Impact of 3-Amino-1,2,4-Triazole (3-AT)-Derived Increase in Hydrogen Peroxide Levels on Inflammation and Metabolism in Human Differentiated Adipocytes

<div><p>Obesity is characterized by an excessive accumulation of fat in adipose tissue, which is associated with oxidative stress and chronic inflammation. Excessive H₂O₂ levels are degraded by catalase (CAT), the activity of which is decreased in obesity. We investigated the effects of inhibition of catalase activity on metabolism and inflammation by incubating human differentiated adipocytes with 10 mM 3-amino-1,2,4-triazole (3-AT) for 24 h. As expected, the treatment decreased CAT activity and increased intracellular H₂O₂ levels significantly. Glutathione peroxidase (GPX) activity was also reduced, and the gene expression levels of the antioxidant enzymes <i>GPX4</i> and peroxiredoxins (1, 3 and 5) were inhibited. Interestingly, this occurred along with lower mRNA levels of the transcription factors nuclear factor (erythroid 2-like 2) and forkhead box O, which are involved in redox homeostasis. However, superoxide dismutase activity and expression were increased. Moreover, 3-AT led to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and increased tumor necrosis alpha and interleukin 6 protein and gene expression levels, while lowering peroxisome proliferator-activated receptor gamma (<i>PPAR</i>γ) mRNA and protein levels. These alterations were accompanied by an altered glucose and lipid metabolism. Indeed, adipocytes treated with 3-AT showed reduced basal glucose uptake, reduced glucose transporter type 4 gene and protein expression, reduced lipolysis, reduced AMP-activated protein kinase activation and reduced gene expression of lipases. Our results indicate that increased H₂O₂ levels caused by 3-AT treatment impair the antioxidant defense system, lower <i>PPARγ</i> expression and initiate inflammation, thus affecting glucose and lipid metabolism in human differentiated adipocytes.</p></div

Forward and reverse primer sequences used in the qPCR assays.

<p>Forward and reverse primer sequences used in the qPCR assays.</p

Diagram of the conditions observed in 3-AT-treated adipocytes.

<p>The increased hydrogen peroxide production leads to lower <i>PPARγ</i> expression and NF-κB activation, while decreasing the expression of important enzymes in metabolism, all of which finally reduce glucose uptake and lipolysis. AMPK: 5'-AMP-activated protein kinase; ATGL: adipose triglyceride lipase; CAT: catalase; FABP4: fatty-acid binding protein 4; GLUT4: glucose transporter type 4; GPX: glutathione peroxidase; HSL: hormone sensitive lipase; IL-6: interleukin 6; NRF2: nuclear factor, erythroid 2-like 2; NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B-cells 2; P: enzyme phosphorylation; PPARγ: peroxisome proliferator-activated receptor gamma; SOD: superoxide dismutase; TNF-α: tumor necrosis factor alpha. The black arrows indicate an increase or decrease in activity, expression or protein levels. The bold arrows indicate causal relationships between findings, and the light grey arrows indicate potential relationships according to the revised literature.</p

Effects of 3-amino-1,2,4-triazole (3-AT) on the antioxidant system in human differentiated adipocytes.

<p>A: Superoxide dismutase activity (SOD) expressed as U/mg protein in the presence or absence of 3-AT. B: mRNA expression of <i>SOD1</i> normalized to hypoxanthine-guanine phosphoribosyltransferase-1 (<i>HPRT1</i>) mRNA levels. C: Glutathione peroxidase (GPX) activity expressed as nmol/min/mg protein in the presence or absence of 3-AT. D: mRNA expression of glutathione peroxidase 4 (<i>GPX4</i>) normalized to <i>HPRT1</i> mRNA levels. E-G: mRNA expression of peroxiredoxin 1, 3 and 5 (<i>PRDX1</i>, <i>PRDX3</i>, and <i>PRDX5</i>) normalized to <i>HPRT1</i> mRNA levels in the presence or absence of 3-AT. H-J: mRNA expression of nuclear factor, erythroid 2-like 2 (<i>NRF2</i>), forkhead box O1 (<i>FOXO1</i>) and catenin beta 1 (<i>CTNNB1</i>) normalized to <i>HPRT1</i> mRNA levels in the presence or absence of 3-AT. The fold-changes from three independent experiments were calculated using the Pfaffl method. The data are presented as the means ± SEM of three independent experiments. Significant differences were identified using the Mann-Whitney U test; * <i>P</i><0.05.</p

Effects of 3-amino-1,2,4-triazole (3-AT) on glucose metabolism in human differentiated adipocytes.

<p>A: Basal and insulin-stimulated glucose uptake levels in human differentiated adipocytes with or without 10 mM 3-AT and 1 μM insulin. Significant differences were identified using the Kruskal-Wallis test. B: mRNA and protein levels of glucose transporter 4 (<i>GLUT4)</i> in the presence or absence of 3-AT (10 mM, 24 h). The mRNA levels were normalized to those of hypoxanthine-guanine phosphoribosyltransferase-1 (<i>HPRT1</i>). The results are presented as fold-changes, which were calculated using the Pfaffl method. Protein expression of GLUT4 analyzed by western blot as described in the Methods section. Protein levels were normalized to the internal control (α-tubulin) and expressed as fold-changes. The data from three independent experiments are presented as the means ± SEM. Significant differences were identified using the Mann-Whitney U test. * <i>P</i><0.05.</p

Effect of 3-amino-1,2,4-triazole (3-AT) treatment on inflammation in adipocytes.

