Metabolically-Incorporated Deuterium in Myelin Localized by Neutron Diffraction and Identified by Mass Spectrometry

2021 ◽  
Author(s):  
Anne Baumann ◽  
Andrew R Denninger ◽  
Marek Domin ◽  
Bruno Deme ◽  
Daniel A Kirschner
Keyword(s):  
Mass Spectrometry ◽  
Neutron Diffraction ◽  
De Novo ◽  
Biological Tissues ◽  
De Novo Lipogenesis ◽  
Myelin Proteins ◽  
Ms Analysis ◽  
Lipid Species ◽  
High Level

Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially its biosynthesis and assembly in the context of its normal formation and renewal, and pathological breakdown. To explore further the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating via drinking water nontoxic levels of deuterium (2H; D) into pregnant dams and their developing embryos. All of the mice were sacrificed when the pups were about 60 days old. Myelinated nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed were frozen for mass spectrometry (MS). Analysis of the neutron diffraction patterns of the sciatic nerves from deuterium-fed mice (D mice) versus the controls (H mice) showed no appreciable differences in myelin periodicity, but major differences in the intensities of the Bragg peaks. The neutron scattering density profiles showed an appreciable increase in density at the center of the membrane bilayer in the D mice, particularly in the pups. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that the level of D was greater in the pups compared to their mother: 97.6% +/- 2.0% (n=54; range 89.6% to 99.6%) versus 60.6% +/- 26.4% (n=27; range 11.4% to 97.3%). Deuteration in the mother also varied by lipid species, and among lipid subspecies. Three molecular species of phosphatidylcholine (PC), phosphatidylinositol (PI), and diglycerol (DG) were the most deuterated lipids, and sulfatide (SHexCer), one species of sphingomyelin (SM), and triacylglycerol (TG) were the least deuterated. The distribution pattern of deuterium in the D pups was always bell-shaped, and the average number of D atoms ranged from a low of ~4 in fatty acid (FA), to a high of ~9 in cerebroside (HexCer). By contrast, in D dam only about one third of the lipids had symmetric bell-shaped distributions; most had more complex, overlapping distributions that were weighted toward a lower average number of D atom labels. The average number of D atoms ranged from a low of ~3 to 4 in FA and in one species of SHexCer, to a high of 6 to 7 in HexCer and SM. In D pups, the consistently high level of deuteration can be attributed to their de novo lipogenesis during gestation and continuation of rapid myelination postnatally. In D dam, the widely varying levels of deuteration of lipids likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance in the mature animal. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to myelin proteins, as well as more broadly to the molecular constituents of other biological tissues.

scholarly journals Targeting de novo lipogenesis and the Lands cycle induces ferroptosis in KRAS-mutant lung cancer

2021 ◽  
Author(s):  
Caterina Bartolacci ◽  
Cristina Andreani ◽  
Goncalo Dias do Vale ◽  
Stefano Berto ◽  
Margherita Melegari ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Fatty Acid ◽  
Metabolic Networks ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Genetically Engineered ◽  
De Novo Lipogenesis ◽  
Main Process

Mutant KRAS (KM) is the most common oncogene in lung cancer (LC). KM regulates several metabolic networks, but their role in tumorigenesis is still not sufficiently characterized to be exploited in cancer therapy. To identify metabolic networks specifically deregulated in KMLC, we characterized the lipidome of genetically engineered LC mice, cell lines, patient derived xenografts and primary human samples. We also determined that KMLC, but not EGFR-mutant (EGFR-MUT) LC, is enriched in triacylglycerides (TAG) and phosphatidylcholines (PC). We also found that KM upregulates fatty acid synthase (FASN), a rate-limiting enzyme in fatty acid (FA) synthesis promoting the synthesis of palmitate and PC. We determined that FASN is specifically required for the viability of KMLC, but not of LC harboring EGFR-MUT or wild type KRAS. Functional experiments revealed that FASN inhibition leads to ferroptosis, a reactive oxygen species (ROS)-and iron-dependent cell death. Consistently, lipidomic analysis demonstrated that FASN inhibition in KMLC leads to accumulation of PC with polyunsaturated FA (PUFA) chains, which are the substrate of ferroptosis. Integrating lipidomic, transcriptome and functional analyses, we demonstrated that FASN provides saturated (SFA) and monounsaturated FA (MUFA) that feed the Lands cycle, the main process remodeling oxidized phospholipids (PL), such as PC. Accordingly, either inhibition of FASN or suppression of the Lands cycle enzymes PLA2 and LPCAT3, promotes the intracellular accumulation of lipid peroxides and ferroptosis in KMLC both in vitro and in vivo. Our work supports a model whereby the high oxidative stress caused by KM dictates a dependency on newly synthesized FA to repair oxidated phospholipids, establishing a targetable vulnerability. These results connect KM oncogenic signaling, FASN induction and ferroptosis, indicating that FASN inhibitors already in clinical trial in KMLC patients (NCT03808558) may be rapidly deployed as therapy for KMLC.

