Fatty Acid Palmitate Research Articles

New nano-chemotherapeutic chitosans- garlic oil - antibiotics against diabetic foot virulent Proteus spp

Background and Objectives: Diabetes foot ulcer is recognized to have a major side effect that raises the risk of amputation. Diabetic ulcer bacterial infections caused by virulent and resistant bacteria like Proteus mirabilis lead to serious wounds that are incurable with conventional medications. Materials and Methods: In this study, we evaluated the antibacterial activity of a natural product nanochitosan - garlic oil against ten diabetic foot isolates of Proteus mirabilis. Various chitosans (Crab (CScr) - shrimp (CSsh) - squilla (CSsq)) in nano form were prepared and coated with garlic oil (GO). GC-MS analysis was carried out to determine the main compo- nents of the essential garlic oil. The physicochemical properties of GO-NCSsq were analyzed using dynamic light scattering (DLS), zeta potentials (ZP) and subsequently scan electron microscope (SEM). Additionally, the minimum inhibitory con- centration (MIC) and fraction inhibitory concentration index (FICI) were determined. Results: GC-MS analysis revealed the presence of major palmitic fatty acid. (GO) loaded on nanochitosan squilla (NCSsq) showed high activity. Although SEM showed lower nano-size, average size of the GO-NCSsq was 330.8 nm by DLS and its zeta potential formulation was +39.6 mV. The final formulation represented by GO-NCSsq + Pipercillin (Pi) inhibited the virulence factor of P. mirabilis and reduced the MIC value (p-value > 0.001). Moreover, the killing time at 9 h was found to be significantly higher for GO-NCSsq + pipercillin (Pi) against P. mirabilis. Conclusion: In order to manage diabetic foot infections, GO-NCSsq is a legitimate antibacterial agent that can be coupled with other antibiotics.

9317 Diabetogenic Stress-Mediated Beta Cell Extracellular Vesicle:Autophagy Pathway Crosstalk

Abstract Disclosure: A. Roy: None. G. Ariyaratne: None. A. Matveyenko: None. N. Javeed: None. Diabetogenic stressors including elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity are central to the development of Type 2 Diabetes (T2D). Due to their high protein synthetic burden, β-cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates, and protect β-cell mass and function. Recently, extracellular vesicles (EVs; 30-150 nm sized nanoparticles) have been shown to work in tandem with autophagy to facilitate cellular adaptation and intercellular communication. As diabetogenic stressors have been shown to perturb autophagy via lysosomal defects, our group has noted enhanced EV secretion. Thus, we hypothesize that diabetogenic factors enhance EV secretion through activation of an EV-based secretory autophagy pathway and that re-balancing of both pathways could improve β-cell function in T2D. To test this, MIN6 cells were exposed to free fatty acid palmitate (PAL; 24h), or pro-inflammatory cytokines IL-1β, TNFα, and IFNγ (CYTO; 48h). Expression of autophagic markers, LC3 and p62 were increased in CYTO and PAL conditions suggesting defects in auto/lysosomal function. Upon PAL/CYTO treatment in MIN6 cells, conditioned media EVs were isolated using differential ultracentrifugation. We noted enhanced protein expression of LC3-II and p62 in PAL- and cytoEVs, suggesting a diversion of immature autophagosome components into EVs due to lysosomal inhibition. To restore balance between autophagy:EV flux, we used EV biogenesis inhibitor, GW4869 (GW; 24h) or autophagy inducer, rapamycin (Rapa; 24h) on MIN6 cells exposed to PAL or CYTO. Isolated EVs were subjected to Nanoparticle Tracking Analysis (NTA) which showed a significant reduction in EV concentration with GW or Rapa addition (p<.05). Isolated mouse islet exposed to CYTO+GW/Rapa or PAL+GW/Rapa showed significant improvements in glucose stimulated insulin secretion (vs. CYTO or PAL; p<.05). Taken together, these data implicate activation of EV-based secretory autophagy in response to diabetogenic stressors and thus, potential targets to improve functional β-cell mass. Presentation: 6/2/2024

