Perilipins: A family of five fat-droplet storing proteins that play a significant role in fat homeostasis.

Lipid droplets are organelles with unique spherical structures. They consist of a hydrophobic neutral lipid core that varies depending on the cell type and tissue. These droplets are surrounded by phospholipid monolayers, along with heterogeneous proteins responsible for neutral lipid synthesis and metabolism. Additionally, there are specialized lipid droplet-associated surface proteins. Recent evidence suggests that proteins from the perilipin family (PLIN) are associated with the surface of lipid droplets and are involved in their formation. These proteins have specific roles in hepatic lipid droplet metabolism, such as protecting the lipid droplets from lipase action and maintaining a balance between lipid storage and utilization in specific cells. Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by the accumulation of lipid droplets in more than 5% of the hepatocytes. This accumulation can progress into metabolic dysfunction-associated steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The accumulation of hepatic lipid droplets in the liver is associated with the progression of MASLD and other diseases such as sarcopenic obesity. Therefore, it is crucial to understand the role of perilipins in this accumulation, as these proteins are key targets for developing novel therapeutic strategies. This comprehensive review aims to summarize the structure and characteristics of PLIN proteins, as well as their pathogenic role in the development of hepatic steatosis and fatty liver diseases.

Effects of Probiotics on Gut Microbiota: An Overview.

The role of probiotics in regulating intestinal flora to enhance host immunity has recently received widespread attention. Altering the human gut microbiota may increase the predisposition to several disease phenotypes such as gut inflammation and metabolic disorders. The intestinal microbiota converts dietary nutrients into metabolites that serve as biologically active molecules in modulating regulatory functions in the host. Probiotics, which are active microorganisms, play a versatile role in restoring the composition of the gut microbiota, helping to improve host immunity and prevent intestinal disease phenotypes. This comprehensive review provides firsthand information on the gut microbiota and their influence on human health, the dietary effects of diet on the gut microbiota, and how probiotics alter the composition and function of the human gut microbiota, along with their corresponding effects on host immunity in building a healthy intestine. We also discuss the implications of probiotics in some of the most important human diseases. In summary, probiotics play a significant role in regulating the gut microbiota, boosting overall immunity, increasing the abundance of beneficial bacteria, and helping ameliorate the symptoms of multiple diseases.

Structure, Functions, and Implications of Selected Lipocalins in Human Disease.

The lipocalin proteins are a large family of small extracellular proteins that demonstrate significant heterogeneity in sequence similarity and have highly conserved crystal structures. They have a variety of functions, including acting as carrier proteins, transporting retinol, participating in olfaction, and synthesizing prostaglandins. Importantly, they also play a critical role in human diseases, including cancer. Additionally, they are involved in regulating cellular homeostasis and immune response and dispensing various compounds. This comprehensive review provides information on the lipocalin family, including their structure, functions, and implications in various diseases. It focuses on selective important human lipocalin proteins, such as lipocalin 2 (LCN2), retinol binding protein 4 (RBP4), prostaglandin D2 synthase (PTGDS), and α1-microglobulin (A1M).

Quantification of liver iron overload disease with laser ablation inductively coupled plasma mass spectrometry.

BACKGROUND: Hereditary hemochromatosis is the most frequent, identified, genetic disorder in Caucasians affecting about 1 in 1000 people of Northern European ancestry, where the associated genetic defect (homozygosity for the p.Cys282Tyr polymorphism in the HFE gene) has a prevalence of approximately 1:200. The disorder is characterized by excess iron stores in the body. Due to the incomplete disease penetrance of disease-associated genotype, genetic testing and accurate quantification of hepatic iron content by histological grading of stainable iron, quantitative chemical determination of iron, or imaging procedures are important in the evaluation and staging of hereditary hemochromatosis. METHODS: We here established novel laser ablation inductively coupled plasma mass spectrometry protocols for hepatic metal bio-imaging for diagnosis of iron overload. RESULTS: We demonstrate that these protocols are a significant asset in the diagnosis of iron overload allowing iron measurements and simultaneous determination of various other metals and metalloids with high sensitivity, spatial resolution, and quantification ability. CONCLUSIONS: The simultaneous measurement of various metals and metalloids offers unique opportunities for deeper understanding of metal imbalances. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a highly powerful and sensitive technique for the analysis of a variety of solid samples with high spatial resolution. We conclude that this method is an important add-on to routine diagnosis of iron overload and associated hepatic metal dysbalances resulting thereof.

Latent TGF-ß binding protein-1 deficiency decreases female fertility.

