Novel secreted regulators of glucose and lipid metabolism in the development of metabolic diseases.

The tight regulation of glucose and lipid metabolism is crucial for maintaining metabolic health. Dysregulation of these processes can lead to the development of metabolic diseases. Secreted factors, or hormones, play an essential role in the regulation of glucose and lipid metabolism, thus also playing an important role in the development of metabolic diseases such as type 2 diabetes and obesity. Given the important roles of secreted factors, there has been significant interest in identifying new secreted factors and new functions for existing secreted factors that control glucose and lipid metabolism. In this review, we evaluate novel secreted factors or novel functions of existing factors that regulate glucose and lipid metabolism discovered in the last decade, including secreted isoform of endoplasmic reticulum membrane complex subunit 10, vimentin, cartilage intermediate layer protein 2, isthmin-1, lipocalin-2, neuregulin-1 and neuregulin-4. We discuss their discovery, tissues of origin, mechanisms of action and sex differences, emphasising their potential to regulate metabolic processes central to diabetes. Additionally, we discuss the translational barriers, particularly the absence of identified receptors, that hamper their functional characterisation and further therapeutic development. Ultimately, the identification of new secreted factors may give insights into previously unidentified pathways of disease progression and mechanisms of glucose and lipid homeostasis.

Loss of RREB1 reduces adipogenesis and improves insulin sensitivity in mouse and human adipocytes.

There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1 +/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1 +/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity.

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

A PTER-dependent pathway of taurine metabolism linked to energy balance.

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in biosynthesis of taurine from cysteine as well as the downstream derivatization of taurine into secondary taurine metabolites4,5. One such taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by diverse physiologic perturbations that alter taurine and/or acetate flux, including endurance exercise7, nutritional taurine supplementation8, and alcohol consumption6,9. While taurine N-acetyltransferase activity has been previously detected in mammalian cells6,7, the molecular identity of this enzyme, and the physiologic relevance of N-acetyltaurine, have remained unknown. Here we show that the orphan body mass index-associated enzyme PTER (phosphotriesterase-related)10 is the principal mammalian taurine N-acetyltransferase/hydrolase. In vitro, recombinant PTER catalyzes bidirectional taurine N-acetylation with free acetate as well as the reverse N-acetyltaurine hydrolysis reaction. Genetic ablation of PTER in mice results in complete loss of tissue taurine N-acetyltransferase/hydrolysis activities and systemic elevation of N-acetyltaurine levels. Upon stimuli that increase taurine levels, PTER-KO mice exhibit lower body weight, reduced adiposity, and improved glucose homeostasis. These phenotypes are recapitulated by administration of N-acetyltaurine to wild-type mice. Lastly, the anorexigenic and anti-obesity effects of N-acetyltaurine require functional GFRAL receptors. Together, these data uncover enzymatic control of a previously enigmatic pathway of secondary taurine metabolism linked to energy balance.

Size matters: the biochemical logic of ligand type in endocrine crosstalk.

The endocrine system is a fundamental type of long-range cell-cell communication that is important for maintaining metabolism, physiology, and other aspects of organismal homeostasis. Endocrine signaling is mediated by diverse blood-borne ligands, also called hormones, including metabolites, lipids, steroids, peptides, and proteins. The size and structure of these hormones are fine-tuned to make them bioactive, responsive, and adaptable to meet the demands of changing environments. Why has nature selected such diverse ligand types to mediate communication in the endocrine system? What is the chemical, signaling, or physiologic logic of these ligands? What fundamental principles from our knowledge of endocrine communication can be applied as we continue as a field to uncover additional new circulating molecules that are claimed to mediate long-range cell and tissue crosstalk? This review provides a framework based on the biochemical logic behind this crosstalk with respect to their chemistry, temporal regulation in physiology, specificity, signaling actions, and evolutionary development.

Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma.

Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

Hallmarks of the metabolic secretome.

The identification of novel secreted factors is advancing at an unprecedented pace. However, there is a critical need to consolidate and integrate this knowledge to provide a framework of their diverse mechanisms, functional significance, and inter-relationships. Complicating this effort are challenges related to nonstandardized methods, discrepancies in sample handling, and inconsistencies in the annotation of unknown molecules. This Review aims to synthesize the rapidly expanding field of the metabolic secretome, encompassing the five major types of secreted factors: proteins, peptides, metabolites, lipids, and extracellular vesicles. By systematically defining the functions and detection of the components within the metabolic secretome, this Review provides a primer into the advances of the field, and how integration of the techniques discussed can provide a deeper understanding of the mechanisms underlying metabolic homeostasis and its disorders.

