Fluorescent Fatty Acid Research Articles

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Obesity induces an increase in fatty acid uptake in endothelium of visceral adipose arteries without altering CD36 expression

Obesity is a prevalent metabolic condition and major contributor to the development of cardiovascular disease. Arteries of visceral adipose present with significant endothelial dysfunction whereas subcutaneous adipose arteries remain functional in obese humans and diet-induced obese mice. Obesity is also associated with elevated plasma long chain fatty acids (LCFAs), well-established contributors to endothelial dysfunction, which require a receptor to facilitate their uptake. CD36, a scavenger receptor, facilitates LCFA uptake, which when in excess would contribute to endothelial dysfunction. Therefore, we aimed to determine the role of CD36 in arteries ex vivo in both mouse and human subcutaneous and visceral adipose arteries as well as examine the difference in uptake of LCFAs in arteries from both depots. First, to determine if CD36 contributes to obesity-induced endothelial dysfunction in visceral adipose arteries, mesenteric (visceral) and subcutaneous adipose arteries isolated from lean and diet-induced obese WT and CD36 knockout were mounted for pressure myography. As expected, visceral adipose arteries of obese mice exhibited a blunted response to endothelium-dependent stimuli, whereas subcutaneous adipose arteries remained functional. CD36 ablation restored endothelium-dependent vasodilation in visceral arteries of obese mice, suggesting a critical role for CD36 in obesity-induced endothelial dysfunction. We next aimed to determine if obesity alters endothelial CD36 expression using two distinct approaches: flow cytometry and immunofluorescence staining of en face preparations in isolated mouse and/or human arteries. For flow cytometry, arteries were digested and sorted to identify the endothelial cell population (i.e., CD45-CD31+ cells). En face preparations were stained for VE-cadherin to locate the endothelial cell layer via confocal microscopy. In both experiments, an antibody that detects an extracellular epitope to CD36 was used to detect membrane expression. Obesity did not alter the expression of CD36 expression in visceral adipose arteries relative to lean counterpart controls or subcutaneous arteries from obese mice or humans. We next aimed to determine if LCFA uptake was altered by obesity. Arteries were exposed to a fluorescent fatty acid, and internalized endothelial LCFA uptake was assessed at different time points via confocal microscopy. Visceral arteries from obese mice and humans had elevated LCFA uptake relative to respective controls which may be an underlying mediator for the dichotomy in endothelial dysfunction observed in obesity. Ongoing work in our lab is focused on determining if this is indeed the case and, if so, if this process is mediated by endothelial CD36. National Institutes of Health (NIH) Grant P20GM113125-6564 (IF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Abstract A090: An inflammatory adipose microenvironment promotes ovarian cancer tumor growth and cisplatin resistance

Abstract Epithelial ovarian cancer (EOC) commonly targets the omentum, an adipocyte rich organ of the peritoneum. Omental adipocytes have been shown to promote EOC development and chemoresistance via transfer of free fatty acids (FFAs) and the induction of bi-directional metabolic changes. Furthermore, obesity and adiposity are risk factors for EOC incidence and prognosis. The metastatic peritoneal tumor microenvironment (TME) is highly inflamed, while obesity is often characterized by chronic inflammation of adipose tissue and hyperlipidemia. Given that inflammation of adipocytes is associated with increased release of FFAs, we hypothesized that acute inflammation in the ovarian metastatic TME exacerbates pro-tumorigenic effects of cancer-associated adipocytes. Using a three-dimensional transwell co-culture model, we explore the impact of inflamed adipocytes on EOC activity. Adipocytes generated from human adipose-derived stem cells were inflamed with 100ng/ml TNFα and cultured alongside tumor spheroids generated from EOC cell lines. Cytokine abundance in serum samples from ovarian cancer patients undergoing cisplatin treatment were also analyzed. We found that inflammation of adipocytes increased cellular lipolysis and release of FFAs, as well as inducing de-differentiation and loss of lipid droplets. Upon co-culture with inflamed adipocytes, EOC cells displayed increased proliferation in 2D monolayer and 3D spheroid cultures. Transfer of FFAs from adipocytes to cancer cells was visualized and quantified using a fluorescent fatty acid analog via confocal microscopy and flow cytometry. Co-culture induced expression of fatty acid chaperone protein FABP4 in EOC cell lines, while blocking fatty acid metabolism in cancer cells reversed pro-tumor effects of adipocytes. To understand signaling mechanisms generating bi-directional phenotypic changes, cytokine content of secretome of co-cultures was assessed by Luminex. IL-6 and IL-8 were highly abundant in co-cultures, and increased expression was confirmed by qPCR. Treatment of EOC cell lines with recombinant IL-6 and IL-8 induced increased uptake of FFAs and blocking of IL-6 and IL-8 signaling reversed pro-cancer effects of adipocytes. Serum samples from patients undergoing cisplatin therapy were analyzed and IL-6 and IL-8 levels were found to be associated with resistance to therapy. To further investigate the impact of inflamed adipocyte signaling on therapeutic resistance, co-culture were treated with cisplatin and tumor spheroids were found to be highly resistant in the presence of inflamed adipocytes. STAT3 activity is known to be upregulated by both IL-6 and IL-8 and we found that STAT3 expression was increased in co-cultures. Knockdown of STAT3 in EOC reversed the chemoprotective effects of inflamed adipocytes. These data suggest an important role for inflammation of adipose tissue in the development of therapeutic resistance in EOC and highlight a potential mechanism linking clinically observed associations between obesity and ovarian cancer risk. Citation Format: Michael E. Williams, David Howard, Deyarina Gonzalez, Francesca Taraballi. An inflammatory adipose microenvironment promotes ovarian cancer tumor growth and cisplatin resistance [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A090.

