A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results: We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions: Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models.

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on these widely used in vitro models of human and mouse adipogenesis, and on cell-to-cell variability during this process.

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models.

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process.

G protein-coupled receptor 151 regulates glucose metabolism and hepatic gluconeogenesis.

Human genetics has been instrumental in identification of genetic variants linked to type 2 diabetes. Recently a rare, putative loss-of-function mutation in the orphan G-protein coupled receptor 151 (GPR151) was found to be associated with lower odds ratio for type 2 diabetes, but the mechanism behind this association has remained elusive. Here we show that Gpr151 is a fasting- and glucagon-responsive hepatic gene which regulates hepatic gluconeogenesis. Gpr151 ablation in mice leads to suppression of hepatic gluconeogenesis genes and reduced hepatic glucose production in response to pyruvate. Importantly, the restoration of hepatic Gpr151 levels in the Gpr151 knockout mice reverses the reduced hepatic glucose production. In this work, we establish a previously unknown role of Gpr151 in the liver that provides an explanation to the lowered type 2 diabetes risk in individuals with nonsynonymous mutations in GPR151.

Phosphoproteomic mapping reveals distinct signaling actions and activation of muscle protein synthesis by Isthmin-1.

The secreted protein isthmin-1 (Ism1) mitigates diabetes by increasing adipocyte and skeletal muscle glucose uptake by activating the PI3K-Akt pathway. However, while both Ism1 and insulin converge on these common targets, Ism1 has distinct cellular actions suggesting divergence in downstream intracellular signaling pathways. To understand the biological complexity of Ism1 signaling, we performed phosphoproteomic analysis after acute exposure, revealing overlapping and distinct pathways of Ism1 and insulin. We identify a 53% overlap between Ism1 and insulin signaling and Ism1-mediated phosphoproteome-wide alterations in ~450 proteins that are not shared with insulin. Interestingly, we find several unknown phosphorylation sites on proteins related to protein translation, mTOR pathway, and, unexpectedly, muscle function in the Ism1 signaling network. Physiologically, Ism1 ablation in mice results in altered proteostasis, including lower muscle protein levels under fed and fasted conditions, reduced amino acid incorporation into proteins, and reduced phosphorylation of the key protein synthesis effectors Akt and downstream mTORC1 targets. As metabolic disorders such as diabetes are associated with accelerated loss of skeletal muscle protein content, these studies define a non-canonical mechanism by which this antidiabetic circulating protein controls muscle biology.

Cells need energy to survive and carry out their role in the body. They do this by breaking down molecules, like sugar, into substances that can fuel the creation of new compounds, like proteins or lipids. This process, known as metabolism, involves a series of interconnecting chemical reactions which are organized into pathways. Metabolic pathways contain proteins that catalyze each sequential reaction. Hormones can change the activity of these proteins by adding a chemical group called a phosphate. This reversible modification can majorly impact the metabolism of cells, resulting in changes to the body's tissues. The hormone insulin, for instance, alters a well-known metabolic pathway that triggers skeletal muscle cells to produce more proteins, leading to stronger and larger muscles. In 2021, a group of scientists discovered a molecule made by fat cells, called Isthmin-1, also activates components in this pathway. Similar to insulin, Isthmin-1 encourages muscle and fat cells to take up sugar. However, it also prevents the liver from accumulating excess fat, suggesting Isthmin-1 may trigger a different cascade of molecules to insulin. To investigate this possibility, Zhao et al. ­ including some of the researchers involved in the 2021 study ­ exposed cells grown in the laboratory to Isthmin-1 or insulin and looked for phosphates on all their proteins. This revealed that only 53% of the proteins Isthmin-1 modifies are also altered by insulin. Of the proteins unique to Isthmin-1, several had known roles in making and maintaining proteins in muscle cells. To understand more about the role of this newly discovered pathway, Zhao et al. genetically engineered mice to lack the gene that codes for Isthmin-1. This decreased the size and strength of the mice's muscle fibers and reduced the signals that normally lead to skeletal muscle growth. These findings suggest that Isthmin-1 regulates skeletal muscle size via a metabolic pathway that is slightly different to the one activated by insulin. Many metabolic disorders are associated with muscle loss, like diabetes, and this newly discovered network of proteins could further our understanding of how to prevent and treat these diseases.

Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy.

Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3 days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3 weeks. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress.

TGF-ß is insufficient to induce adipocyte state loss without concurrent PPARγ downregulation.

Cell plasticity, the ability of differentiated cells to convert into other cell types, underlies the pathogenesis of many diseases including the transdifferentiation of adipocytes (fat cells) into myofibroblasts in the pathogenesis of dermal fibrosis. Loss of adipocyte identity is an early step in different types of adipocyte plasticity. In this study, we determine the dynamics of adipocyte state loss in response to the profibrotic cytokine TGF-ß. We use two complementary approaches, lineage tracing and live fluorescent microscopy, which both allow for robust quantitative tracking of adipocyte identity loss at the single-cell level. We find that the intracellular TGF-ß signaling in adipocytes is inhibited by the transcriptional factor PPARγ, specifically by its ubiquitously expressed isoform PPARγ1. However, TGF-ß can lead to adipocyte state loss when it is present simultaneously with another stimulus. Our findings establish that an integration of stimuli occurring in a specific order is pivotal for adipocyte state loss which underlies adipocyte plasticity. Our results also suggest the possibility of a more general switch-like mechanism between adipogenic and profibrotic molecular states.

White Adipocyte Plasticity in Physiology and Disease.

Cellular plasticity is a transformation of a terminally differentiated cell into another cell type, which has been long known to occur in disease and regeneration. However, white adipocytes (fat cells) have only recently been observed to undergo different types of cellular plasticity. Adipocyte transdifferentiation into myofibroblasts and cancer-associated fibroblasts occurs in fibrosis and cancer, respectively. On the other hand, reversible adipocyte dedifferentiation into adipocyte progenitor cells (preadipocytes) has been demonstrated in mammary gland and in dermal adipose tissue. Here we discuss the research on adipocyte plasticity, including the experimental approaches that allowed to detect and study it, the current state of the knowledge, major research questions which remain to be addressed, and the advances required to stimulate adipocyte plasticity research. In the future, the knowledge of the molecular mechanisms of adipocyte plasticity can be utilized both to prevent adipocyte plasticity in disease and to stimulate it for use in regenerative medicine.

High-speed OCT-based ocular biometer combined with an air-puff system for determination of induced retraction-free eye dynamics.

We demonstrate a swept source OCT-based ocular biometer integrated with an air-puff stimulus to study the reaction of the eye to mechanical stimulation in vivo. The system enables simultaneous measurement of the stimulus strength and high-speed imaging of the eye dynamics along the visual axis. We characterize the stimulus and perform optimization of the data acquisition for a proper interpretation of the results. Access to the dynamics of axial eye length allows for a determination of the eye retraction, which is used to correct the air-puff induced displacement of ocular structures. We define the parameters to quantify the reaction of the eye to the air puff and determine their reproducibility in a group of healthy subjects. We observe the corneal deformation process and axial wobbling of the crystalline lens. OCT biometer combined with the air puff is the first instrument with the potential to provide comprehensive information on the biomechanics of ocular components.

Air-Puff-Induced Dynamics of Ocular Components Measured with Optical Biometry.

Purpose: To analyze the dynamics of all optical components of the eye and the behavior of the eyeball under air-puff conditions in vivo. To determine the impact of the intraocular pressure (IOP) on the air-puff-induced deformation of the eye. Methods: Twenty eyes of 20 healthy subjects were included in this study. The dynamics of the ocular components, such as the cornea, the crystalline lens, and the retina, was measured by a prototype swept source optical coherence tomography biometer integrated with the air-puff system. The system allows to acquire a series of axial scans at the same location as a function of time with no transverse scanning. Several parameters were extracted from optical coherence tomography data. The IOP was measured using a Goldmann applanation tonometry. The measurements of the eyes were performed before and 2 hours after administration of IOP-reducing drops, namely, 0.2 % brimonidine tartrate. Results: There is a statistically significant correlation of corneal thickness, vitreous depth, and eye length with IOP. The deformation amplitudes of the cornea and the crystalline lens are inversely proportional to the IOP, but statistical significance is achieved only for the cornea. The crystalline lens is displaced without compression, and the return has the form of wobbling. The reduction of IOP level induces corresponding changes in the extracted parameters. Conclusions: Optical biometry combined with air puff provides comprehensive information on the in vivo behavior of all ocular components, including the crystalline lens. Measurement of the axial length dynamics of during deformation enables correcting the deformation for eye retraction.

Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air-puff optical coherence tomography and corneal hysteresis.

Application of the air-puff swept source optical coherence tomography (SS-OCT) instrument to determine the influence of viscoelasticity on the relation between overall the air-puff force and corneal apex displacement of porcine corneas ex vivo is demonstrated. Simultaneous recording of time-evolution of the tissue displacement and air pulse stimulus allows obtaining valuable information related in part to the mechanical properties of the cornea. A novel approach based on quantitative analysis of the corneal hysteresis of OCT data is presented. The corneal response to the air pulse is assessed for different well-controlled intraocular pressure (IOP) levels and for the progression of cross-linking-induced stiffness of the cornea. Micrometer resolution, fast acquisition and noncontact character of the air-puff SS-OCT measurements have potential to improve the in vivo assessment of mechanical properties of the human corneas.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.

Transcriptional diversity during lineage commitment of human blood progenitors.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

A loss of function screen of identified genome-wide association study Loci reveals new genes controlling hematopoiesis.

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits.

Prognostic and predictive biomarkers: tools in personalized oncology.

Oncology indispensably leads us to personalized medicine, which allows an individual approach to be taken with each patient. Personalized oncology is based on pharmacogenomics and the effect of genetic differences in individuals (germline and somatic) on the way cancer patients respond to chemotherapeutics. Biomarkers detected using molecular biology tools allow the molecular characterization of cancer signatures and provide information relevant for personalized treatment. Biomarkers can be divided into two main subgroups: prognostic and predictive. The aim of the application of prognostic biomarkers, which provide information on the overall cancer outcome in patients, is to facilitate cancer diagnosis, usually with no need for putting invasive methods into use. Predictive biomarkers help to optimize therapy decisions, as they provide information on the likelihood of response to a given chemotherapeutic. Among the prognostic factors that identify patients with different outcome risks (e.g., recurrence of the disease), the following factors can be distinguished: somatic and germline mutations, changes in DNA methylation that lead to the enhancement or suppression of gene expression, the occurrence of elevated levels of microRNA (miRNA) capable of binding specific messenger RNA (mRNA) molecules, which affects gene expression, as well as the presence of circulating tumor cells (CTCs) in blood, which leads to a poor prognosis for the patient. Biomarkers for personalized oncology are used mainly in molecular diagnostics of chronic myeloid leukemia, colon, breast and lung cancer, and recently in melanoma. They are successfully used in the evaluation of the benefits that can be achieved through targeted therapy or in the evaluation of toxic effects of the chemotherapeutic used in the therapy.

Nornicotine impairs endothelial cell-cell adherens junction complexes in EA.hy926 cell line via structural reorganization of F-actin.

The aim of the study was to estimate the effect of nornicotine on endothelial EA.hy926 cells in the context of its impact on cell-cell junctions. The objective of the study was to determine the relationship between junctional proteins and F-actin after treating the cells with nornicotine. After 24 h of cell exposure to 0.08, 0.12, and 0.16 ng/mL nornicotine, analysis was performed of cell death, cell migration, ultrastructure, and colocalization of beta-catenin/F-actin and zonula occludens (ZO)-1/F-actin. Our study did not reveal any alterations in EA.hy926 cell line survival following treatment with nornicotine. However, nornicotine exerted disparate effects on cell migration and led to changes in both the ultrastructure and organization of cell-cell junctional complexes and F-actin. Moreover, the cell migration observed in the experiments performed in the present work negatively correlated with the number of Weibel-Palade bodies seen through transmission electron microscopy (TEM). Moreover, the mechanism of cell migration promotion was VEGF-independent, and the decrease in the number of Weibel-Palade bodies resulted from nornicotine-induced F-actin depolymerization. In conclusion, the present study demonstrated that low concentrations of nornicotine do not affect cell survival, but promote cell movement and impair adherens junctions through changes in F-actin organization. Our results indicate for the first time the effect of nornicotine on endothelial EA.hy926 cells and suggest that nornicotine may induce transmigration pathways and, consequently, facilitate the transendothelial migration of monocytes associated with atherosclerosis.

SMIM1 underlies the Vel blood group and influences red blood cell traits.

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.