Mesenchymal stromal cells can block palmitate training of macrophages via COX-2 and IL-1Ra

Innate training of macrophages can be beneficial for the clearance of pathogens. However, for certain chronic conditions innate training can have detrimental effects due to an excessive production of pro-inflammatory cytokines. Obesity is a condition that is associated with a range of increased pro-inflammatory training stimuli including the free fatty acid palmitate. Mesenchymal stromal cells (MSCs) are powerful immunomodulators and known to suppress inflammatory macrophages via a range of soluble factors. We show that palmitate training of murine bone-marrow derived macrophages and human monocyte-derived macrophages (MDMs) results in an increased production of TNFα and IL-6 upon stimulation with LPS and is associated with epigenetic remodelling. Palmitate training led to metabolic changes, however, MSCs did not alter the metabolic profile of human MDMs. Using a transwell system, we demonstrated that human bone marrow MSCs block palmitate training in both murine and human macrophages suggesting the involvement of secreted factors. MSC disruption of the training process occurs through more than one pathway. Suppression of palmitate enhanced TNFα production is associated with COX-2 activity in MSCs, while secretion of IL-1Ra by MSCs is required to suppress palmitate enhanced IL-6 production in MDMs.

The novel TFEB agonist desloratadine ameliorates hepatic steatosis by activating the autophagy-lysosome pathway.

The autophagy-lysosome pathway plays an essential role in promoting lipid catabolism and preventing hepatic steatosis in non-alcoholic fatty liver disease (NAFLD). Transcription factor EB (TFEB) enhances the autophagy-lysosome pathway by regulating the expression of genes related to autophagy and lysosome biogenesis. Therefore, targeting TFEB provides a novel strategy for the treatment of lipid metabolic diseases. In this study, the antiallergic drug desloratadine was screened and identified as a novel TFEB agonist. Desloratadine effectively induced translocation of TFEB to the nucleus and promoted autophagy and lysosome biogenesis. Desloratadine-induced TFEB activation was dependent on AMPK rather than mTORC1. Moreover, desloratadine treatment enhanced clearance of lipid droplets in cells induced by fatty acids oleate and palmitate. Furthermore, high-fat diet (HFD) induced obesity mouse model experiments indicated treatment with desloratadine markedly reduced the body weight of HFD-fed mice, as well as the levels of hepatic triglycerides and total cholesterol, serum glutamic pyruvic transaminase and glutamic-oxaloacetic transaminase. Oil red O staining showed the liver fat was significantly reduced after desloratadine treatment, and H&E staining analysis demonstrated hepatocellular ballooning was improved. In addition, autophagy and lysosomal biogenesis was stimulated in the liver of desloratadine treated mice. Altogether, these findings demonstrate desloratadine ameliorates hepatic steatosis through activating the TFEB-mediated autophagy-lysosome pathway, thus desloratadine has an exciting potential to be used to treat fatty liver disease.

High-Intensity Interval Training, Caloric Restriction, or Their Combination Have Beneficial Effects on Metabolically Acquired Peripheral Neuropathy.

Peripheral neuropathy (PN) is a prevalent and debilitating complication of obesity, prediabetes, and type 2 diabetes, which remains poorly understood and lacks disease-modifying therapies. Fortunately, diet and/or exercise have emerged as effective treatment strategies for PN. Here, we examined the impact of caloric restriction (CR) and high-intensity interval training (HIIT) interventions, alone or combined (HIIT-CR), on metabolic and PN outcomes in high-fat diet (HFD) mice. HFD feeding alone resulted in obesity, impaired glucose tolerance, and PN. Peripheral nerves isolated from these mice also developed insulin resistance (IR). CR and HIITCR, but not HIIT alone, improved HFD-induced metabolic dysfunction. However, all interventions improved PN to similar extents. When examining the underlying neuroprotective mechanisms in whole nerve, we found that CR and HIIT-CR activate the fuel-sensing enzyme AMP-activated protein kinase (AMPK). We then performed complimentary in vitro work in Schwann cells, the glia of peripheral nerve. Treating primary Schwann cells with the saturated fatty acid palmitate to mimic prediabetic conditions caused IR, which was reversed by the AMPK activator, AICAR. Together, these results enhance our understanding of PN pathogenesis, the differential mechanisms by which diet and exercise may improve PN, and Schwann cell-specific contributions to nerve insulin signaling and PN progression.

