bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metformin Kills Breast Cancer Cells by Inducing NADH Accumulation, Pyruvate Depletion and Reductive Stress
Authors: Iglesias Clara L. , Ratti Bianca A. , Kang Christie , de Abreu Andre L. , Bonini Marcelo G. 
Published: November 2023

AMP‐activated kinase (AMPK) is a central metabolic regulator activated by deficits in nutrient supply. The role of AMPK in cancer has been contentious with both oncogenic and tumor suppressive roles being proposed. Studies by our group indicated that in breast cancer, AMPK activation promotes tumor evolution towards more aggressive phenotypes. These studies, however, were heavily criticized based on the argument that metformin, a biguanidine drug used to treat diabetes, activates AMPK and kills breast cancer cells. Hence, we set up to test the alternative hypothesis that AMPK activation by metformin relieves metformin toxicity by alleviating energy deficits, which are severe enough to cause cell death despite of AMPK activation. To examine this possibility we used AMPK‐competent (MCF7ev) and AMPK‐deficient (MCF7shAMPK) MCF7 cells, which are a estrogen receptor positive breast cancer cell line. Our results showed that MCF7shAMPK are slightly more sensitive to metformin than parental cells particularly at higher doses (20 mM). In addition, we found that pyruvate suppresses metformin toxicity towards parental cells but not MCF7shAMPK. Combined with metabolomics studies that revealed significant AMPK‐dependent distortions in amino acid metabolism caused by metformin our results indicate that complex I inhibition with the accumulation of NADH are the likely causes of metformin toxicity. This is alleviated by pyruvate (a NADH oxidant) as long as AMPK is functional and activates NADH oxidation by lactate dehydrogenase. We also conclude that metformin may be best utilized to treat tumors deficient in AMPK activity.Support or Funding InformationNIH Grant Number: 3R01HL125356 ‐ 03W1
Succinate Drives Aggressiveness in Breast Cancer
Authors: Kang Christie , Bonini Marcelo 
Published: November 2023

Succinate is an intermediate metabolite in the Kreb cycle which accumulates in hypoxia. It is also becoming clear that succinate is active in signaling directly or indirectly affecting nuclear factor activity and gene expression. We hypothesize that the accumulation of succinate in cancer promotes more aggressive phenotypes by either increasing the production of mitochondrial reactive oxygen species (ROS) or driving hypoxia‐inducible factor (HIF) signaling. We further hypothesize that sirtuin3 (sirt3), an established tumor suppressor, counteracts succinate's push towards more malignant phenotypes and that the loss of sirt3 in certain tumors enhances the pro‐carcinogenic effects of succinate accumulation. In support of the hypothesis, we found that MDA‐MB‐231 (triple negative) and MCF7 (estrogen receptor positive, ER+) cell lines had increased levels of hydrogen peroxide (H2O2) when treated with dimethyl succinate for two weeks. The rise in H2O2 levels was accompanied by a significant increase in cancer stem cell population particularly in MDA‐MB‐231. In addition, we found that sirt3 expression and activity are determinant of the effects of succinate in promoting cancer stem cell (more aggressive) phenotypes in MDA‐MB‐231. Together, our results indicate that the accumulation of succinate, which happens in hypoxia, promotes breast cancer phenotypes associated with treatment resistance, poorer patient outcomes and lower survival rates.Support or Funding Information1RO1 HL125356 and 67263‐RT‐REP
Attenuation of Mitochondrial Dysfunction During Hepatic Steatosis by Avocado Oil Supplementation
Authors: Garcia‐Berumen Claudia isabel , Ortiz‐Avila Omar , Márquez‐Ramírez Cristian Adrián , Calderón‐Cortés Elizabeth , Montoya‐Pérez Rocío , Rodriguez‐Orozco Alain R. , Molina Alfredo Saavedra , Cortés‐Rojo Christian 
Published: November 2023

