Pintado, M. E.

Relvas, J. B.

Rodrigues, A.

Rodríguez-Alcalá, L. M.

Salsinha, A. S.

Socodato, R.

English

Repositório Institucional da Universidade Católica Portuguesa

Study of inflammation mediated by fatty acids in a microglia cell model – the obesity perspectiveSalsinha, AS1,2, Rodrigues, A2, Socodato, R2, Relvas, JB2, Rodríguez-Alcalá, LM1, Pintado, ME11. Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina –Laboratório Associado, Escola Superior de Biotecnologia, Rua de Diogo Botelho, 1327, 4169-005 Porto, Portugal2. Instituto de Investigação e Inovação em Saúde and Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto – Rua Alfredo Allen, 208, 4200-135 Porto, PortugalIntroduction ObjectivesMethodsConclusionsAcknowledgementsObesity has currently reached a worldwide pandemic level, being responsible forthe development of non-communicable diseases. The strategies for body weightcontrol have been targeting mainly adipose tissue. Nevertheless, severalinvestigations suggested that brain plays a major role in obesity development:saturated fatty acids (SFAs) bind to a specific receptor (TLR4) in the hypothalamus,triggering inflammatory processes resulting in overconsumption and food addiction.Some concerns arise here since the current western diet, associated with high-fatand - fructose consumption, often provides considerable amounts of SFAs.On the other hand, omega 3 from fish oils were reported as having anti-inflammatory properties in brain and revert the diet-induced obesity effects. Suchprocesses involve the inhibition of the NFkB pathway (one of the most importantinflammatory pathways). In addition, both CLA and CLNA (punicic acid) fatty acidsisomers have shown to reduce body fat mass in animal models and possess anti-inflammatory properties.Despite promising results regarding a potential beneficial effect of CLA and CLNAisomers in obesity, namely on the peripheral tissues, few studies have specificallytargeted the antiobesogenic effect of these isomers in hypothalamus inflammation.Microglia is the main cellular component of the brain innate immune system and akey player in both regulation and protection of the central nervous systemhomeostasis. Therefore, excessive activation of microglia and inflammation-mediatedneurotoxicity are implicated in the progression of several neurological disorders.This work aimed to analyze a human microglia cell line activation by a solutionmimicking the western pattern diet (palmitic acid, a SFA, and fructose), specificallytargeting the NFkB pathway and other important processes such as reactive oxygenspecies (ROS) production and LynSrc activation, which is an important neurotoxicityindicator. Furthermore, the prevention of such effects by omega-3, CLA and CLNAwas evaluated.The incubation of the human microglia cell line cultures with the solution mimicking the western pattern diet (Fructose+Palmitic acid) showed an activation of the NFkB pathway(Figure 2) by a significative IkBa degradation (decreased signal), an increased ROS production (Figure 3) and LynSrc activation (Figure 4), an importante neurotoxicity indicator. Bypre-incubating the cells with individual omega 3, CLA and CLNA solutions, the IkBa degradation was significantly lowered (increased signal), ROS production was decreased andLynSrc activation was suppressed in comparison to cells where no pre-incubation was performed. Thus, these results point out to an anti-inflammatory, antioxidante and inhibitioneffect of LynSrc activation of CLNA, CLA and omega 3 fatty acids under the studied conditions.Figure 2. CLNA and CLA fatty acids prevent NFkBpathway activation by Fructose and Palmitic acid.Results of the quantification of human microglia expressing themiRFP703-IkBa sensor for the selected time points (0, 5, 10 and 15minutes). A decreased signal means a bigger IkBa degradation andhigher NFkB pathway activation. Error bar represents the SEMcalculated from >15 cells from three independent cultures. Two-wayANOVA in relation to Frut+PA, where no pre-incubation with theselected fatty acids was performed. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.Author Ana Sofia Salsinha would like to thank FCT for her PhD grant (SFRH/BD/136857/2018). The authors would also like to thank the scientific collaboration under the FCTproject UID/Multi/50016/2019.Table 1. Composition of the tested Fatty acids solutions.Figure 1. Experimental design schematic representation.Solution ID Fatty acidsOtherComponentsProportionFinal concentration/ solutionStimulus (western pattern diet)Palmitic Acid (C16:0) Fructose 2:1100 μMOmega 3EPA (C20:5 n-3) -1:1DHA (C22:6 n-3) -CLARumenic acid (C18:2 c9t11) -1:1C18:2 t10c12 CLA isomer -CLNA Punicic Acid C18:3 c9t11c13 - -ResultsFigure 3. CLNA, CLA and omega 3 fatty acids preventROS production by Fructose and Palmitic acid. Results ofthe quantification of human microglia expressing the HSP-FRET sensorfor the selected time points (0, 5, 10 and 15 minutes). Error barrepresents the SEM calculated from >15 cells from three independentcultures. Two-way ANOVA in relation to Frut+PA, where no pre-incubation with the selected fatty acids was performed. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.Figure 4. CLNA prevents Src Tyrosine kinaseactivation by Fructose and Palmitic acid. Results of thequantification of human microglia expressing the LynSrc-FRET sensorfor the selected time points (0, 5, 10 and 15 minutes). Error barrepresents the SEM calculated from >15 cells from three independentcultures. Two-way ANOVA in relation to Frut+PA, where no pre-incubation with the selected fatty acids was performed. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.05 min10 min15 min0.00.20.40.60.80.880.900.920.940.960.981.001.021.041.061.08IkBa degradationFrut+PAOmega 3 + [Frut+PA]CLA + [Frut+PA]CLNA + [Frut+PA]✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱05 min10 min15 min0.00.20.40.60.80.900.920.940.960.981.001.021.041.061.081.101.12Donor/FRET ratio change(ROS production)Frut+PAOmega 3 + [Frut+PA]CLA + [Frut+PA]CLNA + [Frut+PA]✱✱✱✱✱✱✱✱✱✱✱✱✱05 min10 min15 min0.800.850.900.951.001.051.101.15Donor/FRET ratio change(LynSrc biosensor)Frut+PAOmega 3 + [Frut+PA]CLA + [Frut+PA]CLNA + [Frut+PA]✱✱✱✱✱✱✱✱Palmitic acid + Fructose solution(Western Pattern Diet)HMC3(Human microglia cell line) miRFP703-IkBa (NFkB pathway activation)Pre-incubation of HMC3Live imagingInflammatory Stimulus30 minutesLeica DMI6000B invertedmicroscopeOmega 3 CLAIkBa degradation quantificationNFkB pathway activation (indirect measure)CLNAHSP-FRET (ROS production)LynSrc-FRET (Src tyrosine kinase activation)ROS productionSrc tyrosine kinase activationIts activation is essential for the production of inflammatory mediators by microglia

Study of inflammation mediated by fatty acids in a microglia cell model - the obesity perspective

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Study of inflammation mediated by fatty acids in a microglia cell model - the obesity perspective

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