Characteristics of Arabica and Robusta Spent Coffee Grounds Oil Extract with Different Solvents

Abstract High coffee consumption contributes to waste, such as spent coffee grounds (SCG). SCG contains up to 20 % oil, with the main oil content being linoleic and palmitic fatty acids. So, SCG has the potential to be used in valuable products. This study aims to characterise Arabica and Robusta SCG oil using maceration methods with different solvents (hexane, ethanol, and petroleum benzene) to obtain maximum coffee oil as an alternative. Arabica and Robusta SCG characterisation comprises the composition content, oil yield, fatty acid profile, and viscosity. The results showed that Arabica SCG oil yield was higher than Robusta, with hexane becoming the most efficient solvent to extract the oil, with 28,87% and 25,57%, respectively. The lowest free fatty acid (FFA) found in Arabica SCG oil extract using hexane is 1,95%, which consists of palmitic acid (9.41%), linoleic acid (5.37%), and stearic acid (4.7%). The highest viscosity is Arabica SCG oil extract using hexane, which is caused by the large amount of long-chain fatty acids extracted by the solvent, such as palmitic acid and linoleic acid. The FFA content of Arabica SCG oil extract using hexane has the potential to be edible oil (cooking oil) and biodiesel.

Supercritical CO2 Extraction of Natural Compounds from Capuchin (Tropaeolum majus) Leaves and Seeds

This study investigated the supercritical CO2 extraction of oils from capuchin (Tropaeolum majus) seeds (4.34% moisture content) and leaves (5.26% moisture content) and analyzed the effects of varying temperature and pressure conditions. The extraction yields were 3% for the seeds and 2% for the leaves. The seed extracts were rich in oleic, linoleic, and palmitic fatty acids, whereas the leaf extracts contained a high concentration of octacosanol (73.37%). Kinetic analysis revealed distinct mass transfer mechanisms during extraction, and the Sovová model effectively described the extraction kinetics, showing good agreement with experimental data (ADD% < 4%). An analysis of variance (ANOVA) demonstrated the impact of temperature and pressure on the yields, with temperature being the most influential factor. The experimental conditions ranged from 22 to 28 MPa and from 313.15 to 333.15 K. This study contributes to the understanding of capuchin oil extraction and its potential applications in various fields, owing to the presence of bioactive compounds.

Nigella sativa, active principle thymoquinone, alleviates palmitate-induced cytotoxicity, inflammation and bioenergetic disruptions in macrophages: An invitro study model

Thymoquinone (TQ) is one of the main phytochemical bioactive ingredients in Nigella sativa, with reported immunity-boosting properties. The current study evaluated the anti-inflammatory effect of TQ against inflammation brought on by free fatty acid Palmitate (PA) using macrophages raw 264.7 cell line. Data revealed that TQ significantly improved the viability of basal and PA stimulated Macrophages at concentrations of 50 and 100 μg/mL. Also, TQ significantly reduced nitric oxide and triglyceride levels in PA-stimulated macrophages at concentrations of 50 and 100 μg/mL. The pro-inflammatory cytokines studies revealed that PA significantly increased the release of the cytokines TNF-α, MHGB-1, IL-1β, and IL-6. TQ at concentrations 25, 50, and 100 μg/ml significantly decreases the release of the studied cytokines in PA-stimulated macrophages to variable extents with parallel inhibition to their corresponding gene expression. Bioenergetic assays showed that PA significantly decreased cellular ATP, mitochondrial complexes I and III activities and mitochondrial membrane potential with a subsequent significant increase in lactate production. At the same time, TQ can alleviate the effect of PA on macrophages' bioenergetics parameters to variable extent based on TQ concentration. To conclude, TQ could mitigate palmitate-induced inflammation and cytotoxicity in macrophages by improving macrophage viability and controlling cytokine release with improved PA-induced bioenergetics disruption.

