The world is suffering under a huge burden of obesity and type 2 diabetes. The discovery of new pathways controlling systemic energy homeostasis is badly needed. Adipose tissue is the major depot for lipid storage but also can become inflamed during obesity, contributing to certain dysfunctions in obesity. Brown fat dissipates chemical energy in the form of heat and represents an endogenous pathway to improve metabolic diseases. The past term of this MERIT award have allowed the discovery of key molecules and pathways in several aspects of adipose cell biology. We showed previously that PPAR??gets modified at serine 273 and this phosphorylation is both a cause of metabolic dysfunction in vivo and a key target for the PPAR??ligand drugs. We show here that we have created a germ-line mutation in mice whereby serine 273 of PPAR??is replaced by alanine, rendering it non-phosphorylatable. This mutant strain of animals represents a crucial opportunity to test the role of this modification in normal basal physiology and its role in obesity and insulin resistance. It also allows us to ask how much of the therapeutic effects of the PPAR??ligand drugs occur via the reduction in this phosphorylation event. New data shows that PPAR??docks the coregulator Thrap 3 only when it is phosphorylated at S273. We will study this interaction, using a variety of biochemical and biophysical techniques, including hydrogen-deuterium exchange. Importantly, we will create a series of deletions and mutations in both molecules that will better define this functional interface. This will also allow the creation of dominant negative alleles of Thrap 3. Th Thrap 3 /PPAR??interaction will be disrupted transgenically, either through the use of tissue-selective expression of a dominant-negative version of Thrap 3 or by making a tissueselective knock-out at the Thrap 3 locus. Animals bearing such transgenes will be perturbed with diet induced obesity, and analyzed by both MRI and hyperinsulinemic-euglycemic clamps. Our previous work has highlighted the crucial role that the coregulator PRDM16 plays in the fate and function of brown and beige adipose cells. In preliminary data, we have purified PRDM16 complexes from cultures enriched in beige fat cells and identified potentially interesting molecules, including LSD1, a lysine specific demethylase with a wellstudied pharmacology. We will identify important molecules in the PRDM16 complexes, especially those that distinguish beige and brown cells. To do this, we will purify PRDM16 complexes and perform comparative, quantitative proteomics by Mass Spectrometry after isobaric tagging. The function of proteins identified will be altered genetically by both gain and loss of function methods, in vitro and in vivo. Enzymes present in these complxes will be probed chemically, where the pharmacology is developed, as in the case of LSD1. The overall goal of this project will be to elucidate pathways of basic science that can inform novel therapeutic approaches to metabolic diseases such as obesity, diabetes and hepatic steatosis.

Public Health Relevance

Obesity and type 2 diabetes represent a huge burden on the health of the American population. In this grant, we propose experiments to discover molecules and molecular interactions from white and brown fat cells that may inform future therapeutic approaches to treat these disorders more effectively.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK031405-33
Application #
8759397
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Haft, Carol R
Project Start
1982-07-01
Project End
2019-06-30
Budget Start
2014-08-01
Budget End
2015-06-30
Support Year
33
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02215
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Long, Jonathan Z; Roche, Alexander M; Berdan, Charles A et al. (2018) Ablation of PM20D1 reveals N-acyl amino acid control of metabolism and nociception. Proc Natl Acad Sci U S A 115:E6937-E6945
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Chen, Yi; Zeng, Xing; Huang, Xuan et al. (2017) Crosstalk between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity. Cell 171:836-848.e13
Kazak, Lawrence; Chouchani, Edward T; Lu, Gina Z et al. (2017) Genetic Depletion of Adipocyte Creatine Metabolism Inhibits Diet-Induced Thermogenesis and Drives Obesity. Cell Metab 26:693
Bertholet, Ambre M; Kazak, Lawrence; Chouchani, Edward T et al. (2017) Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling. Cell Metab 25:811-822.e4
Cohen, Paul; Spiegelman, Bruce M (2016) Cell biology of fat storage. Mol Biol Cell 27:2523-7
Zeng, Xing; Jedrychowski, Mark P; Chen, Yi et al. (2016) Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation. Genes Dev 30:1822-36

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