Roles of Activin A and Gpnmb in Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as nonalcoholic fatty liver disease [NAFLD]) and metabolic dysfunction-associated steatohepatitis (MASH, formerly known as nonalcoholic steatohepatitis [NASH]) are leading chronic liver diseases, driving cirrhosis, hepatocellular carcinoma, and mortality. MASLD/MASH is associated with increased senescence proteins, including Activin A, and senolytics have been proposed as a therapeutic approach. To test the role of Activin A, we induced hepatic expression of Activin A in a murine MASLD/MASH model. Surprisingly, overexpression of hepatic Activin A dramatically mitigated MASLD, reducing liver steatosis and inflammation as well as systemic fat accumulation, while improving insulin sensitivity. Further studies identified a dramatic decrease in the lipid-associated macrophages marker glycoprotein NMB (Gpnmb) by Activin A, and Gpnmb knockdown in the same model produced similar benefits and transcriptional changes to Activin A expression. These studies reveal a surprising protective role for Activin A in MASLD and the potential for SASP proteins to have context-specific beneficial effects. Moreover, they implicate both Activin A and Gpnmb as potential therapeutic targets for this condition.

A class of secreted mammalian peptides with potential to expand cell-cell communication.

Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

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.

A class of secreted mammalian peptides with potential to expand cell-cell communication.

Peptide hormones and neuropeptides are fundamental signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we demonstrate the endogenous presence of a sequence diverse class of orphan, blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic regulation in blood plasma by diverse environmental and physiologic stimuli. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of multiple mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide that reduces food intake and body weight. Capped peptides therefore define a largely unexplored class of circulating molecules with potential to regulate cell-cell communication in mammalian physiology.

Organism-wide, cell-type-specific secretome mapping of exercise training in mice.

There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training.

Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

AlphaFold2 enables accurate deorphanization of ligands to single-pass receptors.

Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying novel ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold as a screening approach to identify extracellular ligand-binding pairs using a structural library of single-pass transmembrane receptors. Key to the approach is the optimization of AlphaFold input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. Importantly, the predictions were performed on ligand-receptor pairs not used for AlphaFold training. We demonstrate high discriminatory power and a success rate of close to 90 % for known ligand-receptor pairs and 50 % for a diverse set of experimentally validated interactions. These results demonstrate proof-of-concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

Protocol for in vivo measurement of basal and insulin-stimulated glucose uptake in mouse tissues.

Here, we present an in vivo protocol for measuring basal and insulin-stimulated glucose uptake in tissues from mice. We describe steps for administering 2-deoxy-D-[1,2-3H]glucose in the presence or absence of insulin via intraperitoneal injections. We then detail tissue collection, tissue processing to measure 3H counts on a scintillation counter, and data interpretation. This protocol can be applied to other glucoregulatory hormones, genetic mouse models, and other species. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2021).1.

Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution.

Non-alcoholic fatty liver disease is a heterogeneous disease with unclear underlying molecular mechanisms. Here, we perform single-cell RNA sequencing of hepatocytes and hepatic non-parenchymal cells to map the lipid signatures in mice with non-alcoholic fatty liver disease (NAFLD). We uncover previously unidentified clusters of hepatocytes characterized by either high or low srebp1 expression. Surprisingly, the canonical lipid synthesis driver Srebp1 is not predictive of hepatic lipid accumulation, suggestive of other drivers of lipid metabolism. By combining transcriptional data at single-cell resolution with computational network analyses, we find that NAFLD is associated with high constitutive androstane receptor (CAR) expression. Mechanistically, CAR interacts with four functional modules: cholesterol homeostasis, bile acid metabolism, fatty acid metabolism, and estrogen response. Nuclear expression of CAR positively correlates with steatohepatitis in human livers. These findings demonstrate significant cellular differences in lipid signatures and identify functional networks linked to hepatic steatosis in mice and humans.

A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents.

Pain signals are relayed to the brain via a nociceptive system, and in rare cases, this nociceptive system contains genetic variants that can limit the pain response. Here, we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and, further, whether we could target this region with a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N-knockin mouse using CRISPR/Cas9, we discovered that the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral responses to noxious chemical stimuli and less hypersensitivity to nerve injury, while their response to noxious heat remained intact. Furthermore, blocking this K710 region in WT rodents using a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and returned pain hypersensitivity induced by nerve injury to baseline levels. These findings identify K710 TRPV1 as a discrete site that is crucial for the control of nociception and provide insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

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.

New players of the adipose secretome: Therapeutic opportunities and challenges.

Adipose tissue is a functional endocrine organ comprised of adipocytes and other cell types that are known to secrete a multiplicity of adipose-derived factors, including lipids and proteins. It is well established that adipose tissue and its secretome can impact systemic energy homeostasis. The endocrine and paracrine effects of adipose-derived factors have been widely studied over the last several decades. Owing to technological advances in genomics and proteomics, several additional adipose-derived protein factors have recently been identified. By learning from previous efforts, the next challenge will be to leverage these discoveries for the prevention or treatment of metabolic disorders. Here, we discuss recently discovered adipose-derived proteins secreted from white or brown adipose tissue and the opportunities and challenges of translating these biological findings into disease therapeutics.

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.