Abstract 3058: Cofilin 1 Mediates Fatty Acid-induced Foam Cell Formation And Inflammation In Mouse Bone Marrow-derived Macrophages

Background: Macrophages contribute to the development of atherosclerosis, in part through the accumulation of lipid droplets and subsequent transformation into highly inflammatory, lipid laden foam cells. Our previous work suggested that the RAGE/DIAPH1 axis plays a key role in regulating lipid metabolism, as deletion of Diaph1 in hepatocytes reduced nuclear translocation of SREBP1 through the actin cytoskeleton. Cofilin 1 (CFL1) is an actin polymerization molecule which has also been linked to the nuclear translocation of SRBP1, as well as triglyceride synthesis through activation of phospholipase D1. In the aforementioned study by our group, Diaph1 deletion was found to increase the Serine 3 phosphorylation (i.e., inactivation) of Cfl1 . Hypothesis: Accordingly, we hypothesized that CFL1 plays a role in lipid droplet accumulation in macrophages, potentially increasing the risk of atherosclerotic development. Methods: To investigate its role in macrophage lipid handling, we silenced Cfl1 in bone marrow derived macrophages (BMDMs) extracted from male C57BL/6J mice, using silencing RNAs and scramble controls. BMDMs were treated with a mixture of fatty acids palmitate and oleate (250 uM). The effects of Cfl1 knockdown on lipid accumulation were assessed using immunohistochemical staining (Bodipy). Quantitative (q)PCR was used to interrogate the mechanisms underlying these effects, and their impact on inflammation. Results: Cfl1- silenced (75% knockdown shown by qPCR) vs. scramble cells exhibited a significant decrease in the ratio of Bodipy to DAPI % area (p=0.0001). By qPCR, compared to scramble controls, BMDMs subjected to silencing of Cfl1 and incubated with fatty acids (palmitate-oleate) displayed significantly lower mRNA expression of Cd36 (p=0.0018) ; Ppargc1a (p=0.004) , Il6 (p<0.0001), and Tnfa (p=0.013). Conclusions: Our results indicate that silencing of Cfl1 leads to a decrease in lipid accumulation and markers of inflammation in BMDMs. Collectively, our data suggest that the effects of Cfl1 silencing on lipid handling result from decreased fatty acid uptake, which limits fatty acid oxidation on account of fatty acid availability in the cell. Studies are underway to probe the implications of these findings in atherosclerosis.

Non-esterified fatty acid palmitate facilitates oxidative endoplasmic reticulum stress and apoptosis of β-cells by upregulating ERO-1α expression

Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to β-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1β, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic β-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent β-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated β-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic β-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and β-cell failure.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Insufficient TRPM5 Mediates Lipotoxicity-induced Pancreatic β-cell Dysfunction.

While the reduction of transient receptor potential channel subfamily M member 5 (TRPM5) has been reported in islet cells from type 2 diabetic (T2D) mouse models, its role in lipotoxicity-induced pancreatic β-cell dysfunction remains unclear. This study aims to study its role. Pancreas slices were prepared from mice subjected to a high-fat-diet (HFD) at different time points, and TRPM5 expression in the pancreatic β cells was examined using immunofluorescence staining. Glucose-stimulated insulin secretion (GSIS) defects caused by lipotoxicity were mimicked by saturated fatty acid palmitate (Palm). Primary mouse islets and mouse insulinoma MIN6 cells were treated with Palm, and the TRPM5 expression was detected using qRT-PCR and Western blotting. Palm-induced GSIS defects were measured following siRNA-based Trpm5 knockdown. The detrimental effects of Palm on primary mouse islets were also assessed after overexpressing Trpm5 via an adenovirus-derived Trpm5 (Ad-Trpm5). HFD feeding decreased the mRNA levels and protein expression of TRPM5 in mouse pancreatic islets. Palm reduced TRPM5 protein expression in a time- and dose-dependent manner in MIN6 cells. Palm also inhibited TRPM5 expression in primary mouse islets. Knockdown of Trpm5 inhibited insulin secretion upon high glucose stimulation but had little effect on insulin biosynthesis. Overexpression of Trpm5 reversed Palm-induced GSIS defects and the production of functional maturation molecules unique to β cells. Our findings suggest that lipotoxicity inhibits TRPM5 expression in pancreatic β cells both in vivo and in vitro and, in turn, drives β-cell dysfunction.