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic “waste products” such as fatty acids resulting from sex pheromone inactivation.

Design of a serum albumin sensitive probe for cell imaging and drug delivery by modifying a fluorescent agent

Protein-ligand interaction serves as a critical foundation for intramolecular charge transfer (ICT) probes to bind and recognize proteins. Many fluorescent agents are reported to have favorable protein binding abilities but lack protein sensitivity. Herein, a new serum albumin sensitive ICT probe (named MBD) was synthesized by modifying an existing fluorescent agent MPM. MBD not only inherited the favorable human serum albumin (HSA) binding ability of MPM but also newly obtained favorable serum albumin responsiveness. These properties were maintained after MBD formed a complex with water-soluble pillar[5]arene (WP5). Typical commercial drugs, fluorescent amino acids, fatty acids, and metal ions had negligible effects on the HSA responsiveness of the complex. The WP5-MBD complex possessed strong anti-interference ability in binding and recognizing HSA. Moreover, the WP5-MBD complex self-assembled into supramolecular fluorescent nanoparticles in solution. Such nanoparticles possessed good cell imaging and traceable drug delivery properties, which hold great potential for cancer treatment and diagnosis.

Construct Fluorescent Solid Lipid Nanoparticles from Bacterial Outer Membrane Vesicles to Study their Properties and Potential Applications as In Vivo and Invitro Imaging Agents.

In this study, the new solid lipid nanoparticles were created by combining fluorescent dye, fatty acid, lipid, and bacterial outer membranes. The synthesised particles were roughly 95-100nm in size. Vero cells cultivated with these nanoparticles showed no cytotoxicity in 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay. In the cell uptake studies, the vero cell line was employed. Cell lines absorbed fluorescent solid lipid nanoparticles (FSL NPs) better, according to the findings. The confocal microscopy results revealed a significant accumulation of FSL NPs in the cytoplasm over time. The results of small animal imaging employing BALB/c mice revealed that the nanoparticles generated provided high contrast signals. Overall, the OMVs-based FSL NPs system offers a unique imaging tool for studying intracellular interactions as well as a viable tool for drug delivery.

