Project description:Non-shivering thermogenesis in adipocytes is mediated by brown adipose tissue, purportedly through the sole action of uncoupling protein 1 (UCP1). The physiological relevance of UCP1-dependent thermogenesis has primarily been inferred from the attenuation of thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue beyond UCP1, such as reduced electron transport chain abundance. We show here that these secondary changes also encompass reduced expression of genes regulating fuel liberation, changes that would attenuate the capacity of any thermogenic pathway. Therefore, the quantitative contribution of UCP1-dependent and -independent thermogenesis is not fully understood. To mitigate the potentially confounding ancillary changes to brown adipose tissue of germline Ucp1-/- mice, we constructed mice with inducible adipocyte-selective disruption of Ucp1. We find that, while germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase B (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold-intolerance, indicative of a negative genetic interaction and thus a parallel thermogenic function. We find no evidence for impairments in insulation or non-shivering thermogenesis in skeletal muscle that would drive this phenotype. Furthermore, following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature adipocytes that coordinately restore UCP1 and CKB to brown adipose tissue, providing further evidence of their parallel thermogenic relationship. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy – through UCP1 and CKB – to promote cold-induced energy dissipation.

Project description:8 fractions for the TMT with file number of 748186_F1 to F8 was used for this section. Protein lysates were extracted from 3 WT and 3 FAM195A KO mice using RIPA lysis buffer. Protein concentration was determined and 25 ug of protein were processed. After trypsin digestion samples were then labelled with TMT reagent. A reverse-phase fractionation spin column was used according to the manufacturers directions to fractionate the sample into 8 fractions. Fractions were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. The mass spectrometer operated in positive ion mode with a source voltage of 1.6 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and top speed mode was used for SPS-MS3 analysis with a cycle time of 2.5 sec. MS2 was performed with CID with a collision energy of 35%. The top 10 fragments were selected for MS3 fragmentation using HCD, with a collision energy of 55%. Raw MS data files were analyzed using Proteome Discoverer v2.2 (Thermo), with peptide identification performed using Sequest HT searching against themouse protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys and TMT labelling of N-termini and Lys side-chains were set as a fixed modification, with oxidation of Met set as a variable modification. Description for sample PCF-JC-2191-07-2020 ( 783251-2) and PCF-JC-2227-07-2020 (787223-4) Differentiated brown adipocytes stably expressing FAM195A-miniTurbo were supplemented with 50 uM biotin (2 h) or left untreated. Cell lysates were extracted in 6 M urea RIPA. Biotinylated proteins were obtained after incubation of lysates with streptavidin beads. Samples were run for 1 cm in a protein gel. The area of the gel containing proteins was submitted for analysis. Gel band samples were digested overnight with trypsin following reduction and alkylation with DTT and iodoacetamide. The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters) and the resulting samples were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 um i.d., 75-cm long EasySpray column and eluted with a gradient from 0-28% buffer B over 90 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.8 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-7. Dynamic exclusion was set for 25 sec after an ion was selected for fragmentation. The Proteomics Core Facility at UTSW performed all proteomic analysis reported.

Project description:Adipose tissues play a pivotal role in energy homeostasis. They are existent in two forms: white adipose tissue and brown adipose tissue (BAT). Being the primary source of non-shivering thermogenesis of mammals, BAT plays an irreplaceable role in maintaining body temperature . Damaged DNA binding protein 1 (DDB1) is usually recognized as a component of the CUL4-DDB1 E3 complex. We have previously shown that DDB1 functions independently of CUL4 to promote adipogenesis. In the thesis, we focus on the function of DDB1 in mature adipocytes. We crossed Ddb1f/f mice with Adipoq-Cre or Ucp1-Cre mice to generate adipose tissue or BAT-specific mice, designated Ddb1-AKO and Ddb1-BKO mice respectively. At 8-12 weeks, BAT in these mice was servely whitened with decreased mitochdiral content. The thermogenic genes were also significantly downregulated at both RNA and protein levels. To further characterize the effect of DDB1 on thermogenesis, we first examined the bdy temperature of the pulps on postnatal day 3, and found that depletion of DDB1 caused a significant decrease in surface temperature.When the adult mice were subjected to cold challenge, both Ddb1-AKO and Ddb1-BKO mice showed dramatically decreased body temperature compared with control mice. At the end of the experiment, Ddb1-AKO and Ddb1-BKO mice had higher contents in BAT triglycerides and lower blood glucose level. Furthermore, the oxygen comsumption in Ddb1-AKO and Ddb1-BKO mice failed to respond to epinephrine injection. . All these results above demonstrated that lacking of DDB1 in brown adipocytes leads to a destruction in thermogenesis of BAT. We have also examined the phenotypes of Ddb1-AKO and Ddb1-BKO mice on high-fat-diet feeding. Compared with control mice, these mice showed decreased body weight, but significantly glucose intolerance and increased triglycierde content in liver, indicating that these mice have partial lipodystrophy. To study whether the effect of DDB1 was dependent on CUL4, we generated adipose tissue-specific Cul4a or CUl4b knockout mice, and found that these mice have no visible defect in BAT morphology compared with control mice, indicating a CUL4-independent function of DDB1. RNA-Seq analysis revealed that DDB1 is required for cold-induced expression of around 900 genes. ChIP-Seq analysis revelaed that these genes were subjected to regulation of promoter-proximally paused RNA polymerase II (Pol II), and that DDB1 is required for the release of paused Pol II upon cold stimulation. Our findings have thus revealed that DDB1 plays an important role in maintaining the theremogenic function of BAT by regulating the expression of thermogenic genes upon cold stimulation. Our studies will shed insights into transcriptional regulation of thermogenic gene

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	SRR13449409.fastq.gz	Fastqsanger.gz
	SRR13449410.fastq.gz	Fastqsanger.gz
	SRR13449411.fastq.gz	Fastqsanger.gz
	SRR13449412.fastq.gz	Fastqsanger.gz
	SRR13449413.fastq.gz	Fastqsanger.gz

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Regulation of cold-induced thermogenesis by the stress granule protein FAM195A

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