Phytochemistry, Mineral Estimation, Nutritional, and the In Vitro Anti-Sickling Potentials of Oil Extracted from the Seeds of Mucuna Flagellipes

Abstract. Sickle cell disease is an inherited blood disorder indicative of red blood cells becoming sickle-shaped. The study investigated the in vitro anti-sickling potentials of the seed oil of M. flagellipes. The phytochemistry (fatty acids, vitamins, and minerals) was also determined using standard protocols. Finally, nutritional calculations on the oil were performed to determine its suitability for nutritional purposes. The result showed high zinc content (780 ± 2.50 µg/ 100 mg), while low iron content was observed (170 ± 1.30 µg/ 100 mg). Vitamin analysis showed the presence of vitamins A, E and K with values of 220 ± 1.60, 370 ± 2.20, and 197 ± 0.23 µg/100 mg respectively. The fatty acid profile revealed oleic (31.87 %) and linoleic (18.30 %) fatty acids as the major unsaturated fat in the oil, while palmitic fatty acid (5.91 %) was the major saturated fatty acid. Nutritional calculations showed high PUFA/SFA (2.07), MUFA/SFA (3.62), and UI (68.47). However, the index of thrombogenicity (0.07) and atherogenicity (0.11) was low in the seed oil. Finally, the in vitro anti-sickling potentials of the seed oil showed the oil inhibited and reversed sickling in a dose-dependent manner. Hbs polymerization was also inhibited and Fe2+/Fe3+ was upregulated following treatment with the seed oil. Collectively, the oil showed good anti-sickling potentials, which can be labelled to the presence of zinc, vitamins and unsaturated fat. The nutritional calculations suggest that the seed oil is cardio-friendly and does not pose any nutritional threat.

Proteolytic activation of fatty acid synthase signals pan-stress resolution

Chronic stress and inflammation are both outcomes and major drivers of many human diseases. Sustained responsiveness despite mitigation suggests a failure to sense resolution of the stressor. Here we show that a proteolytic cleavage event of fatty acid synthase (FASN) activates a global cue for stress resolution in Caenorhabditis elegans. FASN is well established for biosynthesis of the fatty acid palmitate. Our results demonstrate FASN promoting an anti-inflammatory profile apart from palmitate synthesis. Redox-dependent proteolysis of limited amounts of FASN by caspase activates a C-terminal fragment sufficient to downregulate multiple aspects of stress responsiveness, including gene expression, metabolic programs and lipid droplets. The FASN C-terminal fragment signals stress resolution in a cell non-autonomous manner. Consistent with these findings, FASN processing is also seen in well-fed but not fasted male mouse liver. As downregulation of stress responses is critical to health, our findings provide a potential pathway to control diverse aspects of stress responses.

Abstract A039: Carnitine palmitoyltransferase IA: an emerging potential metabolic target to counteract HER2-targeted therapy resistance in HER2-positive breast cancer