Nonalcoholic Fatty Liver Disease (NFLD) is a clinical syndrome characterized by excessive fat accumulation in the liver and hepatic cellular degeneration even in the absence of alcohol consumption. Mechanisms of alcoholic liver disease have been extensively studied; however, the events that cause progression to hepatitis and fibrosis/cirrhosis are still undefined. It has been recently recognized that mitochondrial dysfunction and mitochondrial oxidant production may play a critical role in the development of NAFLD and progression to nonalcoholic steatohepatitis (NASH). We have recently reported that avocado oil intake has protective effects against mitochondrial dysfunction during diabetes by increasing the resistance of the respiratory chain (RC) to oxidative stress, decreasing ROS production and enhancing glutathione availability Therefore, we aimed to test whether avocado oil protects from the alterations elicited by diabetes on RC function in liver mitochondria from rats with NAFLD. Mitochondria from rats with NAFLD exhibited decreased respiration, impaired activity of complex III and diminished reduction of cytochromes b and c+c1. All these effects were corrected when avocado oil was supplemented after the establishment of NAFLD but not before the induction of the disease. These data suggest that avocado oil could be beneficial to decrease NAFLD progression by improving mitochondrial function.Support or Funding InformationAcknowledgments: this work was supported by a Coordinación de la Investigación Científica‐UMSNH grant (CCR).
Comparative Effects of Avocado Oil and a Thiol Reductant on Impaired Complex I Activity in Mitochondria from Diabetic Rats
Authors: Hernandez Manjury Jatziry , Flores‐Ledesma Claudia Guadalupe , Sánchez‐Briones Luis Alberto , Alonso‐Velázquez Celeste , Aguilar‐Toral Roxana , Campos‐García Jesús , Saavedra‐Molina Alfredo , Cortés‐Rojo Christian 
Published: November 2023

Diabetes is a chief factor for the progression of non‐alcoholic liver disease, which in turn, has been closely linked to mitochondrial dysfunction and excessive ROS production. Complex I may have a prominent role in the development of liver disease because its inhibition may drive to impaired hepatic fatty acid oxidation via decreased reoxidation of the NADH generated during beta‐oxidation. Complex I activity decreases by oxidation of specific thiol groups of the protein. Previously, we have demonstrated that avocado oil intake increases the concentration of reduced glutathione (GSH) in liver mitochondria from diabetic rats. As GSH is essential to maintain thiol groups of complex I in a reduced state, in the present study we aimed to compare the effects of avocado oil and beta‐mercaptoethanol, a thiol reductant, on complex I activity of liver mitochondria from type I‐diabetic rats. Respiration in state 3 was impaired in diabetic rats when a complex I substrate was tested. This was attributed to a decrease in complex I activity in diabetic rats. 90‐days avocado oil intake prevented these defects. When mitochondria from diabetic animals were incubated with beta‐mercaptoethanol, it was observed a full recovery of complex I activity. Besides, upregulated activity of catalase was detected in mitochondria from diabetic animals, indicating an increase in the mitochondrial production of H2O2. All these results suggest that diabetes decreases mitochondrial respiration due to an increment in H2O2 levels, causing the inhibition of complex I due to the oxidation of the free thiol groups of the enzyme, with avocado oil preventing complex I impairment probably by preventing thiol oxidation because enhancement of mitochondrial levels of GSH.Support or Funding InformationAcknowledgments: this work was supported by a Coordinación de la Investigación Científica ‐ UMSNH grant (CCR).
Effects of 3D culture and aqueous cinnamon extract on 3T3‐L1 adipogenesis
Authors: Wells Emily , Dinsmore Olivia , Aulthouse Amy , Kinder David , Marchiano Joe , Stockert Amy 
Published: November 2023

