Formation and activation of thermogenic fat.

Thermogenic fat cells that convert chemical energy into heat are present in both mice and humans. Recent years have witnessed great advances in our understanding of the regulation of these adipocytes and an increased appreciation of the potential these cells have to counteract obesity. We summarize recent efforts to understand the formation of these fat cells and critically review genetic models and other experimental tools currently available to further investigate the development and activation of both classical brown and inducible beige fat cells. We also discuss recent discoveries about the epigenetic regulation of these adipocytes, and finally present emerging evidence revealing the metabolic impacts of thermogenic fat in humans.

Conditional Dependency of LP-184 on Prostaglandin Reductase 1 is Synthetic Lethal in Pancreatic Cancers with DNA Damage Repair Deficiencies.

The greater efficacy of DNA-damaging drugs for pancreatic adenocarcinoma (PDAC) relies on targeting cancer-specific vulnerabilities while sparing normal organs and tissues due to their inherent toxicities. We tested LP-184, a novel acylfulvene analog, for its activity in preclinical models of PDAC carrying mutations in the DNA damage repair (DDR) pathways. Cytotoxicity of LP-184 is solely dependent on prostaglandin reductase 1 (PTGR1), so that PTGR1 expression robustly correlates with LP-184 cytotoxicity in vitro and in vivo. Low-passage patient-derived PDAC xenografts with DDR deficiencies treated ex vivo are more sensitive to LP-184 compared with DDR-proficient tumors. Additional in vivo testing of PDAC xenografts for their sensitivity to LP-184 demonstrates marked tumor growth inhibition in models harboring pathogenic mutations in ATR, BRCA1, and BRCA2. Depletion of PTGR1, however, completely abrogates the antitumor effect of LP-184. Testing combinatorial strategies for LP-184 aimed at deregulation of nucleotide excision repair proteins ERCC3 and ERCC4 established synergy. Our results provide valuable biomarkers for clinical testing of LP-184 in a large subset of genetically defined characterized refractory carcinomas. High PTGR1 expression and deleterious DDR mutations are present in approximately one third of PDAC making these patients ideal candidates for clinical trials of LP-184.

Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio.

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As(III)) produces organic arsenicals, including MMA(III), MMA(V) and DMA(V) with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As(III) to DMA(V) as an end product and produced MMA(III) and MMA(V) as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As(III) as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans.

Pentavalent methylated arsenicals are substrates of human AQP9.

Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (As(III)) and organic monomethylarsonous acid (MAs(III)). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAs(V) and DMAs(V), remains unknown in mammals. These products of arsenic metabolism, in particular DMAs(V), are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAs(V) and DMAs(V) in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of As(III), MAs(III), MAs(V) and DMAs(V) via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAs(V) and DMAs(V) but not trivalent As(III) and MAs(III), indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic As(III) inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.

Zinc- and bicarbonate-dependent ZIP8 transporter mediates selenite uptake.

Selenite (HSeO3-) is a monovalent anion of the essential trace element and micronutrient selenium (Se). In therapeutic concentrations, HSeO3- has been studied for treating certain cancers, serious inflammatory disorders, and septic shock. Little is known, however, about HSeO3- uptake into mammalian cells; until now, no mammalian HSeO3- uptake transporter has been identified. The ubiquitous mammalian ZIP8 divalent cation transporter (encoded by the SLC39A8 gene) is bicarbonate-dependent, moving endogenous substrates (Zn2+, Mn2+, Fe2+ or Co2+) and nonessential metals such as Cd2+ into the cell. Herein we studied HSeO3- uptake in: human and mouse cell cultures, shRNA-knockdown experiments, Xenopus oocytes, wild-type mice and two transgenic mouse lines having genetically altered ZIP8 expression, and mouse erythrocytes ex vivo. In mammalian cell culture, excess Zn2+ levels and/or ZIP8 over-expression can be associated with diminished viability in selenite-treated cells. Intraperitoneal HSeO3- causes the largest ZIP8-dependent increases in intracellular Se content in liver, followed by kidney, heart, lung and spleen. In every model system studied, HSeO3- uptake is tightly associated with ZIP8 protein levels and sufficient Zn2+ and HCO3- concentrations, suggesting that the ZIP8-mediated electroneutral complex transported contains three ions: Zn2+/(HCO3-)(HSeO3-). Transporters having three different ions in their transport complex are not without precedent. Although there might be other HSeO3- influx transporters as yet undiscovered, data herein suggest that mammalian ZIP8 plays a major role in HSeO3- uptake.

Jen1p: a high affinity selenite transporter in yeast.

Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.

Trivalent arsenicals and glucose use different translocation pathways in mammalian GLUT1.

Rat glucose transporter isoform 1 or rGLUT1, which is expressed in neonatal heart and the epithelial cells that form the blood-brain barrier, facilitates uptake of the trivalent arsenicals arsenite as As(OH)3 and methylarsenite as CH3As(OH)2. GLUT1 may be the major pathway for arsenic uptake into heart and brain, where the metalloid causes cardiotoxicity and neurotoxicity. In this paper, we compare the translocation properties of GLUT1 for trivalent methylarsenite and glucose. Substitution of Ser(66), Arg(126) and Thr(310), residues critical for glucose uptake, led to decreased uptake of glucose but increased uptake of CH3As(OH)2. The K(m) for uptake of CH3As(OH)2 of three identified mutants, S66F, R126K and T310I, were decreased 4-10 fold compared to native GLUT1. The osmotic water permeability coefficient (P(f)) of GLUT1 and the three clinical isolates increased in parallel with the rate of CH3As(OH)2 uptake. GLUT1 inhibitors Hg(II), cytochalasin B and forskolin reduced uptake of glucose but not CH3As(OH)2. These results indicate that CH3As(OH)2 and water use a common translocation pathway in GLUT1 that is different to that of glucose transport.