AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity

Renal proximal tubules are a primary site of injury in metabolic diseases. In obese patients and animal models, proximal tubular epithelial cells (PTECs) display dysregulated lipid metabolism, organelle dysfunctions, and oxidative stress that contribute to interstitial inflammation, fibrosis and ultimately end-stage renal failure. Our research group previously pointed out AMP-activated protein kinase (AMPK) decline as a driver of obesity-induced renal disease. Because PTECs display high macroautophagic/autophagic activity and rely heavily on their endo-lysosomal system, we investigated the effect of lipid stress on autophagic flux and lysosomes in these cells. Using a model of highly differentiated primary PTECs challenged with palmitate, our data placed lysosomes at the cornerstone of the lipotoxic phenotype. As soon as 6 h after palmitate exposure, cells displayed impaired lysosomal acidification subsequently leading to autophagosome accumulation and activation of lysosomal biogenesis. We also showed the inability of lysosomal quality control to restore acidic pH which finally drove PTECs dedifferentiation. When palmitate-induced AMPK activity decline was prevented by AMPK activators, lysosomal acidification and the differentiation profile of PTECs were preserved. Our work provided key insights on the importance of lysosomes in PTECs homeostasis and lipotoxicity and demonstrated the potential of AMPK in protecting the organelle from lipid stress

Liver transcriptomics analysis unravels the sequentiality of liver steatosis mechanisms in metabolic dysfunction-associated steatohepatitis

Introduction: Hepatic steatosis is secondary to a complex metabolic dysregulation of lipid homeostasis. Pathophysiological mechanisms are numerous and include dysregulations in lipid uptake, de novo synthesis, oxidation and secretion. Specific drugs targeting lipid metabolism are currently evaluated in clinical trials with inconsistent results. We suggest that the pathophysiology of steatosis may evolve with MAFLD progression and the mixed effects may reveal a mismatch between the deregulated mechanisms and the drug used. Aim: To analyse the evolution of gene expression of key lipid metabolism genes during MASLD-MASH disease progression in mice. Methods: Foz/foz mice (FOZ) were fed normal diet (ND)(3.39 kcal/g, 16% of lipids, A03 Research Diets) or high-fat diet (HFD)(5.24 kcal/g, 60% of lipids, D12492 Research Diets). Liver tissue was harvested at baseline (control group, N=5) and after 4 (FOZ ND=3, HFD=4), 12 (FOZ ND=6, HFD=8) or 32 (FOZ ND=6, HFD=6) weeks. Formalin-fixed paraffin-embedded liver samples were used for histological scoring (SAF score) using hematoxylin & eosin staining. Total RNA from liver samples was extracted and purified using Qiagen® mini kits prior to bulk RNA sequencing (Macrogen Europe). Sequencing data were normalized according to timepoint and diet and analyzed RStudio. Differential gene expression and enrichment analysis (Gene Ontology database, biological process) were performed as follows: FOZ ND week 0 (control group) versus FOZ HFD week 4, FOZ ND week 0 versus FOZ HFD week 12, FOZ ND week 0 versus FOZ HFD week 32. Differentially expressed genes (DEG) were defined by a p adjusted value (padj) 1. Enrichment results are presented by Normalized Enrichment Score (NES) and p adjusted value. Results: FOZ mice fed HFD reached maximal liver weight to body weight ratio at 32 weeks (p < 0.001). Insulin resistant is present at 4 weeks of HFD as assessed by the HOMA-IR index (p < 0.01). FOZ mice fed HFD exhibited the whole MASH spectrum with fluctuating transcriptomics changes over time. At week 4, liver histology highlighted isolated steatosis with 284 DEG involved in 685 enriched pathways including fatty acid biosynthetic process (NES = 13.9, padj = 1.2e-4)(Pparɣ, Hacd4, Obp2a), lipid transport (NES = 9.7, padj = 8.0e-8)(Pparɣ, Vldlr, Pltp), lipid storage (NES = 21.5, padj = 3.0e-6)(Cav1, Clstn3, Mup1) and fatty acid oxidation (NES = 9.4, padj = 0.02)(Pparɣ, Obp2a, Sox9). At week 12, liver histology reported steatohepatitis with 469 DEG involved in 504 enriched pathways including lipid transport (NES = 14.7, padj = 3.6e-15)(Cd36, Pparɣ, Pparδ, Pltp), lipid storage (NES = 24.2, padj = 1.66e-7)(Fitm1, Bltp1, Cidec) and fatty acid oxidation (NES = 22.2, padj = 2.7e-7)(Acaa1b, Ehhadh, Pparδ). At week 32, fibrosing steatohepatitis was observed with 750 DEG involved in 1167 enriched pathways including lipid transport (NES = 11.3, padj = 6.3e-21)(Cd36, Apom, Pparδ), lipid storage (NES = 24, padj = 1.4e-15)(Fitm1, Bltp1, Cidec; Pparɣ) and fatty acid oxidation (NES = 11, padj = 7.4e-7)(Hao2, Pparɣ, Pparδ). At the gene level, the lipid metabolism key regulator Pparɣ was downregulated in a time-dependent manner (Log2FC = -1.6, padj = 9e-5 at week 4; Log2FC = -1.8, padj = 9e-6 at week 12 and Log2FC = -2.35, padj = 6.6e-9 at week 32). Fatty acid oxidation related genes are all downregulated at week 12, such as Obp2a (Log2FC = -3.17, padj = 0.003) and Acaa1b (Log2FC = -1.64, padj = 9.9e-9). Lipid storage genes expression also greatly change over time. Cidec and Fitm1 are involved in lipid droplet homeostasis preventing lipotoxicity of free fatty acids. Both Cidec and Fitm1 are significantly downregulated at week 12 (Log2FC = -2.13, padj = 3e-6; Log2FC = -1.37, padj = 1.68e-7 respectively) and at week 32 (Log2FC = -2.7, padj = 2e-5; Log2FC = -1.02, padj = 2e-4 respectively). Conclusions: we report the dynamic changes of pathophysiological mechanisms potentially involved in hepatic steatosis occurring in a MASH mouse model over time using bulk RNAseq. These time-dependent shifts in gene expression of potential therapeutic targets may significantly influence the response to MASLD treatments