The four latent transforming growth factor-ß (TGF-ß) binding proteins LTBP1-4 are extracellular matrix-associated proteins playing a critical role in the activation of TGF-ß. The LTBP1 gene forms two major transcript variants (i.e. Ltbp1S and Ltbp1L) that are derived from different promoters. We have previously shown the importance of LTBP1 in vivo by using three different Ltbp1 null mice that were either deleted for exons 1 and 2 (Ltbp1L knockout), exon 5 (Ltbp1ΔEx5), or exon 8 (Ltbp1ΔEx8). While the Ltbp1L knockout and the Ltbp1ΔEx8 are perinatal lethal and die of cardiovascular abnormalities, the Ltbp1ΔEx5 is viable because it expresses a short form of Ltbp1L that lacks 55 amino acids (Δ55 variant of Ltbp1) formed by splicing out exon 5, while lacking the Ltbp1S variant. Since only the Ltbp1ΔEx5 mouse is viable, we have used this model to address aspects of puberty, fertility, age-dependent reproduction, and ovary function. We report for the first time a function of LTBP1 in female reproduction. The Ltbp1ΔEx5 females showed impaired fertility associated with delayed sexual maturity (p = 0.0074) and ovarian cyst formation in females older than 40 weeks (p = 0.0204).

Simultaneous monitoring of cerebral metal accumulation in an experimental model of Wilson's disease by laser ablation inductively coupled plasma mass spectrometry.

BACKGROUND: Neuropsychiatric affection involving extrapyramidal symptoms is a frequent component of Wilson's disease (WD). WD is caused by a genetic defect of the copper (Cu) efflux pump ATPase7B. Mouse strains with natural or engineered transgenic defects of the Atp7b gene have served as model of WD. These show a gradual accumulation and concentration of Cu in liver, kidneys, and brain. However, still little is known about the regional distribution of Cu inside the brain, its influence on other metals and subsequent pathophysiological mechanisms. We have applied laser ablation inductively coupled plasma mass spectrometry and performed comparative metal bio-imaging in brain sections of wild type and Atp7b null mice in the age range of 11-24 months. Messenger RNA and protein expression of a panel of inflammatory markers were assessed using RT-PCR and Western blots of brain homogenates. RESULTS: We could confirm Cu accumulation in brain parenchyma by a factor of two in WD (5.5 µg g(-1) in the cortex) vs. controls (2.7 µg g(-1)) that was already fully established at 11 months. In the periventricular regions (PVR) known as structures of prominent Cu content, Cu was reduced in turn by a factor of 3. This corroborates the view of the PVR as efflux compartments with active transport of Cu into the cerebrospinal fluid. Furthermore, the gradient of Cu increasing downstream the PVR was relieved. Otherwise the architecture of Cu distribution was essentially maintained. Zinc (Zn) was increased by up to 40% especially in regions of high Cu but not in typical Zn accumulator regions, a side effect due to the fact that Zn is to some degree a substrate of Cu-ATPases. The concentrations of iron (Fe) and manganese (Mn) were constant throughout all regions assessed. Inflammatory markers TNF-α, TIMP-1 and the capillary proliferation marker α-SMA were increased by a factor of 2-3 in WD. CONCLUSIONS: This study confirmed stable cerebral Cu accumulation in parenchyma and discovered reduced Cu in cerebrospinal fluid in Atp7b null mice underlining the diagnostic value of micro-local analytical techniques.

Vitamin A: too good to be bad?

Vitamin A is a micronutrient important for vision, cell growth, reproduction and immunity. Both deficiency and excess consuming of vitamin A cause severe health consequences. Although discovered as the first lipophilic vitamin already more than a century ago and the definition of precise biological roles of vitamin A in the setting of health and disease, there are still many unresolved issues related to that vitamin. Prototypically, the liver that plays a key role in the storage, metabolism and homeostasis of vitamin A critically responds to the vitamin A status. Acute and chronic excess vitamin A is associated with liver damage and fibrosis, while also hypovitaminosis A is associated with alterations in liver morphology and function. Hepatic stellate cells are the main storage site of vitamin A. These cells have multiple physiological roles from balancing retinol content of the body to mediating inflammatory responses in the liver. Strikingly, different animal disease models also respond to vitamin A statuses differently or even opposing. In this review, we discuss some of these controversial issues in understanding vitamin A biology. More studies of the interactions of vitamin A with animal genomes and epigenetic settings are anticipated in the future.

Lipocalin 2 receptors: facts, fictions, and myths.

