The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A previously unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-sequencing of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.
Alan C Rupp, Abigail J. Tomlinson, Alison H. Affinati, Warren T. Yacawych, Allison M. Duensing, Cadence True, Sarah R. Lindsley, Melissa A. Kirigiti, Alexander J. MacKenzie, Joseph Polex-Wolf, Chien Li, Lotte B. Knudsen, Randy J. Seeley, David P. Olson, Paul Kievit, Martin G. Myers, Jr
Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o G-protein-coupled-receptors by Gβγ subunit inhibition of two mechanisms: decreased calcium entry and direct interaction of Gβγ with Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) decreasing Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact, and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. SNAP25Δ3/Δ3 mice exhibit enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in SNAP25Δ3/Δ3 mice challenged with high fat diet. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in SNAP25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neuron exocytosis from SNAP25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combatting metabolic disease.
Ryan P. Ceddia, Zack Zurawski, Analisa Thompson Gray, Feyisayo Adegboye, Ainsley McDonald-Boyer, Fubiao Shi, Dianxin Liu, Jose Maldonado, Jiesi Feng, Yulong Li, Simon Alford, Julio E. Ayala, Owen P. McGuinness, Sheila Collins, Heidi E. Hamm
The liver has a high demand for phosphatidylcholine (PC) particularly in overnutrition where reduced phospholipid levels have been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Whether other pathways exist in addition to de novo PC synthesis that contribute to hepatic PC pools remains unknown. Here, we identified the lysophosphatidylcholine (LPC) transporter Mfsd2a as critical for maintaining hepatic phospholipid pools. Hepatic Mfsd2a expression was induced in patients having NAFLD and in mice in response to dietary fat via glucocorticoid receptor action. Mfsd2a liver-specific deficiency in mice (L2aKO) led to a robust NASH-like phenotype within just two weeks of dietary fat challenge associated with reduced hepatic phospholipids containing linoleic acid. Reducing dietary choline intake in L2aKO mice exacerbated liver pathology and deficiency of liver phospholipids containing polyunsaturated fatty acids (PUFA). Treating hepatocytes with LPC containing oleate and linoleate, two abundant blood-derived LPCs, specifically induced lipid droplet biogenesis and contributed to phospholipid pools, while LPC containing the omega-3 fatty acid DHA promoted lipid droplet formation and suppressed lipogenesis. This study revealed that PUFA containing LPCs drive both hepatic lipid droplet formation, suppress lipogenesis and sustain hepatic phospholipid pools--processes that are critical for protecting the liver from excess dietary fat.
Cheen Fei Chin, Dwight L.A. Galam, Liang Gao, Bryan C. Tan, Bernice H. Wong, Geok-Lin Chua, Randy Y.J. Loke, Yen Ching Lim, Markus R. Wenk, Miao Shan Lim, Wei-Qiang Leow, George B.B. Goh, Federico Torta, David L. Silver
The Rad50 interacting protein 1 (Rint1) is a key player in vesicular trafficking between the ER and Golgi apparatus. Biallelic variants in RINT1 cause infantile-onset episodic acute liver failure (ALF). Here, we describe 3 individuals from 2 unrelated families with novel biallelic RINT1 loss-of-function variants who presented with early onset spastic paraplegia, ataxia, optic nerve hypoplasia, and dysmorphic features, broadening the previously described phenotype. Our functional and lipidomic analyses provided evidence that pathogenic RINT1 variants induce defective lipid–droplet biogenesis and profound lipid abnormalities in fibroblasts and plasma that impact both neutral lipid and phospholipid metabolism, including decreased triglycerides and diglycerides, phosphatidylcholine/phosphatidylserine ratios, and inhibited Lands cycle. Further, RINT1 mutations induced intracellular ROS production and reduced ATP synthesis, affecting mitochondria with membrane depolarization, aberrant cristae ultrastructure, and increased fission. Altogether, our results highlighted the pivotal role of RINT1 in lipid metabolism and mitochondria function, with a profound effect in central nervous system development.
Nathalie Launay, Montserrat Ruiz, Laura Planas-Serra, Edgard Verdura, Agustí Rodríguez-Palmero, Agatha Schlüter, Leire Goicoechea, Cristina Guilera, Josefina Casas, Felix Campelo, Emmanuelle Jouanguy, Jean-Laurent Casanova, Odile Boespflug-Tanguy, Maria Vazquez Cancela, Luis González Gutiérrez-Solana, Carlos Casasnovas, Estela Area-Gomez, Aurora Pujol
Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle–specific deletion of Cdk4’s target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber–specification that is of relevance to metabolic and muscular diseases.
Young Jae Bahn, Hariom Yadav, Paolo Piaggi, Brent S. Abel, Oksana Gavrilova, Danielle A. Springer, Ioannis Papazoglou, Patricia M. Zerfas, Monica C. Skarulis, Alexandra C. McPherron, Sushil G. Rane
Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity towards increased purine synthesis in MLL3/4 knockout (KO) mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA sequencing identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag (TMT) proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4 KO cells. Mechanistically, compensation by MLL1/COMPASS underlied these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.