Hiring subsidies and temporary work agencies

This paper evaluates a hiring subsidy for lower-educated youths in Flanders (Belgium) that reduced labour costs by approximately 13% for a period of two years, starting in 2016. Using a donut Regression Discontinuity Design, we find no evidence that the subsidy improved the job finding rate of eligible job seekers in 2016-19, a period marked by a tight labour market. We then investigate the role of temporary work agencies, which disproportionately employ the target group and obtain 25% to 34% of the subsidies. Using Difference-in-Differences regressions, we demonstrate that agencies did not raise wages of eligible agency workers in response to the policy. Remarkably, despite a 3.3% labour cost reduction, full-time equivalent employment of eligible workers in these agencies decreased by 9.2% over the three years following the reform. Our findings highlight how an active labour market policy affects agency employment

On the combustion of Fe-Al and Fe-Si powders for sustainable energy storage

Recently, metal fuels have been proposed as sustainable energy carriers with high energy density. In particular, iron powder holds great promise due to its relatively low cost, safety, abundance, and easy retrofit of power plants. So far, research and development have been focused on the use of pure Fe powder for this sustainable fuel application. Yet, the use of impure Fe coming from low-cost Fe sources such as recycled scraps would strongly increase the economic competitiveness of iron fuel compared to fossil fuels or other high-energy density carriers such as ammonia. The influence of these impurities on the combustion process has however not yet been studied in detail. In this work, the combustion behaviour of Fe-Al and Fe-Si powders will be presented, with a focus on their ignition process and on their microstructural and chemical evolution after combustion. Chemically homogeneous metallic powders with different concentrations of Al and Si (up to 7 wt%) will be produced by casting followed by gas atomization. The ignition efficiency of each powder will be compared to that of pure Fe for identical combustion parameters, based on the relative amount of unoxidized, metallic phase in the combusted products measured by x-ray diffraction. The ignition temperature for each composition will be additionally measured using a solid particle ignition reactor, and linked in situ with the particle diameter using high-speed optical diagnostics. The ignition efficiency of each composition will be discussed in light of their solid-state oxidation reactions. Isothermal solid-state oxidation kinetics will be measured by thermogravimetry analysis (TGA) at different oxidation temperatures (700°C- 1000°C) and coupled with microstructural characterization to identify the limiting mechanisms governing the oxidation process. Advanced microstructural characterization of the combusted powders will also be presented to unveil the impact of Al and Si on the combustion behaviour of alloyed Fe powders. In particular, the global chemical, porosity and phase evolution will be characterized using Inductive Coupled Plasma (ICP), gas pycnometer, surface area BET measurements, and x-ray diffraction (XRD). The local microstructural, morphological and chemical evolution of the different phases in the micro- and nano-particles will be presented using scanning electron microscopy (SEM, STEM) coupled with Energy Dispersive Spectroscopy (EDS) and Electron Backscatter Diffraction (EBSD). The microstructural and chemical evolution of the powders will be discussed with thermodynamic simulations of the combustion process using Thermo-Calc

Gut dysbiosis is linked to severe steatosis and enhances its diagnostic performance in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the leading cause of chronic liver disease globally, with rising prevalence linked to metabolic syndrome (MetS). Excessive liver fat accumulation (steatosis) worsens disease progression and MASLD prognosis. Moreover, gut microbiota dysbiosis might promote steatosis, accelerating the disease progression to severe stages. Identifying gut microbiota signatures specific to steatosis severity might improve its diagnosis and inform personalised interventions in MASLD. This study aimed to characterise associations between gut microbiota composition and hepatic steatosis severity in a cohort of patients with MASLD/MetS. Ultimately, we aimed to assess the potential for microbiota features to enhance the diagnosis of severe steatosis. A cross-sectional cohort of 61 patients with MetS with extensive clinical history was recruited at different stages of MASLD. Transient elastography was used to evaluate liver fibrosis and steatosis severity. Participants' faecal microbiota were profiled using 16S rRNA gene sequencing. Statistical analyses first identified correlations between microbiota profiles and patients' phenotypes, while disentangling important confounders such as medication. Identified features were then used to build predictive models for diagnosing severe steatosis. High steatosis severity was distinctly associated with a higher prevalence of the inflammation-associated Bacteroides 2 (Bact2)-enterotype, accompanied by a lower proportion of beneficial commensals (eg, ) and a higher proportion of opportunistic bacteria (eg, ). Patients harbouring a Bact2-enterotype reached severe steatosis at lower Fatty Liver Index (FLI) thresholds. Using Bact2-carrier status together with FLI in a predictive model significantly improved the classification of severe steatosis (accuracy 90%, receiver operating characteristics 96%) when compared with FLI alone. Gut microbiota composition and dysbiosis (defined as Bact2-enterotype) are distinctly associated with steatosis severity in MASLD/MetS. Patient stratification by microbiota composition enhances the diagnostic classification of severe steatosis in MASLD, suggesting a potential for personalised interventions in patients with microbiota dysbiosis