Imbalanced lipid homeostasis caused by membrane αKlotho deficiency contributes to the acute kidney injury to chronic kidney disease transition

After acute kidney injury (AKI), renal tubular epithelial cells (RTECs) are pathologically characterized by intracellular lipid droplet (LD) accumulation, which are involved in RTEC injury and kidney fibrosis. However, its pathogenesis remains incompletely understood. The protein, αKlotho, primarily expressed in RTECs, is well known as an anti-aging hormone wielding versatile functions, and its membrane form predominantly acts as a co-receptor for fibroblast growth factor 23. Here, we discovered a connection between membrane αKlotho and intracellular LDs in RTECs. Fluorescent fatty acid (FA) pulse-chase assays showed that membrane αKlotho deficiency in RTECs, as seen in αKlotho homozygous mutated (kl/kl) mice or in mice with ischemia-reperfusion injury (IRI)-induced AKI, inhibited FA mobilization from LDs by impairing adipose triglyceride lipase (ATGL)-mediated lipolysis and lipophagy. This resulted in LD accumulation and FA underutilization. IRI-induced alterations were more striking in αKlotho deficiency. Mechanistically, membrane αKlotho deficiency promoted E3 ligase peroxin2 binding to ubiquitin-conjugating enzyme E2 D2, resulting in ubiquitin-mediated degradation of ATGL which is a common molecular basis for lipolysis and lipophagy. Overexpression of αKlotho rescued FA mobilization by preventing ATGL ubiquitination, thereby lessening LD accumulation and fibrosis after AKI. This suggests that membrane αKlotho is indispensable for the maintenance of lipid homeostasis in RTECs. Thus, our study identified αKlotho as a critical regulator of lipid turnover and homeostasis in AKI, providing a viable strategy for preventing tubular injury and the AKI-to-chronic kidney disease transition.

Lipidomic analysis identifies long-chain acylcarnitine as a target for ischemic stroke

IntroductionLipid metabolism dysfunction is widely involved in the pathological process of acute ischemic stroke (AIS). The coordination of lipid metabolism between neurons and astrocytes is of great significance. However, the full scope of lipid dynamic changes and the function of key lipids during AIS remain unknown. Hence, identifying lipid alterations and characterizing their key roles in AIS is of great importance. MethodsUntargeted and targeted lipidomic analyses were applied to profile lipid changes in the ischemic penumbra and peripheral blood of transient middle cerebral artery occlusion (tMCAO) mice as well as the peripheral blood of AIS patients. Infarct volume and neurological deficits were assessed after tMCAO. The cell viability and dendritic complexity of primary neurons were evaluated by CCK8 assay and Sholl analysis. Seahorse, MitoTracker Green, tetramethyl rhodamine methyl ester (TMRM), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX were used as markers of mitochondrial health. Fluorescent and isotopic free fatty acid (FFA) pulse-chase assays were used to track FFA flux in astrocytes. ResultsLong-chain acylcarnitines (LCACs) were the lipids with the most dramatic changes in the ischemic penumbra and peripheral blood of tMCAO mice. LCACs were significantly elevated on admission in AIS patients and associated with poor outcomes in AIS patients. Increasing LCACs through a bolus administration of palmitoylcarnitine amplified stroke injury, while decreasing LCACs by overexpressing carnitine palmitoyltransferase 2 (CPT2) ameliorated stroke injury. Palmitoylcarnitine aggravated astrocytic mitochondrial damage after OGD/R, while CPT2 overexpression in astrocytes ameliorated cocultured neuron viability. Further study revealed that astrocytes stimulated by OGD/R liberated FFAs from lipid droplets into mitochondria to form LCACs, resulting in mitochondrial damage and lowered astrocytic metabolic support and thereby aggravated neuronal damage. ConclusionLCACs could accumulate and damage neurons by inducing astrocytic mitochondrial dysfunction in AIS. LCACs play a crucial role in the pathology of AIS and are novel promising diagnostic and prognostic biomarkers for AIS.

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

Metabolic distribution of fatty acid to organelles is an essential biological process for energy homeostasis as well as for the maintenance of membrane integrity, and the metabolic pathways are strictly regulated in response to environmental stimuli. Herein, we report a fluorescent fatty acid probe, which bears an azapyrene dye that changes its absorption and emission features depending on the microenvironment polarity of the organelle into which it is transported. Owing to the environmental sensitivity of this dye, the distribution of the metabolically incorporated probe in non-polar lipid droplets, medium-polarity membranes, and the polar aqueous regions, can be visualized in different colors. Based on density scatter plots of the fluorophore, we demonstrate that the degradation of triacylglycerols in lipid droplets occurs predominantly via lipolysis rather than lipophagy in nutrition-starved hepatocytes. This tool can thus be expected to significantly advance our understanding of the lipid metabolism in living organisms.

Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.