scholarly journals AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo

2018 ◽  
Vol 19 (9) ◽  
pp. 2826 ◽  
Author(s):  
Marc Foretz ◽  
Patrick Even ◽  
Benoit Viollet
Keyword(s):  
Lipid Metabolism ◽  
De Novo ◽  
Fat Oxidation ◽  
De Novo Lipogenesis ◽  
Acid Oxidation ◽  
Ampk Activation ◽  
Hepatic Lipid ◽  
Adenoviral Delivery ◽  
Body Production

The energy sensor AMP-activated protein kinase (AMPK) is a key player in the control of energy metabolism. AMPK regulates hepatic lipid metabolism through the phosphorylation of its well-recognized downstream target acetyl CoA carboxylase (ACC). Although AMPK activation is proposed to lower hepatic triglyceride (TG) content via the inhibition of ACC to cause inhibition of de novo lipogenesis and stimulation of fatty acid oxidation (FAO), its contribution to the inhibition of FAO in vivo has been recently questioned. We generated a mouse model of AMPK activation specifically in the liver, achieved by expression of a constitutively active AMPK using adenoviral delivery. Indirect calorimetry studies revealed that liver-specific AMPK activation is sufficient to induce a reduction in the respiratory exchange ratio and an increase in FAO rates in vivo. This led to a more rapid metabolic switch from carbohydrate to lipid oxidation during the transition from fed to fasting. Finally, mice with chronic AMPK activation in the liver display high fat oxidation capacity evidenced by increased [C14]-palmitate oxidation and ketone body production leading to reduced hepatic TG content and body adiposity. Our findings suggest a role for hepatic AMPK in the remodeling of lipid metabolism between the liver and adipose tissue.

Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans

1991 ◽  
Vol 261 (4) ◽  
pp. E479-E486 ◽  
Author(s):  
M. K. Hellerstein ◽  
C. Kletke ◽  
S. Kaempfer ◽  
K. Wu ◽  
C. H. Shackleton
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Density Lipoprotein ◽  
Gas Chromatography Mass Spectrometry ◽  
Invasive Technique ◽  
Acetyl Coa ◽  
Precursor Pool ◽  
Chromatography Mass Spectrometry ◽  
Mass Isotopomer

Measurement of hepatic fatty acid (FA) and cholesterol synthesis has been limited by lack of access to the precursor pool, cytosolic acetyl-CoA. We present a method for inferring the enrichment of the true hepatic lipogenic precursor pool in humans using the frequency distribution of mass isotopomers within enriched circulating polymers of acetyl-CoA [very low-density lipoprotein (VLDL)-palmitate, VLDL-stearate]. Human subjects were infused intravenously (n = 16) with [1-13C]- or [2-13C]acetate. Oral sulfamethoxazole (SMX) was administered concurrently, and the acetylated conjugate (SMX acetate) was used to estimate independently the hepatic cytosolic acetyl-CoA enrichment. Isotopomer frequencies in VLDL-FA were determined by gas chromatography-mass spectrometry, whereas high-performance liquid chromatography-mass spectrometry was used to measure enrichments in SMX acetate. Based on the excess M2/excess M1 ratio in VLDL-FA, calculated acetyl-CoA enrichments were 5.59 +/- 0.33 molar percent excess (MPE), whereas SMX acetate enrichments were 5.38 +/- 0.31 MPE (the 2 methods were not significantly different). Mass isotopomer-calculated and SMX acetate-measured estimates of acetyl-CoA enrichments correlated very closely in individual subjects (r2 = 0.93; P less than 0.0001). De novo hepatic lipogenesis can be measured using isotopomer-calculated precursor enrichments compared with measured incorporation in specific isotopomers of VLDL-FA. In summary, excess isotopomer frequencies in secreted lipids provide a non-invasive technique for estimating hepatic cytosolic acetyl-CoA enrichments in humans in vivo and correlate closely with enrichments observed using the xenobiotic probe technique. Isotopomeric distributions represent a new strategy for accurate measurement of macromolecule synthesis that may be applicable to other classes of molecules besides lipids.

Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel

2000 ◽  
Vol 278 (4) ◽  
pp. F613-F619 ◽  
Author(s):  
Alexander Shigaev ◽  
Carol Asher ◽  
Hedva Latter ◽  
Haim Garty ◽  
Eitan Reuveny
Keyword(s):  
De Novo ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Fourfold Increase ◽  
Tight Epithelia ◽  
High Level

Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

scholarly journals Advanced glycation end products promote hepatosteatosis by interfering with SCAP-SREBP pathway in fructose-drinking mice

2013 ◽  
Vol 305 (6) ◽  
pp. G398-G407 ◽  
Author(s):  
Raffaella Mastrocola ◽  
Massimo Collino ◽  
Mara Rogazzo ◽  
Claudio Medana ◽  
Debora Nigro ◽  
...  
Keyword(s):  
Advanced Glycation End Products ◽  
De Novo ◽  
Regulatory Element ◽  
Plasma Lipid ◽  
De Novo Lipogenesis ◽  
Advanced Glycation ◽  
Sugary Drinks ◽  
Glycation End Products ◽  
End Products

Clinical studies have linked the increased consumption of fructose to the development of obesity, dyslipidemia, and impaired glucose tolerance, and a role in hepatosteatosis development is presumed. Fructose can undergo a nonenzymatic reaction from which advanced glycation end products (AGEs) are derived, leading to the formation of dysfunctional, fructosylated proteins; however, the in vivo formation of AGEs from fructose is still less known than that from glucose. In the present study C57Bl/6J mice received 15% (wt/vol) fructose (FRT) or 15% (wt/vol) glucose (GLC) in water to drink for 30 wk, resembling human habit to consume sugary drinks. At the end of the protocol both FRT- and GLC-drinking mice had increased fasting glycemia, glucose intolerance, altered plasma lipid profile, and marked hepatosteatosis. FRT mice had higher hepatic triglycerides deposition than GLC, paralleled by a greater increased expression and activity of the sterol regulatory element-binding protein 1 (SREBP1), the transcription factor responsible for the de novo lipogenesis, and of its activating protein SCAP. LC-MS analysis showed a different pattern of AGE production in liver tissue between FRT and GLC mice, with larger amount of carboxymethyl lysine (CML) generated by fructose. Double immunofluorescence and coimmunoprecipitation analysis revealed an interaction between CML and SCAP that could lead to prolonged activation of SREBP1. Overall, the high levels of CML and activation of SCAP/SREBP pathway associated to high fructose exposure here reported may suggest a key role of this signaling pathway in mediating fructose-induced lipogenesis.

scholarly journals Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

2021 ◽  
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Paula Greer ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
High Level ◽  
The Impact

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

scholarly journals The Effect of Fructose Feeding on Intestinal Triacylglycerol Production and De Novo Fatty Acid Synthesis in Humans

Nutrients ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1781
Author(s):  
Simon Steenson ◽  
Fariba Shojaee-Moradie ◽  
Martin B. Whyte ◽  
Kim G. Jackson ◽  
Julie A. Lovegrove ◽  
...  
Keyword(s):  
De Novo ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Acid Synthesis ◽  
Acute Effect ◽  
De Novo Lipogenesis ◽  
Fructose Intake ◽  
High Fructose ◽  
Catabolic Rate

A high fructose intake exacerbates postprandial plasma triacylglycerol (TAG) concentration, an independent risk factor for cardiovascular disease, although it is unclear whether this is due to increased production or impaired clearance of triacylglycerol (TAG)-rich lipoproteins. We determined the in vivo acute effect of fructose on postprandial intestinal and hepatic lipoprotein TAG kinetics and de novo lipogenesis (DNL). Five overweight men were studied twice, 4 weeks apart. They consumed hourly mixed-nutrient drinks that were high-fructose (30% energy) or low-fructose (<2% energy) for 11 h. Oral 2H2O was administered to measure fasting and postprandial DNL. Postprandial chylomicron (CM)-TAG and very low-density lipoprotein (VLDL)-TAG kinetics were measured with an intravenous bolus of [2H5]-glycerol. CM and VLDL were separated by their apolipoprotein B content using antibodies. Plasma TAG (p < 0.005) and VLDL-TAG (p = 0.003) were greater, and CM-TAG production rate (PR, p = 0.046) and CM-TAG fractional catabolic rate (FCR, p = 0.073) lower when high-fructose was consumed, with no differences in VLDL-TAG kinetics. Insulin was lower (p = 0.005) and apoB48 (p = 0.039), apoB100 (p = 0.013) and non-esterified fatty acids (NEFA) (p = 0.013) were higher after high-fructose. Postprandial hepatic fractional DNL was higher than intestinal fractional DNL with high-fructose (p = 0.043) and low-fructose (p = 0.043). Fructose consumption had no effect on the rate of intestinal or hepatic DNL. We provide the first measurement of the rate of intestinal DNL in humans. Lower CM-TAG PR and CM-TAG FCR with high-fructose consumption suggests lower clearance of CM, rather than elevated production, may contribute to elevated plasma TAG, possibly due to lower insulin-mediated stimulation of lipoprotein lipase.