<p>A: phospho-NFκB p65 protein levels analyzed by western blot. B: mRNA expression of tumor necrosis factor-α (<i>TNFA</i>) normalized to hypoxanthine-guanine phosphoribosyltransferase-1(<i>HPRT1</i>) mRNA levels. C: mRNA expression of interleukin 6 (<i>IL-6</i>) normalized to <i>HPRT1</i> mRNA levels. The fold-changes from three independent experiments were calculated using the Pfaffl method. D: TNF-α protein levels analyzed by western blot using a specific antibody, normalized to α-tubulin. E: IL-6 protein levels analyzed by XMap technology (Luminex) as indicated in the methods section. The data from three independent experiments are presented as the means ± SEM. Significant differences were identified using the Mann-Whitney U test; * <i>P<</i>0.05.</p

Effects of 3-amino-1,2,4-triazole (3-AT) on lipid metabolism.

<p>A: Glycerol levels (μM) in cell supernatants after treatment with 3-AT (10 mM, 24 h). B: Hormone sensitive lipase (<i>HSL</i>) gene expression. C: Adipose triglyceride lipase (<i>ATGL</i>) gene expression. D: Perilipin (<i>PLIN</i>) gene expression. The mRNA levels were normalized to those of hypoxanthine-guanine phosphoribosyltransferase-1 <i>(HPRT1</i>), and data from three independent experiments are presented as the means ± SEM of the fold-changes calculated using the Pfaffl method. E: Protein Kinase A (PKA) activity in the cell lysates of adipocytes treated with or without 3-AT (10 mM, 24 h) and 0.1 mM cAMP. F: 5'-AMP-activated protein kinase catalytic subunit alpha (AMPKα) protein levels. The cell lysates were prepared and then analyzed by western blot using specific antibodies against total AMPKα and phospho-AMPKα (Thr172) as described in the Methods section. The data are presented as the ratio of phosphor-AMPKα/total-AMPKα to no treatment fold-change, and the bars represent the means ± SEM of three separate experiments. Significant differences were identified using the Mann-Whitney U test; * <i>P<</i>0.05, ** <i>P<</i>0.01.</p

BiSKY Team an aerospace-focused interdisciplinary student project

BiSKY Team is an aerospace-focused student group from the University of the Basque Country (UPV/EHU, Bilbao, Spain) where it is a recognized teaching project. It was born in 2018 and its main activities deal with the development of the technologies involved in the design, manufacture and launch of suborbital rockets. This team is currently the only Spanish university team involved in the research and construction of hybrid engine rockets. The primary objective of the team is to enable young science and engineering students to acquire expertise in the aerospace field, and several transversal skills as well, by designing and constructing space vehicles. Further purposes include promoting science and engineering among high school students and children and to also reduce the existing disparity between male and female involvement in science, technology, engineering and mathematics (STEM). Besides, the project will make interesting contributions to space science by providing researchers with the means to test their experiments in zero gravity and high-altitude vacuum. BiSKY Team is divided into several specialized groups: i) Aerodynamics and Recovery, ii) Propulsion, iii) Avionics, iv) Flight Control and Simulation, v) Business and Management and vi) Structure. This interdisciplinary project makes the collaboration among all groups crucial. In this regard, the team stands for respectful cooperation between all its students. BiSKY Team is an example of a multidisciplinary student project that implements innovative technologies allowing not only its members, but also members of other student research groups and training centres, to enter the competitive sector of aerospace engineering and space science research. Even if this is a university student-developed and managed project, vocational training schools’ involvement is also considered. Close contacts with research and technological institutions as well as industrial companies are pursued looking for technical advice and financial support. Within the operations of the team, several phases are being undertaken in order to acquire the expertise necessary to design, manufacture and launch a hybrid engine rocket that reaches an altitude of 100 kilometres, also acknowledged as the Karman Line. The phases include the development of two engine test stands, a flight simulator and the complete avionics for the rockets and test stands. The expertise gained through the implementation of the mentioned technology is being applied in the hybrid engine rockets of the so-called Cosmox family, whose primary mission is to reach space and allow the research experiments to be carried out. The design of the Cosmox and future families of rockets is iterative, giving continuity to the project by allowing next generations students to get involved

Pro-inflammatory cytokine production in DCs after exposure to <i>L. paracasei</i>, <i>Salmonella</i> or both.

<p>Dendritic cells (DCs) were incubated with the probiotic <i>L. paracasei</i> CNCM I-4034 (Prob) or its cell-free supernatant (CFS), <i>Salmonella</i> (<i>S.typhi</i>), or both. <i>E. coli</i> lipopolysaccharide (LPS, 20 ng/ml) was used as a positive control. Negative-control cultures contained unstimulated DCs. IL-1β, IL-6, IL-8, IL-12(p40) and IL-12(p70) production was measured. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to <i>S.typhi.</i></p

Expression levels of TLR signalling pathway in DCs treated with <i>L. paracasei</i>, <i>Salmonella</i> or both.

<p>Comparison of the expression of <i>CASP8, TAK-1, JNK, IRF-3</i> and <i>MAPK14</i> in DCs in the presence of the probiotic (Prob), its supernatant (CFS), <i>Salmonella</i> (<i>S.typhi</i>) or a combination. LPS, 20 ng/ml, was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to the expression in control cells. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to <i>S.typhi.</i></p