CircEZH2 Regulates Milk Fat Metabolism through miR-378b Sponge Activity.

Simple SummaryHeat stress has seriously threatened the performance and health of dairy cows and has become one of the most important factors restricting the development of the dairy industry. In our previous study, we found that heat stress markedly altered the expression patterns of circRNAs in dairy cow’s mammary gland tissue, and heat-induced circRNAs participated in the regulation of milk fat metabolism through competing endogenous RNA (ceRNA) networks. Therefore, we evaluated the roles of heat-induced circEZH2 in the regulation of milk fat metabolism in this study. In more detail, we found that circEZH2 affects the proliferation, apoptosis, and lipid metabolism of mammary gland epithelial cells, and successfully verified the targeting relationship of circEZH2-bta-miR378b-LPL and circEZH2-bta-miR378b-CD36. This experiment expands the basic data on the role of circRNA in milk fat regulation, and provides a theoretical basis for alleviating heat stress in dairy cows.In this study, we evaluated the roles of heat-induced circEZH2 in the regulation of milk fat metabolism. CircEZH2 overexpression increased HC11 cell proliferation and decreased apoptosis. These changes were accompanied by increased expression of proliferation marker proteins (PCNA, Cyclin D, and Cyclin E) and the anti-apoptotic protein Bcl2, while expression of the pro-apoptotic proteins Bax and cleaved-caspase was reduced. SiRNA-mediated silencing of EZH2 in HC11 cells had the opposite effects. CircEZH2 overexpression promoted the uptake of a fluorescent fatty acid (Bodipy) as well as expression of the fatty acid transport-related protein CD36, lipolysis-related protein LPL, and unsaturated fatty acid metabolism-related proteins FADS1 and SCD1. Dual luciferase reporter assays verified the targeting relationship of the two ceRNA networks, circEZH2-miR378b-LPL and circEZH2-miR378b-CD36. This information provides further clarification of the role of circRNAs in milk fat regulation in addition to a theoretical basis for alleviating the effects of heat stress on milk production by dairy cows.

A near-infrared fluorescent long-chain fatty acid toward optical imaging of cardiac metabolism in living mice.

Non-invasive fatty acid (FA) metabolic imaging is crucial for the evaluation of cardiac function in the heart. Currently, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are widely employed for cardiac metabolic imaging both in pre-clinical and clinical studies. Although SPECT and PET enable highly sensitive cardiac metabolic imaging, there are several disadvantages such as the high cost of instruments and radioactive tracer synthesis. In contrast, near-infrared (NIR) optical imaging using fluorescent FAs provides a simple and useful platform for in vivo imaging of cardiac metabolism. In this work, we synthesized a NIR fluorescence labelled long-chain fatty acid (LCFA) for real-time imaging of cardiac metabolism in vivo. A NIR fluorescence labelled LCFA was designed as an analogue of β-methyl [123I] iodophenyl-pentanedecanoic acid (123I-BMIPP), which is widely used for the diagnosis of heart diseases in clinical practice. As a NIR fluorescent label, we used an Alexa 680 fluorophore that emits over 700 nm. By conjugation of Alexa 680 to Amino-BMPP (15-(4-(3-aminopropyl)phenyl)-3-methylpentadecanoic acid), we prepared a NIR fluorescent BMIPP analogue, Alexa680-BMPP. NIR fluorescence imaging showed that Alexa680-BMPP is taken up by the mouse heart tissue after intravenous injection, showing that Alexa680-BMPP can act as a fluorescent LCFA analogue. Among Alexa680 conjugated FA analogues including short and middle chain NIR fluorescent FAs, Alexa680-BMPP was most efficiently taken up by heart tissues. For fasted and fed mice, the difference in the degree of the uptake of Alexa680-BMPP in their heart tissues was clearly observed by in vivo and ex vivo NIR fluorescence imaging. Herein, we present the synthesis of a NIR fluorescent LCFA, Alexa680-BMPP, and its capability for real-time optical imaging of cardiac metabolism in living mice.

Single-Cell Sorting of Non-human Primate Adipocytes with Large-particle Flow Cytometry.