The human 25-kDa Lipocalin 2 (LCN2) was first identified and purified as a protein that in part is associated with gelatinase from neutrophils. This protein shows a high degree of sequence similarity with the deduced sequences of rat α2-microglobulin-related protein and the mouse protein 24p3. Based on its typical lipocalin fold, which consists of an eight-stranded, anti-parallel, symmetrical ß-barrel fold structure it was initially thought that LCN2 is a circulating protein functioning as a transporter of small lipophilic molecules. However, studies in Lcn2 null mice have shown that LCN2 has bacteriostatic properties and plays a key role in innate immunity by sequestering bacterial iron siderophores. Numerous reports have further shown that LCN2 is involved in the control of cell differentiation, energy expenditure, cell death, chemotaxis, cell migration, and many other biological processes. In addition, important roles for LCN2 in health and disease have been identified in Lcn2 null mice and multiple molecular pathways required for regulation of Lcn2 expression have been identified. Nevertheless, although six putative receptors for LCN2 have been proposed, there is a fundamental lack in understanding of how these cell-surface receptors transmit and amplify LCN2 to the cell. In the present review we summarize the current knowledge on LCN2 receptors and discuss inconsistencies, misinterpretations and false assumptions in the understanding of these potential LCN2 receptors.

Isolation of Bovine and Human Milk Extracellular Vesicles.

Extracellular vesicles such as exosomes are small-sized, bilayered extracellular biovesicles generated by almost every cell and released into the surrounding body fluids upon the fusion of multivesicular bodies and the plasma membrane. Based on their origin, they are enriched with a variety of biologically active components including proteins, lipids, nucleic acids, cellular metabolites, and many other constituents. They can either attach or fuse with the membrane of a target cell, or alternatively be taking up via endocytosis by a recipient cell. In particular, milk exosomes have been recently shown to be a fundamental factor supporting infant growth, health, and development. In addition, exosomes derived from different cell types have been shown to possess regenerative, immunomodulatory, and anti-inflammatory properties, suggesting that they are a potential therapeutic tool in modulating the pathogenesis of diverse diseases. Therefore, efficient protocols for the isolation of milk exosomes in a high quantity and purity are the basis for establishing clinical applications. Here, we present an easy-to-follow protocol for exosome isolation from bovine and human milk. Electron microscopic analysis and nanoparticle tracking analysis reveal that the protocols allow the isolation of highly enriched fractions of exosomes. The purified exosomes express the typical exosomal protein markers, CD81 and ALIX.

A Beginner's Guide to Cell Culture: Practical Advice for Preventing Needless Problems.

The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update.

In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.

Phalloidin Staining for F-Actin in Hepatic Stellate Cells.

During the development of liver fibrosis, hepatic stellate cells undergo a transition from a quiescent phenotype into a proliferative, fibrogenic, and contractile, α-smooth muscle actin-positive myofibroblast. These cells acquire properties that are strongly associated with the reorganization of the actin cytoskeleton. Actin possesses a unique ability to polymerize into filamentous actin (F-actin) form its monomeric globular state (G-actin). F-actin can form robust actin bundles and cytoskeletal networks by interacting with a number of actin-binding proteins that provide important mechanical and structural support for a multitude of cellular processes including intracellular transport, cell motility, polarity, cell shape, gene regulation, and signal transduction. Therefore, stains with actin-specific antibodies and phalloidin conjugates for actin staining are widely used to visualize actin structures in myofibroblasts. Here we present an optimized protocol for F-actin staining for hepatic stellate cells using a fluorescent phalloidin.

Induction of Obstructive Cholestasis in Mice.

Experimental bile duct ligation (BDL) in rodents causes cholestatic liver injury characterized by structural and functional alterations that include periportal biliary fibrosis. These changes are time-dependent and based on excess accumulation of bile acids in the liver. This in turn causes damage of hepatocytes and functional loss, leading to recruitment of inflammatory cells. Liver resident pro-fibrogenic cells facilitate extracellular matrix synthesis and remodeling. The proliferation of bile duct epithelial cells provokes a ductular reaction characterized by bile duct hyperplasia. Experimental BDL surgery is technically simple and quick to perform and reliably generates progressive liver damage with a predictable kinetics. The cellular, structural, and functional alterations induced in this model are similar to that in humans suffering from diverse forms of cholestasis including primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). Therefore, this extrahepatic biliary obstruction model is used in many laboratories worldwide. Nevertheless, BDL can result in significant variations and high mortality rates when surgery is carried out by untrained or inexperienced personnel. Here we present a detailed protocol to achieve a robust experimental obstructive cholestasis in mice.

