The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.

Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.

Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis.

Hypermetabolism following severe burn injuries is associated with adipocyte dysfunction, elevated beige adipocyte formation, and increased energy expenditure. The resulting catabolism of adipose leads to detrimental sequelae such as fatty liver, increased risk of infections, sepsis, and even death. While the phenomenon of pathological white adipose tissue (WAT) browning is well-documented in cachexia and burn models, the molecular mechanisms are essentially unknown. Here, we report that adipose triglyceride lipase (ATGL) plays a central role in burn-induced WAT dysfunction and systemic outcomes. Targeting adipose-specific ATGL in a murine (AKO) model resulted in diminished browning, decreased circulating fatty acids, and mitigation of burn-induced hepatomegaly. To assess the clinical applicability of targeting ATGL, we demonstrate that the selective ATGL inhibitor atglistatin mimics the AKO results, suggesting a path forward for improving patient outcomes.

Adipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism.

Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells.

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

Acetyl-CoA carboxylase (ACC) regulates lipid synthesis; however, its role in inflammatory regulation in macrophages remains unclear. We generated mice that are deficient in both ACC isoforms in myeloid cells. ACC deficiency altered the lipidomic, transcriptomic, and bioenergetic profile of bone marrow-derived macrophages, resulting in a blunted response to proinflammatory stimulation. In response to lipopolysaccharide (LPS), ACC is required for the early metabolic switch to glycolysis and remodeling of the macrophage lipidome. ACC deficiency also resulted in impaired macrophage innate immune functions, including bacterial clearance. Myeloid-specific deletion or pharmacological inhibition of ACC in mice attenuated LPS-induced expression of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β, while pharmacological inhibition of ACC increased susceptibility to bacterial peritonitis in wild-type mice. Together, we identify a critical role for ACC in metabolic regulation of the innate immune response in macrophages, and thus a clinically relevant, unexpected consequence of pharmacological ACC inhibition.

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3-/- preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical β-catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of β-catenin can fully rescue adipogenesis and UCP1 expression in the STAT3-/- adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3-/- adipocytes, potentially explaining the increased levels and activity of β-catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/β-catenin pathway during the early stages of in-vitro adipogenesis.

The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart-adipose tissue-immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin-/- mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes' recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.

Macrophages control tissue homeostasis and inflammation by sensing and responding to environmental cues. However, the metabolic adaptation of macrophages to oxidative tissue damage and its translation into inflammatory mechanisms remains enigmatic.

Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis.

Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific rictor knockout (FRic(-/-)) mice.

Failure to reorganize the endoplasmic reticulum (ER) in mitosis results in chromosome missegregation. Here, we show that accurate chromosome segregation in human cells requires cell cycle-regulated ER membrane production. Excess ER membranes increase the viscosity of the mitotic cytoplasm to physically restrict chromosome movements, which impedes the correction of mitotic errors leading to the formation of micronuclei. Mechanistically, we demonstrate that the protein phosphatase CTDNEP1 counteracts mTOR kinase to establish a dephosphorylated pool of the phosphatidic acid phosphatase lipin 1 in interphase. CTDNEP1 control of lipin 1 limits the synthesis of fatty acids for ER membrane biogenesis in interphase that then protects against chromosome missegregation in mitosis. Thus, regulation of ER size can dictate the biophysical properties of mitotic cells, providing an explanation for why ER reorganization is necessary for mitotic fidelity. Our data further suggest that dysregulated lipid metabolism is a potential source of aneuploidy in cancer cells.