Fluorescence-activated cell sorting enables separation and analysis of heterogeneous cell populations based on size, granularity, and fluorescence intensity. Cell sorting has been widely used for isolation of cells that are ∼10 to 25 μm in diameter. By contrast, cell sorting of unilocular adipocytes isolated from white adipose tissue imposes a significant technological challenge. The combination of their large size (up to 200 μm) and the fragile nature of lipid-laden adipocytes requires the use of specialized flow cytometers equipped with a large nozzle and capable of using low pressure to reduce shear forces during the cell sorting process. Furthermore, isolation of single adipocytes is rarely performed due to the lack of specialized cell sorters that can dispense single adipocytes into individual wells. Conducting cell sorting on single adipocytes would enable analyses of the cell-autonomous heterogeneity in nutrient uptake and metabolism observed in white adipose tissue. In this protocol, we describe single-cell sorting of rhesus macaque adipocytes labeled with fluorescent fatty acid and live-cell indicators using large-particle flow cytometry. This methodology represents a valuable resource for basic and translational studies aimed at understanding the development and function of adipocytes. © 2021 Wiley Periodicals LLC. Basic Protocol: Single-cell flow sorting of adipocytes.

OAB-029: Bone marrow adipocytes induce metabolic reprogramming of multiple myeloma cells

<h3>Background</h3> Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of MM. However, the associated molecular mechanisms have remained largely unknown. We hypothesize that BMAd support MM cells through metabolic reprogramming. <h3>Methods</h3> BM aspirates of MGUS, SMM, and NDMM patients were used to obtain mature BMAd or stromal cells (BMSCs). Murine BMSC cell-line OP9, MM cell-line 5TGM1, and human-origin MM cell-lines MM.1S, OPM2 were obtained from ATCC. Cell proliferation was assessed by CyQUANT Assay; lipolysis by Lipolysis Assay (Sigma); lipid uptake by flow cytometry (FCM) of fluorescent fatty acids (FA) BODIPY-C12 and -C16, or LipidTox. In-vivo efficacy of FA treatment was assessed in a MM CB17 SCID mouse plasmacytoma model. <h3>Results</h3> In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS/SMM and NDMM donors significantly increased the proliferation of MM.1S MM cells. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad, further confirmed by increased glycerol content in conditioned media in the presence of MM cells. Moreover, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by FCM. The addition of acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are transported into cells through FATP (1-6) or CD36 receptors. Bioinformatics analysis of public Oncomine database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model decreased tumor volume significantly. <h3>Conclusion</h3> We show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression.

UCTD and SLE patients show increased levels of oxidative and DNA damage together with an altered kinetics of DSB repair.

Immunological tolerance is a critical feature of the immune system; its loss might lead to an abnormal response of lymphocytes causing autoimmune diseases. One of the most important groups belonging to autoimmune disorders is the connective tissue diseases (CTD). CTD are classified among systemic rheumatic diseases and include pathologies such as systemic lupus erythematosus (SLE), and undifferentiated CTD (UCTD). In this study, we evaluated oxidative and genome damage in peripheral blood lymphocytes from patients with SLE and UCTD, further classified on the basis of disease activity and the presence/absence of a serological profile. Oxidative damage was evaluated in cell membrane using the fluorescent fatty acid analogue BODIPY581/591 C11. The percentage of oxidised lymphocytes in both SLE and UCTD patients was higher than in the control group, and the oxidative stress correlated positively with both disease activity and autoantibody profile. The γH2AX focus assay was used to quantify the presence of spontaneous double strand breaks (DSBs), and to assess the abilities of DSBs repair system after T cells were treated with mitomycin C (MMC). Subjects with these autoimmune disorders showed a higher number of γH2AX foci than healthy controls, but no correlation with diseases activity and presence of serological profile was observed. In addition, patients displayed an altered response to MMC-induced DSBs, which led their peripheral cells to greatly increase apoptosis. Taken together our results confirmed an interplay among oxidative stress, DNA damage and impaired DNA repair, which are directly correlated to the aggressiveness and clinical progression of the diseases. We propose the evaluation of these molecular markers to better characterise SLE and UCTD, aiming to improve the treatment plan and the quality of the patients' life.