Flow Cytometry: A Blessing and a Curse.

Flow cytometry is a laser-based technology generating a scattered and a fluorescent light signal that enables rapid analysis of the size and granularity of a particle or single cell. In addition, it offers the opportunity to phenotypically characterize and collect the cell with the use of a variety of fluorescent reagents. These reagents include but are not limited to fluorochrome-conjugated antibodies, fluorescent expressing protein-, viability-, and DNA-binding dyes. Major developments in reagents, electronics, and software within the last 30 years have greatly expanded the ability to combine up to 50 antibodies in one single tube. However, these advances also harbor technical risks and interpretation issues in the identification of certain cell populations which will be summarized in this viewpoint article. It will further provide an overview of different potential applications of flow cytometry in research and its possibilities to be used in the clinic.

Cellular Mechanisms of Liver Fibrosis.

The liver is a central organ in the human body, coordinating several key metabolic roles. The structure of the liver which consists of the distinctive arrangement of hepatocytes, hepatic sinusoids, the hepatic artery, portal vein and the central vein, is critical for its function. Due to its unique position in the human body, the liver interacts with components of circulation targeted for the rest of the body and in the process, it is exposed to a vast array of external agents such as dietary metabolites and compounds absorbed through the intestine, including alcohol and drugs, as well as pathogens. Some of these agents may result in injury to the cellular components of liver leading to the activation of the natural wound healing response of the body or fibrogenesis. Long-term injury to liver cells and consistent activation of the fibrogenic response can lead to liver fibrosis such as that seen in chronic alcoholics or clinically obese individuals. Unidentified fibrosis can evolve into more severe consequences over a period of time such as cirrhosis and hepatocellular carcinoma. It is well recognized now that in addition to external agents, genetic predisposition also plays a role in the development of liver fibrosis. An improved understanding of the cellular pathways of fibrosis can illuminate our understanding of this process, and uncover potential therapeutic targets. Here we summarized recent aspects in the understanding of relevant pathways, cellular and molecular drivers of hepatic fibrosis and discuss how this knowledge impact the therapy of respective disease.

Analyzing the Therapeutic Efficacy of Bis-Choline-Tetrathiomolybdate in the Atp7b-/- Copper Overload Mouse Model.

Bis-choline-tetrathiomolybdate, introduced as WTX101 (now known as ALXN1840), is a first-in-class copper-protein-binding agent for oral therapy of Wilson's disease. In contrast to other decoppering agents such as trientine or D-penicillamine it acts by forming a tripartite complex with copper and albumin, thereby detoxifying excess liver and blood copper through biliary excretion. Preclinical animal experimentation with this drug was typically done with the alternative ammonium salt of tetrathiomolybdate, which is expected to have identical properties in terms of copper binding. Here, we comparatively analyzed the therapeutic efficacy of ALXN1840, D-penicillamine and trientine in lowering hepatic copper content in Atp7b-/- mouse. Liver specimens were subjected to laser ablation inductively conductively plasma mass spectrometry and electron microscopic analysis. We found that ALXN1840 caused a massive increase of hepatic copper and molybdenum during early stages of therapy. Prolonged treatment with ALXN1840 reduced hepatic copper to an extent that was similar to that observed after administration of D-penicillamine and trientine. Electron microscopic analysis showed a significant increase of lysosomal electron-dense particles in the liver confirming the proposed excretory pathway of ALXN1840. Ultrastructural analysis of mice treated with dosages comparable to the bis-choline-tetrathiomolybdate dosage used in an ongoing phase III trial in Wilson's disease patients, as well as D-penicillamine and trientine, did not show relevant mitochondrial damage. In contrast, a high dose of ALXN1840 applied for four weeks triggered dramatic structural changes in mitochondria, which were notably characterized by the formation of holes with variable sizes. Although these experimental results may not be applicable to patients with Wilson's disease, the data suggests that ALXN1840 should be administered at low concentrations to prevent mitochondrial dysfunction and overload of hepatic excretory pathways.

All You Can Feed: Some Comments on Production of Mouse Diets Used in Biomedical Research with Special Emphasis on Non-Alcoholic Fatty Liver Disease Research.