Extracts of cinnamon have long been used medicinally and recent research has solidified its use for controlling blood glucose levels. The correlation between type 2 diabetes and obesity make research in carbohydrate and lipid metabolism essential. We utilized the 3T3‐L1 cells as a model to examine degrees of differentiation to mature adipocytes by examining lipid droplet accumulation at various time points post treatment. Lipids from micro‐droplets were exchanged with oil red O as a marker of adipogenesis, and where appropriate the lipid volume was estimated and assayed. Culture periods were extended up to two weeks to reach mature adipocytes. A reduction in cell size was observed in both monolayer and three‐dimensional culture, but the size changes appear more significant when grown in three‐dimensional culture. A comparison was also made between a monolayer 3T3‐L1 culture and an agarose 3D culture, neither of which was treated to cinnamon. Using western blot analysis, adipogenesis markers were used to determine if growth in 3D culture is inducing differentiation into adipocytes. Cinnamon treated cells appear to have more lipid droplet accumulation, yet the lipid volume did not correlate directly with the reduction in cell size, suggesting a true change in metabolic signaling rather than an osmotic contraction. In three‐dimensional culture, there was an obvious cluster of lipid droplets, yet in monolayer this was not as obvious due to the single layer of cells. Monolayer cells were counted in each quadrant of the culture to determine the percent of cells that had rounded morphology compared to those that remained flat and fibroblast‐like. Of the rounded cells, the portion containing at least 50% space fill with lipid droplets were considered adipogenic. In monolayer, an estimate of lipid droplet volume was not reliable and a quantitation of extracted dye was necessary.Support or Funding InformationDepartments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University.
The Ebola Virus Matrix Protein VP40 Interacts Selectively with Plasma Membrane Lipids to Promote Viral Egress
Authors: Vecchio Kathryn Del , Shwarz Anna , Saphire Erica Ollmann , Stahelin Robert 
Published: November 2023

Filoviruses are filamentous viruses and include Ebola (EBOV) and Marburg (MARV), which are morphologically identical but antigenically distinct. Mortality rates can be has high as 90% and to date there are no FDA approved vaccines or small molecules for treatment outside the realm of emergency situations under the compassionate use clauses of FDA policy.EBOV harbors a genome of 7 proteins, the most abundantly expressed in mature virions is Viral Protein 40 (VP40) also known as the matrix protein. VP40 is required for the assembly and budding of EBOV and alone VP40 can form virus like particles (VLPs) from the plasma membrane of host cells. Recent work by the Stahelin and Ollmann Saphire labs indicates that VP40 adopts multiple different structures to elicit different functions in the viral life cycle. Cellular data demonstrates that each structure adopts a specific function, one for budding form the plasma membrane of human cells and one for regulation of viral transcription. This work investigates how distinct VP40 structures assemble in the presence of synthetic lipid vesicles and at the inner leaflet of the plasma membrane in live cells. This project aims to determine lipid composition requirements for functionality of VP40 mutants vs. wild‐type VP40, and to elucidate the function of VP40 oligomerization with site‐specific mutants.The C‐terminal domains of the VP40 homodimer exhibit a highly conserved basic patch which is thought to promote interaction with anionic lipids. Preliminary data confirms that VP40 binds with nanomolar affinity to liposomes containing phosphatidylserine (PS), an anionic lipid found within the inner leaflet of the plasma membrane, and has specificity for this anionic lipid in particular. Cellular data from the Stahelin lab provides precedent that VP40 requires PS in order to bud from the plasma membrane. Lipidomics analysis of mammalian cells transfected with VP40 has aided in the investigation of the species and saturation of lipid populations of in the plasma membrane versus lipid populations in VLPs. Preliminary data suggest that VP40 selects for not only specific species of lipids, but for particular saturation states of lipids within the plasma membrane.Support or Funding InformationThis work has been supported by NIH grant AI081077 and NIH grant T32GM075762.
Optimization of ESI Parameters for Comprehensive Lipid Analysis
Authors: Gathungu Rose M , Larrea Pablo , Sniatynski Matthew J , Kristal Bruce S 
Published: November 2023

