Acute Deletion of the Glucocorticoid Receptor in Hepatocytes Disrupts Postprandial Lipid Metabolism in Male Mice.

Hepatic lipid metabolism is highly dynamic, and disruption of several circadian transcriptional regulators results in hepatic steatosis. This includes genetic disruption of the glucocorticoid receptor (GR) as the liver develops. To address the functional role of GR in the adult liver, we used an acute hepatocyte-specific GR knockout model to study temporal hepatic lipid metabolism governed by GR at several preprandial and postprandial circadian timepoints. Lipidomics analysis revealed significant temporal lipid metabolism, where GR disruption results in impaired regulation of specific triglycerides, nonesterified fatty acids, and sphingolipids. This correlates with increased number and size of lipid droplets and mildly reduced mitochondrial respiration, most noticeably in the postprandial phase. Proteomics and transcriptomics analyses suggest that dysregulated lipid metabolism originates from pronounced induced expression of enzymes involved in fatty acid synthesis, ß-oxidation, and sphingolipid metabolism. Integration of GR cistromic data suggests that induced gene expression is a result of regulatory actions secondary to direct GR effects on gene transcription.

Wrapping anisotropic microgel particles in lipid membranes: Effects of particle shape and membrane rigidity.

Cellular engulfment and uptake of macromolecular assemblies or nanoparticles via endocytosis can be associated to both healthy and disease-related biological processes as well as delivery of drug nanoparticles and potential nanotoxicity of pollutants. Depending on the physical and chemical properties of the system, the adsorbed particles may remain at the membrane surface, become wrapped by the membrane, or translocate across the membrane through an endocytosis-like process. In this paper, we address the question of how the wrapping of colloidal particles by lipid membranes can be controlled by the shape of the particles, the particle-membrane adhesion energy, the membrane phase behavior, and the membrane-bending rigidity. We use a model system composed of soft core-shell microgel particles with spherical and ellipsoidal shapes, together with phospholipid membranes with varying composition. Confocal microscopy data clearly demonstrate how tuning of these basic properties of particles and membranes can be used to direct wrapping and membrane deformation and the organization of the particles at the membrane. The deep-wrapped states are more favorable for ellipsoidal than for spherical microgel particles of similar volume. Theoretical calculations for fixed adhesion strength predict the opposite behavior-wrapping becomes more difficult with increasing aspect ratio. The comparison with the experiments implies that the microgel adhesion strength must increase with increasing particle stretching. Considering the versatility offered by microgels systems to be synthesized with different shapes, functionalizations, and mechanical properties, the present findings further inspire future studies involving nanoparticle-membrane interactions relevant for the design of novel biomaterials and therapeutic applications.

The ROC skin model: A robust skin equivalent for permeation and live cell imaging studies.

Rat epidermal keratinocyte (REK) Organotypic culture (ROC) is an epidermis model that is robust and inexpensive to develop and maintain, and it has in previous studies been shown to have permeability characteristics close to those of human skin. Here, we characterize the model further by structural comparison to native human and rat skin and by investigating functional characteristics of lipid packing, polarity, and permeability coefficients. We show that the ROC model has structural similarities to native human skin and observe human skin-like permeability coefficients for testosterone and mannitol. We develop a transwell device that allows live cell microscopy of the tissue at the air-liquid interface and establish transgenic cell lines expressing different fluorescently tagged proteins. This enables showing the migration of keratinocytes during the first days after seeding, finding that keratinocytes have a higher mean migration rate in the earlier days of development. Collectively, our results show that the ROC model is an inexpensive and robust epidermis model that works reproducibly across laboratories.

Effects of recombinant human gastric lipase and pancreatin during in vitro pediatric gastro-intestinal digestion.

The aim of the study was to implement a gastric digestion step using recombinant human gastric lipase (rHGL) in an in vitro pediatric gastro-intestinal digestion model to achieve a physiologically relevant gastric contribution to total gastro-intestinal lipid digestion. A commercial infant formula (NAN Comfort stage 1 (NAN1)) with 3.4% lipid and an in-lab prepared oil-in-water emulsion, emulsified with soy phosphatidylcholine (SPCemul), with 3.5% lipid (oil-blend containing Akonino NS, MEG-3 and ARASCO oils) were subjected to in vitro gastro-intestinal digestion. To achieve a physiologically relevant level of gastric digestion, 50 min of in vitro gastric digestion, using either 0, 3.75 or 7.5 TBU mL-1 rHGL, was followed by 90 min of in vitro intestinal digestion, using either 0 or 26.5 TBU mL-1 pancreatic triglyceride lipase (PTL) from porcine pancreatin. The digestion of the substrates was assessed using titration-based quantification supported by HPLC-ELSD analysis. In vitro gastric digestion of NAN1 and SPCemul with either 3.75 or 7.5 TBU mL-1 rHGL contributed with 10-27% of the total gastro-intestinal digestion, corresponding to the reported contribution in human infants. At the end of the gastro-intestinal digestion (t = 140 min), the combined lipolytic effect of rHGL and PTL was additive during digestion of SPCemul, but not for the digestion of NAN1, as all lipase activity combinations resulted in a similar degree of NAN1 digestion. The effect of gastric digestion with rHGL on total digestion therefore appeared to be substrate dependent. To conclude, a gastric digestion step using rHGL resulting in physiologically relevant gastric contribution to the observed gastro-intestinal digestion was successfully implemented into an in vitro pediatric gastro-intestinal digestion model.