scholarly journals Recent Advances and New Perspectives in Capillary Electrophoresis-Mass Spectrometry for Single Cell “Omics”

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 42 ◽  
Author(s):  
Kellen DeLaney ◽  
Christopher Sauer ◽  
Nhu Vu ◽  
Lingjun Li
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Resolving Power ◽  
Ms Analysis ◽  
Large Populations ◽  
Analysis Workflow ◽  
High Level ◽  
Near Future

Accurate clinical therapeutics rely on understanding the metabolic responses of individual cells. However, the high level of heterogeneity between cells means that simply sampling from large populations of cells is not necessarily a reliable approximation of an individual cell’s response. As a result, there have been numerous developments in the field of single-cell analysis to address this lack of knowledge. Many of these developments have focused on the coupling of capillary electrophoresis (CE), a separation technique with low sample consumption and high resolving power, and mass spectrometry (MS), a sensitive detection method for interrogating all ions in a sample in a single analysis. In recent years, there have been many notable advancements at each step of the single-cell CE-MS analysis workflow, including sampling, manipulation, separation, and MS analysis. In each of these areas, the combined improvements in analytical instrumentation and achievements of numerous researchers have served to drive the field forward to new frontiers. Consequently, notable biological discoveries have been made possible by the implementation of these methods. Although there is still room in the field for numerous further advances, researchers have effectively minimized various limitations in detection of analytes, and it is expected that there will be many more developments in the near future.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence

1998 ◽  
Vol 111 (6) ◽  
pp. 759-767
Author(s):  
G. Herrmann ◽  
P. Brenneisen ◽  
M. Wlaschek ◽  
J. Wenk ◽  
K. Faisst ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Molecular Data ◽  
Cellular Aging ◽  
Premature Aging ◽  
Functional Changes ◽  
Uva Irradiation ◽  
High Level

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of &gt;120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

scholarly journals Pivotal Role of Fatty Acid Synthase in c-MYC Driven Hepatocarcinogenesis

2020 ◽  
Vol 21 (22) ◽  
pp. 8467
Author(s):  
Jiaoyuan Jia ◽  
Li Che ◽  
Antonio Cigliano ◽  
Xue Wang ◽  
Graziella Peitta ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Liver Tumors ◽  
Cellular Growth ◽  
De Novo Lipogenesis ◽  
Liver Malignancy ◽  
Genetic Ablation

Hepatocellular carcinoma (HCC) is a deadly form of liver malignancy with limited treatment options. Amplification and/or overexpression of c-MYC is one of the most frequent genetic events in human HCC. The mammalian target of Rapamycin Complex 1 (mTORC1) is a major functional axis regulating various aspects of cellular growth and metabolism. Recently, we demonstrated that mTORC1 is necessary for c-Myc driven hepatocarcinogenesis as well as for HCC cell growth in vitro. Among the pivotal downstream effectors of mTORC1, upregulation of Fatty Acid Synthase (FASN) and its mediated de novo lipogenesis is a hallmark of human HCC. Here, we investigated the importance of FASN on c-Myc-dependent hepatocarcinogenesis using in vitro and in vivo approaches. In mouse and human HCC cells, we found that FASN suppression by either gene silencing or soluble inhibitors more effectively suppressed proliferation and induced apoptosis in the presence of high c-MYC expression. In c-Myc/Myeloid cell leukemia 1 (MCL1) mouse liver tumor lesions, FASN expression was markedly upregulated. Most importantly, genetic ablation of Fasn profoundly delayed (without abolishing) c-Myc/MCL1 induced HCC formation. Liver tumors developing in c-Myc/MCL1 mice depleted of Fasn showed a reduction in proliferation and an increase in apoptosis when compared with corresponding lesions from c-Myc/MCL1 mice with an intact Fasn gene. In human HCC samples, a significant correlation between the levels of c-MYC transcriptional activity and the expression of FASN mRNA was detected. Altogether, our study indicates that FASN is an important effector downstream of mTORC1 in c-MYC induced HCC. Targeting FASN may be helpful for the treatment of human HCC, at least in the tumor subset displaying c-MYC amplification or activation.

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