Abstract HER2 overexpression/amplification (HER2+) occurs in 15-20% of breast cancer (BC) and identifies a clinically aggressive BC subtype. Although the introduction of effective anti-HER2 drugs remarkably improved the prognosis of HER2+ BC patients, primary and acquired tumor resistance to anti-HER2 treatments underscores the need for novel therapies for HER2+ BC patients. Tumor cells exhibit unique metabolic alterations that are responsible for primary or acquired tumor resistance to standard therapies, and which are increasingly emerging as potential targets for novel treatment strategies. In particular, the dysregulation of fatty acid β-oxidation (FAO), a catabolic process that produces energy (ATP and NADPH production) to sustain cancer growth, survival and aggressiveness, is associated with therapy resistance in diverse malignancies including BC. Specifically, carnitine palmitoyltransferase 1A (CPT1A), the key rate-limiting enzyme of mitochondrial FAO, facilitates cancer metabolic adaptation, thus representing a promising target for cancer therapy. In this context, gene set enrichment analysis of profiled matched HER2+ BC samples (n=33) collected before and after trastuzumab-based neo-adjuvant biochemotherapy (trastuzumab plus taxanes) revealed that the Fatty Acids (FA) metabolism gene set was one of the most significantly Hallmark enriched pathway (NES=1.57; FDR=0.0039) in post- compared with pre-treated HER2+ BC samples, implying that trastuzumab plus chemotherapy treatment induces an enhancement of FAO activity. Accordingly, CPT1A transcript levels were found to be inversely associated with lapatinib (L) susceptibility in HER2+ BC cell lines and patient-derived organoids resistant to anti-HER2 therapy versus L-sensitive cell models. Additionally, Seahorse Analysis revealed higher baseline oxidation of the FA palmitate in intrinsically L-resistant HER2+ BC cells as compared with L-sensitive cells, thus supporting the hypothesis that FAO could represent the major lipid metabolic pathway sustaining the energetic needs of HER2+ BC cells resistant to anti-HER2 therapy for survival and growth. Further, the pharmacological dual blockade of HER2 and CPT1A significantly increases cytotoxicity of L-resistant cell models versus single agents, corroborating the candidacy of CPT1A as a potential metabolic vulnerability to be targeted to overcome refractoriness to HER2-targeted therapy in HER2+ BC. Our data support the hypothesis that the inhibition of FAO sensitizes HER2+ BC cells to anti-HER2 drugs, thus representing a potential strategy to overcome drug resistance in HER2+ BC and paving the way to investigate CPT1A inhibitors with anti-HER2 drugs to improve the clinical outcomes of HER2+ BC patients with acquired resistance to standard anti-HER2 therapies. Citation Format: Alma Franceschini, Lorenzo T Castagnoli, Tiziana IINDT Triulzi, Paola Antonia Corsetto, Matteo Dugo, Antonino Belfiore, Andrea Vingiani, Lorena Signati, Serena Mazzucchelli, Fabio Corsi, Francesca Ligorio, Elda Tagliabue, Claudio Vernieri, Serenella M. Pupa. Carnitine palmitoyltransferase IA: an emerging potential metabolic target to counteract HER2-targeted therapy resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A039.

Abiotic stress applied to Tetraselmis subcordiformis reveals enhanced metabolic responses and increased production of Omega-3 fatty acids: A Metabolomic approach

The appeal of high value algal metabolites is certain due to their sustainability, low energy and space requirements. Yet, commercial-level production of such products is limited and has the potential to be enhanced. In microalgae Tetraselmis subcordiformis (T. subcordiformis) was subjected to four different stressors and the stress response was recorded in terms of changes in the metabolic profile of the strain. The stressors applied were nitrogen (N-starvation) and phosphorus starvation (P-starvation), high salinity, and cold stress. Although growth of the strain was impacted by application of all stressors, an increase of 34 % and 14.9 % in the protein content was noted for 100 PSU and P-starvation stress. Moreover, nitrogen content increase by 20 % and 5.8 % for 100 PSU and N-starvation stress. While the most common lipids for algae in high salinity and N-starvation stressors were triglycerides with α-linoleic (18:3), linoleic (18:2), and palmitic (16:0) fatty acids. Overall, compared to all stressors applied high salinity of 100 PSU enhanced the overall metabolic profile of T. subcordiformis.

Effect of Uncaria tomentosa aqueous extract on the response to palmitate-induced lipotoxicity in cultured skeletal muscle cells

BackgroundType 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA).MethodsCells were incubated with PA in different concentrations ranging from 10 to 1000 μM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 μg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe.ResultsCell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 μM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 μM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively.ConclusionThe Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management.