Activation of phospholipase A2 by prostaglandin in vitro.

Prostaglandins are a diverse family of biological active molecules that are synthesized after liberation of arachnidonic or linolenic acid from the plasma membrane by phospholipase A2 (PLA2). Specific prostaglandins may be pro-inflammatory or anti-inflammatory due to a poorly understood biochemical equilibrium. Some of the anti-inflammatory prostaglandins namely, prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) have a cyclopentenone moiety that can react and modify a protein's activity. These two prostaglandins are electrophilic reactive lipid species and are formed as a result of the reaction cascade initiated by PLA2. It was of interest to study the interaction with these prostaglandins as they could either amplify or block this enzyme's activity. We found that the former is true initially as there is a shorter time to activate the protein on the lipid membrane and an overall increase in hydrolysis was observed when PGA1 and PGE1 prostaglandin was added with PLA2 and liposomes. The interfacial activation model was further explored in which there is a modification of the enzyme rather than a modifcation of the substrate. However, after a time the protein was shown to form amyloid like fibrils thereby blocking further hydrolysis. The fibrillization kinetics in the presence of the one of the prostaglandins was monitored using thioflavin T (ThT) and the resulting fibrils were characterized using transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modification of PLA2 by these prostaglandins leading to amyloid like fibrils gives an additional perspective of control of the interfacial activation mechanism of this enzyme.

Plasticity of Epididymal Adipose Tissue in Response to Diet-Induced Obesity at Single-Nucleus Resolution.

Adipose tissues display a remarkable ability to adapt to the dietary status. Here, we have applied single-nucleus RNA-seq to map the plasticity of mouse epididymal white adipose tissue at single-nucleus resolution in response to high-fat-diet-induced obesity. The single-nucleus approach allowed us to recover all major cell types and to reveal distinct transcriptional stages along the entire adipogenic trajectory from preadipocyte commitment to mature adipocytes. We demonstrate the existence of different adipocyte subpopulations and show that obesity leads to disappearance of the lipogenic subpopulation and increased abundance of the stressed lipid-scavenging subpopulation. Moreover, obesity is associated with major changes in the abundance and gene expression of other cell populations, including a dramatic increase in lipid-handling genes in macrophages at the expense of macrophage-specific genes. The data provide a powerful resource for future hypothesis-driven investigations of the mechanisms of adipocyte differentiation and adipose tissue plasticity.

Polycation-based nanoparticles for RNAi-mediated cancer treatment.

Cancer disorders exhibit an increasing high global incidence, in part, to an aging population with a high socio-economic burden. The cellular transition from normal to malignant state is linked to deregulated gene expression. The discovery of microRNA-mediated cellular regulation by the RNA interference (RNAi) pathway and the possibility to engage this pathway with exogenous triggers such as small interfering RNA (siRNA) could offer a new paradigm in anti-cancer intervention with RNAi-based therapeutics. The potential to silence the expression of any cancer-relevant protein with high selectivity promotes RNAi therapeutics as a more effective and safer treatment to traditional approaches. This combined with microRNA-based tumour profiling could pave the way for personalised approaches based on the genetic characteristics of the individual. Clinical translation of this technology, however, depends on the development of systems for effective delivery of the molecular medicine to the target site. Polycation-based nanoparticles (termed polyplexes) constitute an attractive platform for RNAi therapeutic delivery due to flexibility and versatility in design to overcome extracellular and intracellular barriers. In this review we focus on pre-clinical and clinical studies using polycation-based nanocarriers for RNAi mediated anti-cancer intervention after intratumoural or intravenous administration. Potential RNAi targets are highlighted and special attention is given to the enhanced permeability and retention (EPR) effect commonly cited at the predominant mechanism of delivery after systemic administration. The cyclodextrin polymer-based system now in clinical trials offers optimism that polyplexes may potentially be used for RNAi-mediated cancer intervention in the clinic.