Untargeted lipidomics is mainly performed using electrospray ionization mass spectrometry (ESI‐MS) either with or without chromatographic separation. ESI‐MS enables structural annotation and quantitation of lipids, which can then be used to determine the biological relevance of lipids. The use of ESI‐MS requires the optimization of the MS method to ensure detection of as many lipids as possible and the correct annotation of these lipids. Here we look at two factors that affect ESI‐MS that, in turn, influence lipid detection and identification. First, we show that in‐source fragmentation at the ESI source can generate artifacts that can have identical masses to other common lipids leading to the misannotation of the in‐source fragment ions as true lipids. In‐source fragmentation can lead to (i) missing the biologically relevant lipids (i.e., a false negative), and/or; (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). To mitigate this problem, we have conducted a systematic evaluation of ESI source parameters with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in‐source fragmentation. Second, since analysis in both the positive and the negative ionization modes is required for a complete coverage of the lipidome, we show that the optimal conditions for analysis of lipids in the positive and negative MS ionization modes are different. We have evaluated the additive effect on ionization of lipids in both the positive and negative ionization modes. Our results indicate that the optimal additives for negative ionization of lipids are 1 mM ammonium acetate with 0.1 acetic acid and the optimal additives for positive analysis are 5 mM ammonium formate with 0.1% formic acid.Support or Funding InformationNIH
Homeostatic Regulation of Serine Palmitoyltransferase (SPT) is Mediated by a Direct Interaction of Ceramide with the SPT/ORMDL Complex
Authors: Davis Deanna , Wattenberg Binks 
Published: November 2023

Sphingolipids, the large class of lipids based on the sphingosine backbone, have diverse functions in health and disease. The overall rate of synthesis of the sphingolipid pool is determined by serine palmitoyltransferase (SPT), the initiating enzyme in the de novo biosynthetic pathway. SPT is subject to homeostatic regulation such that elevated cellular sphingolipid results in decreased SPT activity. This regulation is mediated by small proteins of the endoplasmic reticulum: the Orms in yeast and ORMDLs in mammalian cells. The Orms and ORMDLs are negative regulators of SPT. Here we describe the mechanism by which sphingolipids are sensed by the ORMDL/SPT system. The mammalian ORMDLs are not phosphorylated under any conditions, in contrast to the yeast Orms, which are regulated by phosphorylation. To characterize regulation of the ORMDL/SPT complex we devised a cell free system that recapitulates that regulation. Using isolated membranes we demonstrate that the triggering sphingolipid is ceramide. Moreover, regulation does not require ATP or cytosolic proteins, eliminating the role of protein expression or post‐translational modification as potential regulatory mechanisms. A distinct spectrum of ceramide structures activate ORMDL regulation of SPT. Strikingly, only the native stereoisomer of ceramide will trigger this regulation, the hallmark of a specific binding interaction. These data strongly suggest that ORMDL dependent regulation of SPT is not due to a complex signal transduction pathway or sphingolipid‐induced changes in membrane properties but instead is due to direct binding of ceramide to a membrane‐bound ceramide sensor, likely ORMDL.Support or Funding InformationUniversity of Louisville Bridge Grant National Institutes of Health‐R01HL131340
Single‐Molecule Analysis of PKB/AKT‐Lipid Interactions
Authors: Singh Nilmani , Arauz Edwin , Aggarwal Vasudha , Ha Taekjip , Chen Jie 
Published: November 2023

