Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20–30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with ^99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with ^99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using ^99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.

Background: High fat diet (HFD) increases the likelihood of dyslipidemia, which can be a serious risk factor for atherosclerosis, diabetes or hepatosteatosis. Although changes in different blood lipid levels were broadly investigated, such alterations in the liver tissue have not been studied before. The aim of the current study was to investigate the effect of HFD on hepatic triglyceride (TG), diglyceride (DG) and ceramide (CER) levels and on the expression of four key genes involved in lipid homeostasis (Pcsk9, Ldlr, Cd36 and Anxa2) in the liver. In addition, the potential role of PCSK9 in the observed changes was further investigated by using PCSK9 deficient mice. Methods: We used two in vivo models: mice kept on HFD for 20 weeks and PCSK9^−/− mice. The amount of the major TGs, DGs and CERs was measured by using HPLC–MS/MS analysis. The expression profiles of four lipid related genes, namely Pcsk9, Ldlr, Cd36 and Anxa2 were assessed. Co-localization studies were performed by confocal microscopy. Results: In HFD mice, hepatic PCSK9 expression was decreased and ANXA2 expression was increased both on mRNA and protein levels, and the amount of LDLR and CD36 receptor proteins was increased. While LDLR protein level was also elevated in the livers of PCSK9^−/− mice, there was no significant change in the expression of ANXA2 and CD36 in these animals. HFD induced a significant elevation in the hepatic levels of all measured TG and DG but not of CER types, and increased the proportion of monounsaturated vs. saturated TGs and DGs. Similar changes were detected in the hepatic lipid profiles of HFD and PCSK9^−/− mice. Co-localization of PCSK9 with LDLR, CD36 and ANXA2 was verified in HepG2 cells. Conclusions: Our results show that obesogenic HFD downregulates PCSK9 expression in the liver and causes alterations in the hepatic lipid accumulation, which resemble those observed in PCSK9 deficiency. These findings suggest that PCSK9-mediated modulation of LDLR and CD36 expression might contribute to the HFD-induced changes in lipid homeostasis.

Disturbances in lipid metabolism related to excessive food intake and sedentary lifestyle are among major risk of various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) has an essential role in these diseases, as it catalyzes the synthesis of unsaturated fatty acids, both supplying for fat storage and contributing to cellular defense against saturated fatty acid toxicity. Recent studies show that increased activity or over-expression of SCD1 is one of the contributing factors for type 2 diabetes mellitus (T2DM). We aimed to investigate the impact of the common missense rs2234970 (M224L) polymorphism on SCD1 function in transfected cells. We found a higher expression of the minor Leu224 variant, which can be attributed to a combination of mRNA and protein stabilization. The latter was further enhanced by various fatty acids. The increased level of Leu224 variant resulted in an elevated unsaturated: saturated fatty acid ratio, due to higher oleate and palmitoleate contents. Accumulation of Leu224 variant was found in a T2DM patient group, however, the difference was statistically not significant. In conclusion, the minor variant of rs2234970 polymorphism might contribute to the development of obesity-related metabolic disorders, including T2DM, through an increased intracellular level of SCD1.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.

Background. tert-butylhydroquinone (tBHQ) is an antioxidant commonly used as a food additive. Studies suggest that tBHQ could modulate immune responses to influenza and SARS-CoV-2 infection. In our transcriptomic analysis we explored the molecular mechanisms behind tBHQ’s modulatory properties and the relationships to respiratory viral infections. Methods. tBHQ was administered per os to BALB/c mice (1.5% [w/w]) for 20 days. Splenic T cells were isolated with magnetic separation and subjected to transcriptomic analysis. Gene-set enrichment analysis and g:Profiler was conducted to provide a functional interpretation of significantly changed genes. Further analysis for AHR/NRF2 binding sites was performed with GeneHancer. Results. In CD4⁺ cells, we found significantly altered expression of 269 genes by tBHQ. Of them, many had relevance in influenza infection such as genes responsible for virus entry (Anxa1/2, Cd14), interferon signaling (Dusp10, Tnfsf13), or prostaglandin synthesis (Ptgs1/2). In SARS-CoV-2 infections, interferon signaling (Ifitm1), proteolytic enzymes (CtsB), and also cell-surface proteins (Cd14, Cd151) were among the prominent alterations after tBHQ exposure. Of these genes, many had one or more binding sites for AHR and NRF2, two major xenosensors triggered by tBHQ. Conclusions. Our results strongly suggest that a common food additive, tBHQ, can modulate virus-dependent processes in both influenza and SARS-CoV-2 infections.

The rise of antimicrobial resistance is a health crisis that severely compromises treatment options for bacterial infections. Metallo-β-lactamases (MBLs) are zinc-dependent hydrolases that are an increasingly important contributor to this crisis by inactivating near all β-lactam antibiotics, including carbapenems. The WHO has identified the widespread dissemination of MBL-producing pathogens as a priority threat. The Verona integron-encoded metallo-β-lactamase-2 (VIM-2) is one of the most prevalent and broadly disseminated MBLs, conferring high-level carbapenem resistance in Gram-negative pathogens. Here we report the α-aminophosphonic acid group as a new scaffold that effectively targets VIM-2 providing a new avenue for restoring the clinical utility of β-lactams. α-Aminophosphonic acid derivatives can be produced in 2–3 steps from commercially available starting materials. The VIM-2 inhibition activity, cytotoxicity, membrane permeability and the NMR and X-ray based characterisation of their VIM-2 binding mode are described. The overall results identify aminophosphonic acid derivatives as a new mode of MBL inhibition.