Zibo Zhao, Kaixiang Cao, Jun Watanabe, Cassandra N. Philips, Jacob M. Zeidner, Yukitomo Ishi, Qixuan Wang, Sarah R. Gold, Katherine Junkins, Elizabeth T. Bartom, Feng Yue, Navdeep S. Chandel, Rintaro Hashizume, Issam Ben-Sahra, Ali Shilatifard
Deciphering the crosstalk between metabolic reprogramming and epigenetic regulation is a promising strategy for cancer therapy. In this study, we discovered that the gluconeogenic enzyme PCK1 fueled the generation of S-adenosylmethionine (SAM) through the serine synthesis pathway. The methyltransferase SUV39H1 catalyzed SAM, which served as a methyl donor to support H3K9me3 modification, leading to the suppression of the oncogene S100A11. Mechanistically, PCK1 deficiency-induced oncogenic activation of S100A11 was due to its interaction with AKT1, which upregulated PI3K/AKT signaling. Intriguingly, the progression of hepatocellular carcinoma (HCC) driven by PCK1 deficiency was suppressed by SAM supplement or S100A11 knockout in vivo and in vitro. These findings reveal the availability of key metabolite SAM as a bridge connecting the gluconeogenic enzyme PCK1 and H3K9 trimethylation in attenuating HCC progression, thus suggesting a potential therapeutic strategy against HCC.
Dongmei Gou, Rui Liu, Xiaoqun Shan, Haijun Deng, Chang Chen, Jin Xiang, Yi Liu, Qingzhu Gao, Zhi Li, Ailong Huang, Kai Wang, Ni Tang
Patients with Autosomal Recessive Microcephaly 15 caused by deficiency in the sodium-dependent lysophosphatidylcholine (LPC) transporter Major Facilitator Superfamily Domain containing 2a (Mfsd2a) present with both microcephaly and hypomyelination, suggesting an important role of LPC uptake by oligodendrocytes in the process of myelination. Here, we demonstrate that Mfsd2a is specifically expressed in oligodendrocyte precursor cells (OPC) and is critical for oligodendrocyte development. Single cell sequencing of the oligodendrocyte lineage revealed that OPCs from OPC-specific Mfsd2a KO mice (2aOKO) underwent precocious differentiation into immature oligodendrocytes (iOLs) and impaired maturation into myelinating oligodendrocytes, correlating with postnatal brain hypomyelination. 2aOKO mice did not exhibit microcephaly, consistent with microcephaly being consequential to absence of LPC uptake at the blood-brain barrier and not from deficiency in OPCs. Lipidomic analysis showed that OPCs and iOLs from 2aOKO mice had significantly decreased phospholipids containing omega-3 fatty acids with an opposite increase in unsaturated fatty acids, that latter being products of de novo synthesis governed by Srebp-1. RNA sequencing indicated activation of the Srebp-1 pathway and defective expression of regulators of oligodendrocyte development. Taken together, these findings indicate that the transport of LPCs by Mfsd2a in OPCs is important for maintaining OPC cell state to regulate postnatal brain myelination.
Vetrivel Sengottuvel, Monalisa Hota, Jeongah Oh, Dwight L. Galam, Bernice H. Wong, Markus R. Wenk, Sujoy Ghosh, Federico Torta, David L. Silver
Renal osteodystrophy (ROD) is a disorder of bone metabolism that affects virtually all patients with chronic kidney disease (CKD), and is associated with adverse clinical outcomes including fractures, cardiovascular events and death. In the present study, we showed that hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor mostly expressed in the liver, is also expressed in bone, and that osseous HNF4α expression was dramatically reduced in patients and mice with ROD. Osteoblast-specific deletion of Hnf4α resulted in impaired osteogenesis in cells and mice. Using multi-omics analyses of bones and cells lacking or overexpressing Hnf4α1 and Hnf4α2, we showed that HNF4α2 is the main osseous Hnf4α isoform that regulates osteogenesis, cell metabolism, and cell death. As a result, osteoblast-specific overexpression of Hnf4α2 prevented bone loss in mice with CKD. Our results showed that HNF4α2 is a transcriptional regulator of osteogenesis, implicated in the development of ROD.
Marta Martinez-Calle, Guillaume Courbon, Bridget Hunt-Tobey, Connor Francis, Jadeah J. Spindler, Xueyan Wang, Luciene M. dos Reis, Carolina Steller Wagner Martins, Isidro B. Salusky, Hartmut H. Malluche, Thomas L. Nickolas, Rosa M.A. Moyses, Aline Martin, Valentin David
Rhythmic intraorgan communication coordinates environmental signals and the cell-intrinsic clock to maintain organ homeostasis. Hepatocyte-specific KO of core components of the molecular clock Rev-erbα and -β (Reverb-hDKO) alters cholesterol and lipid metabolism in hepatocytes as well as rhythmic gene expression in nonparenchymal cells (NPCs) of the liver. Here, we report that in fatty liver caused by diet-induced obesity (DIO), hepatocyte SREBP cleavage–activating protein (SCAP) was required for Reverb-hDKO–induced diurnal rhythmic remodeling and epigenomic reprogramming in liver macrophages (LMs). Integrative analyses of isolated hepatocytes and LMs revealed that SCAP-dependent lipidomic changes in REV-ERB–depleted hepatocytes led to the enhancement of LM metabolic rhythms. Hepatocytic loss of REV-ERBα and β (REV-ERBs) also attenuated LM rhythms via SCAP-independent polypeptide secretion. These results shed light on the signaling mechanisms by which hepatocytes regulate diurnal rhythms in NPCs in fatty liver disease caused by DIO.
Dongyin Guan, Hosung Bae, Dishu Zhou, Ying Chen, Chunjie Jiang, Cam Mong La, Yang Xiao, Kun Zhu, Wenxiang Hu, Trang Minh Trinh, Panpan Liu, Ying Xiong, Bishuang Cai, Cholsoon Jang, Mitchell A. Lazar