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.

Lac-Phe mediates the effects of metformin on food intake and body weight.

Metformin is a widely prescribed anti-diabetic medicine that also reduces body weight. There is ongoing debate about the mechanisms that mediate metformin's effects on energy balance. Here, we show that metformin is a powerful pharmacological inducer of the anorexigenic metabolite N-lactoyl-phenylalanine (Lac-Phe) in cells, in mice and two independent human cohorts. Metformin drives Lac-Phe biosynthesis through the inhibition of complex I, increased glycolytic flux and intracellular lactate mass action. Intestinal epithelial CNDP2+ cells, not macrophages, are the principal in vivo source of basal and metformin-inducible Lac-Phe. Genetic ablation of Lac-Phe biosynthesis in male mice renders animals resistant to the effects of metformin on food intake and body weight. Lastly, mediation analyses support a role for Lac-Phe as a downstream effector of metformin's effects on body mass index in participants of a large population-based observational cohort, the Multi-Ethnic Study of Atherosclerosis. Together, these data establish Lac-Phe as a critical mediator of the body weight-lowering effects of metformin.

Identification of extracellular membrane protein ENPP3 as a major cGAMP hydrolase, cementing cGAMP's role as an immunotransmitter.

cGAMP is a second messenger that is synthesized in the cytosol upon detection of cytosolic dsDNA and passed between cells to facilitate downstream immune signaling. ENPP1, an extracellular enzyme, was the only metazoan cGAMP hydrolase known to regulate cGAMP levels to dampen anti-cancer immunity. Here, we uncover ENPP3 as the second and only other metazoan cGAMP hydrolase under homeostatic conditions. ENPP3 has a tissue expression pattern distinct from that of ENPP1 and accounts for all remaining cGAMP hydrolysis activity in mice lacking ENPP1. Importantly, we also show that as with ENPP1, selectively abolishing ENPP3's cGAMP hydrolase activity results in diminished cancer growth and metastasis of certain tumor types. Both ENPP1 and ENPP3 are extracellular enzymes, suggesting the dominant role that extracellular cGAMP must play as a mediator of cell-cell innate immune communication. Our work clearly shows that ENPP1 and ENPP3 non-redundantly dampen extracellular cGAMP-STING signaling, pointing to ENPP3 as a new target for cancer immunotherapy.

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.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Lac-Phe mediates the anti-obesity effect of metformin.

Metformin is a widely prescribed anti-diabetic medicine that also reduces body weight. The mechanisms that mediate metformin's effects on energy balance remain incompletely defined. Here we show that metformin is a powerful pharmacological inducer of the anorexigenic metabolite Lac-Phe in mice as well as in two independent human cohorts. In cell culture, metformin drives Lac-Phe biosynthesis via inhibition of complex I, increased glycolytic flux, and intracellular lactate mass action. Other biguanides and structurally distinct inhibitors of oxidative phosphorylation also increase Lac-Phe levels in vitro. Genetic ablation of CNDP2, the principal biosynthetic enzyme for Lac-Phe, in mice renders animals resistant to metformin's anorexigenic and anti-obesity effects. Mediation analyses also support a role for Lac-Phe in metformin's effect on body mass index in humans. These data establish the CNDP2/Lac-Phe pathway as a critical mediator of the effects of metformin on energy balance.

Tachycardia-induced metabolic rewiring as a driver of contractile dysfunction.

Prolonged tachycardia-a risk factor for cardiovascular morbidity and mortality-can induce cardiomyopathy in the absence of structural disease in the heart. Here, by leveraging human patient data, a canine model of tachycardia and engineered heart tissue generated from human induced pluripotent stem cells, we show that metabolic rewiring during tachycardia drives contractile dysfunction by promoting tissue hypoxia, elevated glucose utilization and the suppression of oxidative phosphorylation. Mechanistically, a metabolic shift towards anaerobic glycolysis disrupts the redox balance of nicotinamide adenine dinucleotide (NAD), resulting in increased global protein acetylation (and in particular the acetylation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), a molecular signature of heart failure. Restoration of NAD redox by NAD+ supplementation reduced sarcoplasmic/endoplasmic reticulum Ca2+-ATPase acetylation and accelerated the functional recovery of the engineered heart tissue after tachycardia. Understanding how metabolic rewiring drives tachycardia-induced cardiomyopathy opens up opportunities for therapeutic intervention.

Metabolic switch from glycogen to lipid in the liver maintains glucose homeostasis in neonatal mice.

Neonates strive to acquire energy when the continuous transplacental nutrient supply ceases at birth, whereas milk consumption takes hours to start. Using murine models, we report the metabolic switches in the first days of life, with an unexpected discovery of glucose as the universal fuel essential for neonatal life. Blood glucose quickly drops as soon as birth, but immediately rebounds even before suckling and maintains stable afterward. Meanwhile, neonatal liver undergoes drastic metabolic changes, from extensive glycogenolysis before suckling to dramatically induced fatty acid oxidation (FAO) and gluconeogenesis after milk suckling. Unexpectedly, blocking hepatic glycogenolysis only caused a transient hypoglycemia before milk suckling without causing lethality. Limiting lipid supply in milk (low-fat milk, [LFM]) using Cidea-/- mice, however, led to a chronic and severe hypoglycemia and consequently claimed neonatal lives. While fat replenishment rescued LFM-caused neonatal lethality, the rescue effects were abolished by blocking FAO or gluconeogenesis, pointing to a funneling of lipids and downstream metabolites into glucose as the essential fuel. Finally, glucose administration also rescued LFM-caused neonatal lethality, independent on FAO or gluconeogenesis. Therefore, our results show that the liver works as an energy conversion center to maintain blood glucose homeostasis in neonates, providing theoretical basis for managing infant hypoglycemia.

