 SD), vesicle diameter (812 nm ± 57 SD) and vesicles per cell (21.6 ± 4.2 SD) were also quantified. The vesicle percentage of the cell area varied between 24% and 30%. In a blinded study we found no significant effects of cholinergic agents on parameters of vesicle number or vesicle diameter. Unexpectedly, pilocarpine treatment increased olfactory gland size, probably by inducing cell swelling. In conclusion, we have established a quantitative EM assay for olfactory gland activation and provided new data on basic olfactory gland cell characteristics. By using the EM assay, olfactory glands are shown not to be activated by cholinergic agents, which indicates an alternative regulation pathway or constitutive secretion from olfactory glands.
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Li, Yuchuan; Zhou, Yun & Danbolt, Niels Christian
(2012).
The Rates of Postmortem Proteolysis of Glutamate Transporters Differ Dramatically between Cells and between Transporter Subtypes.
Journal of Histochemistry and Cytochemistry.
ISSN 0022-1554.
60(11),
p. 811–821.
doi:
10.1369/0022155412458589.
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Published
Dec. 9, 2014 12:37 PM