Role of methyltransferase Set7 in obesity-related endothelial activation: functions beyond histone methylation

Abstract Introduction Obesity is associated with environmental changes such as hyperglycemia, hyperinsulinemia, inflammation, and elevated free fatty acids, all of which compromise endothelial function1,2. We have previously reported that in individuals with type 2 diabetes, lysine methyltransferase Set7 is involved in hyperglycemia-induced endothelial damage through monomethylation of histone 3 at lysine 4 (H3K4me1)3. Apart from such histone-driven epigenetic modulation, Set7-induced methylation of non-histone proteins is emerging as a novel mechanism underlying the (dys)regulation of many biological processes4. Purpose To investigate the role of Set7-related endothelial damage in cells exposed to different obesity-associated stimuli and to determine its impact on the protein methylome in these settings. Methods Human aortic endothelial cells (HAECs) were exposed to high concentrations of glucose, insulin, TNFa, or fatty acids (palmitate or oleate). Set7 overexpression, silencing, and inhibition studies were also performed in these cells. Gene and protein expression were assessed by RT-PCR and immunoblotting respectively, whereas production of the reactive oxygen species (ROS) was measured by electron spin resonance spectroscopy. Specific antibodies were used for the enrichment of lysine mono-, di-, and tri-methylated proteins and LC-MS/MS proteomic analysis was carried out for their identification. Results Each obesity model in HAECs increased the expression of nearly all the inflammatory markers tested and, except for TNFa, they also increased the expression of Set7. Significant changes in the global levels of H3K4me1, as well as of methylated proteins were also observed in these cells. Studies overexpressing a mutant form of Set7, which is lacking the SET domain, revealed that Set7 methyltransferase activity is required to define the protein methylome in HAECs. Similar results were obtained after Set7 silencing and inhibition. Despite no differences were observed in the production of ROS when modulating Set7 expression in HAECs, overexpression of an active form of Set7 and its silencing/inhibition resulted in a consistent up- and down-regulation, respectively, of several inflammatory mediators and adhesion molecules. Finally, proteomics analysis identified, for the first time, the protein methylome of endothelial cells, comprising almost 2,700 methylated proteins involved in essential biological processes such as RNA metabolism and cytoskeleton. Conclusions Our results indicate that Set7 may contribute to inflammatory changes in response to obesogenic insults. Moreover, our findings uncover a dual role of methyltransferase Set7 in governing the methylation of both histone and non-histone proteins in endothelial cells. These preliminary findings open a new field of research aiming to further elucidate the molecular mechanisms underlying the modulation of the protein methylome in the endothelium and its role in endothelial damage.

Cladodes of Opuntia ficus-indica (L.) as a source of bioactive compounds in dairy products

Recently, the interest in improving livestock product nutraceutical profiles through sustainable feeding systems has increased. In this context, the overall quality and nutraceutical profiles were examined in dairy products obtained from 16 lactating Cinisara cows given an integrated feed in dry season with Opuntia ficus-indica cladodes. Two homogeneous groups of cows (milk yield: 6.3 ± 1.5 kg; body weight: 213 ± 55 kg) were fed with 2 different diets (CON: pasture and wheat bran; OFI: pasture, wheat bran, and cladodes), according to a 2 × 2 Latin square design. The bulk milk produced during the study was used to make Caciotta cheese and was analyzed at 0, 15, and 30 storage days. Milk and cheese samples were analyzed for chemical, physical, and microbiological traits. The nutraceutical and sensorial profiles, as well as the antioxidant capacity, were also determined in the final products. For milk, the urea content in individual samples was reduced in OFI but not in CON. In the cheese, integration of cladodes did not influence the starter cultures development with 2 strains of Streptococcus thermophilus, but it caused a higher content of polyphenols and a consequent greater antioxidant capacity and a change in the fatty acids profile. In particular, the caprylic, capric, lauric, myristic, and palmitic fatty acids were higher, as were the petroselinic, vaccenic, rumenic, and α-linolenic fatty acids. In contrast, the oleic and the γ-linolenic fatty acids were lower for OFI. The cheese from the OFI group showed better overall acceptability, and a higher yellow color, odor intensity, and butter flavor. The multivariate analysis well distinguished the cheeses belonging to the 2 groups. Further investigations should be conducted to formulate well-balanced diets that include cladodes for Cinisara lactating cows, but also to determine the content of other important bioactive compounds in fresh and treated cladodes, as well as their effects on animal welfare and production.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.