The laboratory mouse is the most common used mammalian research model in biomedical research. Usually these animals are maintained in germ-free, gnotobiotic, or specific-pathogen-free facilities. In these facilities, skilled staff takes care of the animals and scientists usually don't pay much attention about the formulation and quality of diets the animals receive during normal breeding and keeping. However, mice have specific nutritional requirements that must be met to guarantee their potential to grow, reproduce and to respond to pathogens or diverse environmental stress situations evoked by handling and experimental interventions. Nowadays, mouse diets for research purposes are commercially manufactured in an industrial process, in which the safety of food products is addressed through the analysis and control of all biological and chemical materials used for the different diet formulations. Similar to human food, mouse diets must be prepared under good sanitary conditions and truthfully labeled to provide information of all ingredients. This is mandatory to guarantee reproducibility of animal studies. In this review, we summarize some information on mice research diets and general aspects of mouse nutrition including nutrient requirements of mice, leading manufacturers of diets, origin of nutrient compounds, and processing of feedstuffs for mice including dietary coloring, autoclaving and irradiation. Furthermore, we provide some critical views on the potential pitfalls that might result from faulty comparisons of grain-based diets with purified diets in the research data production resulting from confounding nutritional factors.

Intrahepatic TH17/TReg Cells in Homeostasis and Disease-It's All About the Balance.

Both acute and chronic hepatic inflammation likely result from an imbalance in the TH1/TH2 cell response and can lead to liver fibrosis and end-stage liver disease. More recently, a novel CD4+ T helper cell subset was described, characterized by the production of IL-17 and IL-22. These TH17 cells 50were predominantly implicated in host defense against infections and in autoimmune diseases. Interestingly, studies over the last 10 years revealed that the development of TH17 cells favors pro-inflammatory responses in almost all tissues and there is a reciprocal relationship between TH17 and TReg cells. The balance between TH17and TReg cells is critical for immune reactions, especially in injured liver tissue and the return to immune homeostasis. The pathogenic contribution of TH17 and TReg cells in autoimmunity, acute infection, and chronic liver injury is diverse and varies among disease etiologies. Understanding the mechanisms underlying TH17 cell development, recruitment, and maintenance, along with the suppression of TReg cells, will inform the development of new therapeutic strategies in liver diseases. Active manipulation of the balance between pathogenic and regulatory processes in the liver may assist in the restoration of homeostasis, especially in hepatic inflammation.

The hepatic lipidome: From basic science to clinical translation.

The liver is the key organ involved in lipid metabolism and transport. Excessive lipid accumulation due to dysregulated lipid metabolism predisposes the liver to steatosis, cirrhosis, and hepatocellular carcinoma. Lipids are generally compartmentalized in specialized organelles called lipid droplets that enable cells to store and release lipids in a regulated manner. However, during flux-in and flux-out of droplets, lipids are converted into toxic species leading to lipid-mediated liver damage. Lipids are categorized into 'toxic' or 'healthy' lipids that are involved in liver disease pathogenesis or resolution, respectively. Lipidomic analysis have revealed unique lipid signature that correlates with the disease progression therefore being used for disease diagnosis. In this comprehensive review, we provide an overview on hepatic lipid homeostasis, lipid compartmentalization mechanisms and lipidomic profiles in different liver diseases. We further discuss promising therapeutics targeting the hepatic lipidome including pro-resolving lipids, liposomes, and small-molecule inhibitors for the treatment of liver diseases.

Accurate Measurement of Copper Overload in an Experimental Model of Wilson Disease by Laser Ablation Inductively Coupled Plasma Mass Spectrometry.

Wilson disease is a rare inherited autosomal recessive disorder. As a consequence of genetic alterations in the ATP7B gene, copper begins to accumulate in the body, particularly in the liver and brain. Affected persons are prone to develop liver cancer and severe psychiatric and neurological symptoms. Clinically, the development of corneal Kayser-Fleischer rings and low ceruloplasmin concentrations (<20 mg/dL) are indicative of Wilson disease. However, the detection of elevated hepatic copper content (>250 µg/g dry weight) alone is still considered as the best but not exclusive diagnostic test for Wilson disease. Presently, specific copper stains (e.g., rhodanine) or indirect staining for copper-associated proteins (e.g., orcein) are widely used to histochemically visualize hepatic copper deposits. However, these procedures only detect lysosomal copper, while cytosolic copper is not detectable. Similarly, elemental analysis in scanning electron microscope with energy dispersive X-ray analysis (EDX) often leads to false negative results and inconsistencies. Here, we tested the diagnostic potential of laser ablation inductively-coupled mass spectrometry (LA-ICP-MS) that allows quantitative analysis of multiple elements. Comparative studies were performed in wild type and the Atp7b null mouse model. We propose LA-ICP-MS as a versatile and powerful method for the accurate determination of hepatic copper in people with Wilson disease with high spatial resolution.