Discovery and Characterization of a Bilirubin Inducible Green Fluorescent Protein From the Moray Eel Gymnothorax zonipectis

Since the initial discovery of Aqueoria victoria’s green fluorescence off the coast of Washington’s Puget Sound, biofluorescent marine organisms have been found across the globe. The variety of colors of biofluorescence as well as the variability in the organisms that exhibit this fluorescence is astounding. The mechanisms of biofluorescence in marine organisms are also variable. To fluoresce, some organisms use fluorescent proteins, while others use small molecules. In eels, green biofluorescence was first identified in Anguilla japonica. The green fluorescence in A. japonica was discovered to be caused by a fatty acid binding protein (UnaG) whose fluorescence is induced by the addition of bilirubin. Members of this class of proteins were later discovered in Kaupichthys eels (Chlopsid FP I and Chlopsid FP II). Here, we report the discovery and characterization of the first member of this class of green fluorescent fatty acid binding proteins from the moray eel Gymnothorax zonipectis. This protein, GymFP, is 15.6 kDa with a fluorescence excitation at 496 nm and an emission maximum at 532 nm upon addition of bilirubin. GymFP is 61% homologous to UnaG and 47% homologous to Chlopsid FP I. Here, we report de novo transcriptome assembly, protein expression, and fluorescence spectroscopic characterization of GymFP. These findings extend the fluorescent fatty acid binding proteins into a third family of true eels (Anguilliformes).

Abstract 1266: Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy

Abstract Dysregulated fatty acid metabolism is thought to be a hallmark of cancer, wherein fatty acids function both as an energy source and as signals for enzymatic and transcriptional networks contributing to malignancy. Fatty acid-binding protein 5 (FABP5) is an intracellular protein that facilitates transport of fatty acids and plays a role in regulating the expression of genes associated with cancer progression such as cell growth, survival, and metastasis. Overexpression of FABP5 has been reported to contribute to an aggressive phenotype and a poor survival correlation in several cancers. Therefore, inhibition of FABP5 is considered as a therapeutic approach for cancers. Phenotypic screening of a library of covalent compounds for selective sensitivity of cancer cells followed by medicinal chemistry optimization resulted in the identification of AUR104 with desirable properties. Chemoproteomic-based target deconvolution revealed FABP5 as the cellular target of AUR104. Covalent adduct formation with Cys43 of FABP5 by AUR104 was confirmed by mass spectrometry. Target occupancy studies using a biotin-tagged AUR104 demonstrated potent covalent binding to FABP5 in both cell-free and cellular conditions. Ligand displacement assay with a fluorescent fatty acid probe confirmed the competitive binding mode of AUR104 with fatty acids. Binding at the fatty acid site and covalent bond formation with Cys43 were also demonstrated by crystallography. Furthermore, AUR104 showed a high degree of selectivity against a broad safety pharmacology panel of enzymes and receptors. AUR104 exhibited potent anti-proliferative activity in a large panel of cell lines derived from both hematological and solid cancers with a high degree of selectivity over normal cells. Anti-proliferative activity in lymphoma cell lines correlated with inhibition of MALT1 pathway activity, cleavage of RelB/Bcl10 and secretion of cytokines, IL-10 and IL-6. AUR104 displayed desirable drug-like properties and dose-dependent oral exposure in pharmacokinetic studies. Oral dosing with AUR104 resulted in dose-dependent anti-tumor activity in DLBCL (OCI-LY10) and NSCLC (NCI-H1975) xenograft models. In a repeated dose MTD studies in rodents and non-rodents, AUR104 showed good tolerability with an exposure multiple of &amp;gt;500 over cellular EC50 for up to 8 hours. In summary, we have identified a novel covalent FABP5 inhibitor with optimized properties that showed anti-tumor activity in in vitro and in vivo models with acceptable safety profile. The data presented here strongly support clinical development of AUR104. Citation Format: Dinesh Chikkanna, Leena Khare Satyam, Sunil Kumar Pnaigrahi, Vinayak Khairnar, Manoj Pothuganti, Lakshmi Narayan Kaza, Narasimha Raju Kalidindi, Vijaya Shankar Nataraj, Aditya Kiran Gatta, Narasimha Rao Krishnamurthy, Sandeep Patil, DS Samiulla, Kiran Aithal, Vijay Kamal Ahuja, Nirbhay Kumar Tiwari, KB Charamannna, Pravin Pise, Thomas Anthony, Kavitha Nellore, Sanjeev Giri, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1266.

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.