Protein kinase B (PKB)/AKT is a serine/threonine kinase with a wide range of essential functions in cell proliferation, survival, differentiation, and metabolism. Growth factor stimulation of phosphoinositide‐3 kinase (PI3K) results in generation of phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P3] or PI(3,4)P2 at the plasma membrane, which recruits AKT. At the membrane AKT is phosphorylated and activated, and subsequently dissociates from the membrane and translocates to cytoplasm or nucleus in order to phosphorylate its targets. Hence, it is postulated that phosphorylation of AKT dampens its affinity for membrane lipids. We have employed a newly developed lipid single‐molecule pulldown (lipid‐SiMPull) assay to study AKT interaction with lipid vesicles and its functional implication. Quantitative and kinetic measurements were made with wild‐type and phosphor‐mimetic or unphosphorylatable mutants of AKT in whole cell lysates for their binding to PIP3 and PIP2 vesicles. Indeed, phosphorylation of AKT drastically decreased its affinity for the lipids. The potential role of phosphotidylserine (PS) in AKT membrane recruitment was also examined. Furthermore, distinct binding behaviors were observed with full‐length AKT and the PH domain alone, suggesting that other domains in AKT modulate its lipid interaction and thus membrane association. Our results provide new insights into the molecular mechanism of AKT activation, and also highlight the unique advantages of the lipid‐SiMPull method in probing protein‐lipid interactions with full‐length proteins in a near‐physiological context.Support or Funding InformationNIH NIGMS R01GM089771
Sphingosine kinase 1 inhibition modulates crosstalk between p53 and autophagic cell death
Authors: Lima Santiago , Takabe Kazuaki , Leopoldino Andreia Machado , Newton Jason , Dent Paul , Milstien Sheldon , Spiegel Sarah 
Published: November 2023

Sphingosine‐1‐phosphate is a highly bioactive lipid that promotes cellular growth and survival important to the etiology of cancer, and levels of the enzyme that produces it, sphingosine kinase 1 (SphK1), are elevated in many human cancers and are associated with higher tumor grade and worse prognosis. The great majority of human cancers have mutations in the tumor suppressor p53, a key regulator of cell cycle arrest and programmed cell death. There are important associations that suggest SphK1 levels are p53‐dependent in tumors and in preclinical cancer models. Therefore, we sought to better understand whether alterations in SphK1 activity might also regulate activation of p53‐dependent processes, such as apoptosis and autophagy. By using an SphK1 isotype specific inhibitor in cancer cell lines with or without p53 expression, we establish that inhibiting SphK1 activity leads to phosphorylation of p53, induction of downstream p53 targets, increased processing of autophagic markers such as LC3 in a p53‐dependent manner, and the formation of autophagic and multi‐vesicular bodies. Moreover, depleting protein levels of master regulators of autophagic flux, Beclin1 and ATG5, dramatically decreased the cytotoxicity of the SphK1 inhibitor only in cells with p53 expression. Hence, our study reveals that SphK1 inhibition is cytotoxic to cancer cells in a p53‐dependent manner via autophagic and apoptotic pathways, suggesting crosstalk between these important players in human cancers.Support or Funding InformationNCI grant K22 CA187314‐01 to S.L. and R01GM043880 to S.S
NPC1‐mediated cholesterol export from lysosomes
Authors: Pfeffer Suzanne R. 
Published: November 2023

Endocytosed, LDL‐derived cholesterol is exported from the interior of lysosomes by a process that requires the action of NPC1 and NPC2 proteins. We have recently shown that LAMP1 and LAMP2 proteins interact with NPC1 and NPC2 and also bind cholesterol (1). LAMPs are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon's disease, an X‐linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone‐mediated autophagy, and its expression improves tissue function in models of aging. We have shown that human LAMP2 binds cholesterol in a manner that buries the cholesterol 3β‐hydroxyl group; it also binds tightly to NPC1 and NPC2 (1). Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly (1). Why do LAMP proteins bind cholesterol and what is the significance of their interaction with NPC1 and NPC2 proteins? Our recent determination of the structure of a complex of NPC1 and NPC2 proteins in collaboration with Xiaochun Li and Günter Blobel (2) provides a model for how NPC2 may deliver cholesterol to NPC1. Current work is exploring the subsequent steps involved in exporting that cholesterol across the lysosomal glycocalyx (3) and into the cytoplasm.Support or Funding InformationThis research was funded by the Ara Parseghian Medical Research Foundation and the NIH (DK37332).