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.

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.

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.