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.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway.

ENPP1 expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer STING pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single cell RNA-seq (scRNA-seq), we show that ENPP1 overexpression drives primary breast tumor growth and metastasis by synergistically dampening extracellular cGAMP-STING mediated antitumoral immunity and activating immunosuppressive extracellular adenosine (eADO) signaling. In addition to cancer cells, stromal and immune cells in the tumor microenvironment (TME) also express ENPP1 that restrains their response to tumor-derived cGAMP. Enpp1 loss-of-function in both cancer cells and normal tissues slowed primary tumor initiation and growth and prevented metastasis in an extracellular cGAMP- and STING-dependent manner. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied total ENPP1 knockout, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Strikingly, we find that breast cancer patients with low ENPP1 expression have significantly higher immune infiltration and improved response to therapeutics impacting cancer immunity upstream or downstream of the cGAMP-STING pathway, like PARP inhibitors and anti-PD1. Altogether, selective inhibition of ENPP1's cGAMP hydrolase activity alleviates an innate immune checkpoint to boost cancer immunity and is therefore a promising therapeutic approach against breast cancer that may synergize with other cancer immunotherapies.

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.

CYP4F2 is a human-specific determinant of circulating N-acyl amino acid levels.

N-acyl amino acids are a large family of circulating lipid metabolites that modulate energy expenditure and fat mass in rodents. However, little is known about the regulation and potential cardiometabolic functions of N-acyl amino acids in humans. Here, we analyze the cardiometabolic phenotype associations and genomic associations of four plasma N-acyl amino acids (N-oleoyl-leucine, N-oleoyl-phenylalanine, N-oleoyl-serine, and N-oleoyl-glycine) in 2351 individuals from the Jackson Heart Study. We find that plasma levels of specific N-acyl amino acids are associated with cardiometabolic disease endpoints independent of free amino acid plasma levels and in patterns according to the amino acid head group. By integrating whole genome sequencing data with N-acyl amino acid levels, we identify that the genetic determinants of N-acyl amino acid levels also cluster according to the amino acid head group. Furthermore, we identify the CYP4F2 locus as a genetic determinant of plasma N-oleoyl-leucine and N-oleoyl-phenylalanine levels in human plasma. In experimental studies, we demonstrate that CYP4F2-mediated hydroxylation of N-oleoyl-leucine and N-oleoyl-phenylalanine results in metabolic diversification and production of many previously unknown lipid metabolites with varying characteristics of the fatty acid tail group, including several that structurally resemble fatty acid hydroxy fatty acids. These studies provide a structural framework for understanding the regulation and disease associations of N-acyl amino acids in humans and identify that the diversity of this lipid signaling family can be significantly expanded through CYP4F-mediated ω-hydroxylation.

CYP4F2 is a human-specific determinant of circulating N-acyl amino acid levels.

N-acyl amino acids are a large family of circulating lipid metabolites that modulate energy expenditure and fat mass in rodents. However, little is known about the regulation and potential cardiometabolic functions of N-acyl amino acids in humans. Here, we analyze the cardiometabolic phenotype associations and genetic regulation of four plasma N-fatty acyl amino acids (N-oleoyl-leucine, N-oleoyl-phenylalanine, N-oleoyl-serine, and N-oleoyl-glycine) in 2,351 individuals from the Jackson Heart Study. N-oleoyl-leucine and N-oleoyl-phenylalanine were positively associated with traits related to energy balance, including body mass index, waist circumference, and subcutaneous adipose tissue. In addition, we identify the CYP4F2 locus as a human-specific genetic determinant of plasma N-oleoyl-leucine and N-oleoyl-phenylalanine levels. In vitro, CYP4F2-mediated hydroxylation of N-oleoyl-leucine and N-oleoyl-phenylalanine results in metabolic diversification and production of many previously unknown lipid metabolites with varying characteristics of the fatty acid tail group, including several that structurally resemble fatty acid hydroxy fatty acids (FAHFAs). By contrast, FAAH-regulated N-oleoyl-glycine and N-oleoyl-serine were inversely associated with traits related to glucose and lipid homeostasis. These data uncover a human-specific enzymatic node for the metabolism of a subset of N-fatty acyl amino acids and establish a framework for understanding the cardiometabolic roles of individual N-fatty acyl amino acids in humans.

An exercise-inducible metabolite that suppresses feeding and obesity.

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

Protocol for cell type-specific labeling, enrichment, and proteomic profiling of plasma proteins in mice.

Secreted polypeptides represent a fundamental axis of intercellular communication. Here, we present a protocol for the cell type-specific biotinylation, enrichment, and proteomic profiling of secreted plasma proteins directly in mice. This protocol uses conditional "turn-on" adeno-associated viruses expressing an endoplasmic reticulum-targeted biotin ligase to globally biotinylate proteins of the secretory pathway in a cell type-specific manner. Biotinylated secreted proteins can be directly purified from blood plasma and analyzed by SDS-PAGE gel or shotgun proteomics. For complete information on the generation and use of this protocol, please refer to Wei et al. (2021).

A gel-like condensation of Cidec generates lipid-permeable plates for lipid droplet fusion.

Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.