SUMMARY Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.

Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Here, through the generation and analysis of a liver-specific Med23-knockout mouse, we found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila. Collectively, our data revealed Mediator MED23 as a novel regulator for energy homeostasis, suggesting potential therapeutic strategies against metabolic diseases.

The early stages of nonalcoholic fatty liver disease (NAFLD) are characterized by the accumulation of fat in the liver (steatosis). This can lead to cell injury and inflammation resulting in nonalcoholic steatohepatitis (NASH). To determine whether lipid profiling of liver tissue can identify metabolic signatures associated with disease presence and severity, we explored liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel sampling tool. Using LESA-MS, lipids were extracted directly from the surface of ultrathin slices of liver tissue prior to detection by high-resolution mass spectrometry (MS). An isotopically labeled internal standard mix was incorporated into the extraction solvent to attain semiquantitative data. Data mining and multivariate statistics were employed to evaluate the generated lipid profiles and abundances. With this approach, we were able to differentiate healthy and NAFLD liver in mouse and human tissue samples, finding several triacylglyceride (TAG) and free fatty acid (FFA) species to be significantly increased. Furthermore, LESA-MS was able to successfully differentiate between simple steatosis and more severe NASH, based on a set of short-chain TAGs and FFAs. We compared the data obtained by LESA-MS to that from liquid chromatography (LC)-MS and matrix-assisted laser desorption ionization MS. Advantages of LESA-MS include rapid analysis, minimal sample preparation, and high lipid coverage. Furthermore, since tissue slices are routinely used for diagnostics in clinical settings, LESA-MS is ideally placed to complement traditional histology. Overall LESA-MS is found to be a robust, fast, and discriminating approach for determining NAFLD presence and severity in clinical samples.

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Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation.he metabolic syndrome is defined by the coexistence of central obesity, deregulated carbohydrate, and lipid metabolism and/or hypertension. Collectively these features increase the risk of developing type 2 diabetes, nonalcoholic fatty liver diseases, and atherosclerosis. These metabolic diseases have additional features in common; they are chronic disorders characterized by a state of persistent inflammation and tissue remodeling. In particular, chronic low-grade inflammation or "metaflammation" is now established as an important causative factor driving metabolic disease. Much progress has been made in our understanding of how metabolic stress or overnutrition induces metaflammation. However, the molecules involved in maintaining chronicity of low-grade inflammation remain unclear.As specialized mediators of host defense, macrophages express danger-sensing pattern recognition receptors (PRRs) that include transmembrane receptors of the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily and intracellular cytosolic receptors such as RIG-I-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors (1, 2). Numerous TLR/IL-1Rs and their downstream mediators have been implicated in the pathogenesis of obesity-associated diabetes (3, 4), fatty liver disease (5, 6), and atherosclerosis (7,8). In some cases, putative nonmicrobial, host-derived sterile ligands have also been identified. For example, TLR4 is a putative sensor for dietary saturated fatty acids (SF...

Interaction of HOXA9/MEIS1/PBX3 is responsible for hematopoietic system transformation in MLL-rearranged (MLL-r) leukemia. Of these genes, HOXA9 has been shown to be critical for leukemia cell survival, while MEIS1 has been identified as an essential regulator for leukemia stem cell (LSC) maintenance. Although significantly high expression of PBX3 was observed in clinical acute myeloid leukemia (AML) samples, the individual role of PBX3 in leukemia development is still largely unknown. In this study, we explored the specific role of PBX3 and its associated regulatory network in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 is significantly correlated with a poor prognosis in AML patients. ChIP-Seq/qPCR analysis in MLL-r mouse models revealed aberrant epigenetic modifications with increased H3K79me2, and decreased H3K9me3 and H3K27me3 levels in LSCs, which may account for the high expression levels of Pbx3. To further examine the role of Pbx3 in AML maintenance and progression, we used the CRISPR/Cas9 system to delete Pbx3 in leukemic cells in the MLL-AF9 induced AML mouse model. We found that Pbx3 deletion significantly prolonged the survival of leukemic mice and decreased the leukemia burden by decreasing the capacity of LSCs and promoting LSC apoptosis. In conclusion, we found that PBX3 is epigenetically aberrant in the LSCs of MLL-r AML and is essential for leukemia development. Significantly, the differential expression of PBX3 in normal and malignant hematopoietic cells suggests PBX3 as a potential prognostic marker and therapeutic target for MLL-r leukemia.

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As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2−/− mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities.