Proteome and phosphoproteome analysis of brown adipocytes reveals that RICTOR loss dampens global insulin/AKT signaling

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-minute time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation due to chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.Fil: Entwisle, Samuel W.. University of Washington; Estados UnidosFil: Martinez Calejman, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Valente, Anthony S.. University of Washington; Estados UnidosFil: Lawrence, Robert T.. University of Washington; Estados UnidosFil: Hung, Chien Min. University Of Massachussets. Medical School; Estados UnidosFil: Guertin, David A.. University Of Massachussets. Medical School; Estados UnidosFil: Villen, Judit. University of Washington; Estados Unido

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease

Published in final edited form as: Nat Metab. 2020 December ; 2(12): 1472–1481. doi:10.1038/s42255-020-00319-x.Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.R01 NS098329 - NINDS NIH HHS; T32 HG00035 - U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI); T32 LM012419 - NLM NIH HHS; T32 HG000035 - NHGRI NIH HHS; R35 GM119536 - NIGMS NIH HHS; F32 NS110109 - NINDS NIH HHS; postdoctoral fellowship - Uehara Memorial Foundation; P30 AG013280 - NIA NIH HHS; postdoctoral fellowship - MEXT | Japan Society for the Promotion of Science (JSPS)Accepted manuscrip

Proteome-Scale Investigation of Protein Modification and Metabolic Regulation in Brown Adipose Tissue

Thesis (Ph.D.)--University of Washington, 2019Brown adipose tissue (BAT) is present in most mammals, and becomes highly metabolically active during cold exposure. Its ability to lower blood glucose and burn calories when it is activated holds great interest for its potential as a therapeutic target for metabolic diseases such as type-2 diabetes. However, in order to leverage this unique functionality of BAT to develop novel therapeutics, it will be important to gain a better systems-wide understand of the cellular consequences of its activation, and the signaling and regulatory networks that control its metabolism. In this dissertation, these questions are explored using mass spectrometry (MS)-based proteomics, through two research projects: a study of protein acetylation and metabolomics in mouse BAT, and a study of signaling dynamics dependent on the mTOR complex 2 subunit RICTOR. To investigate the system-wide molecular effects of different degrees of chronic thermogenesis, we measured protein expression and the levels of protein acetylation using MS, and integrated our analysis with polar metabolite data that had been collected from the same mouse cohort. This analysis uncovered broad increases in mitochondrial protein acetylation after severe cold acclimation, increases to acetylated amino acids and acetylcarnitine, and novel cold-dependent acetylation sites on the uncoupling protein UCP1 that may play a role in regulating protein stability. To investigate the downstream effectors of RICTOR in brown adipocytes, we compared the time course response to insulin of control brown adipocytes and those possessing an inducible knockout of Rictor (Rictor-iKO). We employed MS to measure the global proteome and phosphoproteome, as well as a set of phosphorylation sites in a targeted analysis. This revealed broad differences in the proteome between control and knockout cells including an increased immune response in Rictor-iKO cells, as well as a mild repression of insulin-sensitive sites including the lipogenesis-associated enzyme ATP citrate lyase. These results suggest that RICTOR plays a multifaceted role in cells that extends beyond insulin signaling. In sum, we have investigated the proteome responses to cold acclimation and to the disruption of important signal transduction machinery. The findings have expanded our view of the metabolic flexibility of BAT, and laid the groundwork for future therapeutic strategies to improve BAT function in different contexts

A comparison of non-integrating reprogramming methods

Human induced pluripotent stem cells (hiPSCs1,2,3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation

A comparison of non-integrating reprogramming methods

Human induced pluripotent stem cells (hiPSCs1, 2, 3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.Stem Cell and Regenerative Biolog