Regulation of cold-induced thermogenesis by the RNA binding protein FAM195A.
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ABSTRACT:
SUBMITTER: Cannavino J
PROVIDER: S-EPMC8201964 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Homeothermic vertebrates are capable of surviving in cold environments by maintaining constant body temperature through thermogenesis, in which brown adipose tissue (BAT) produces heat by increasing mitochondrial oxidation along with the uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors that control the expression of UCP1 and genes involved in nutrient oxidation, mitochondrial function, have been extensively studied, only a few other proteins essential for BAT function have been identified. Here we describe the discovery of FAM195A, a disordered domain RNA-binding protein associated with stress granules that is required for cold-dependent thermogenesis in mice. FAM195A expression is enriched in BAT and striated muscles, and mice lacking FAM195A display whitening of BAT and an inability to survive at cold temperatures. In BAT of mice lacking FAM195A, key enzymes involved in branched chain amino acid (BCAA) metabolism, in particular leucine, and fatty acid (FA) oxidation are downregulated, impairing the physiological response to cold stress. Moreover, in vitro knock down of FAM195A impairs expression of leucine oxidation enzymes, revealing a direct role of FAM195A in the regulation of BCAA metabolism during thermogenesis
2021-06-05 | GSE164964 | GEO
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.
2021-01-15 | MSV000086711 | MassIVE
Project description:Insulin resistance is closely associated with metabolic diseases such as type 2 diabetes, dyslipidemia, hypertension and atherosclerosis. Thiazolidinediones (TZDs) have been developed to ameliorate insulin resistance by activation of peroxisome proliferator-activated receptor (PPAR) ?. Although TZDs are synthetic ligands for PPAR?, metabolic outcomes of each TZD are different. Moreover, there are lack of head-to-head comparative studies among TZDs in the aspect of metabolic outcomes. In this study, we analyzed the effects of three TZDs, including lobeglitazone (Lobe), rosiglitazone (Rosi), and pioglitazone (Pio) on metabolic and thermogenic regulation. In adipocytes, Lobe more potently stimulated adipogenesis and insulin-dependent glucose uptake than Rosi and Pio. In the presence of pro-inflammatory stimuli, Lobe efficiently suppressed expressions of pro-inflammatory genes in macrophages and adipocytes. In obese and diabetic db/db mice, Lobe effectively promoted insulin-stimulated glucose uptake and suppressed pro-inflammatory responses in epididymal white adipose tissue (EAT), leading to improve glucose intolerance. Compared to other two TZDs, Lobe enhanced beige adipocyte formation and thermogenic gene expression in inguinal white adipose tissue (IAT) of lean mice, which would be attributable to cold-induced thermogenesis. Collectively, these comparison data suggest that Lobe could relieve insulin resistance and enhance thermogenesis at low-concentration conditions where Rosi and Pio are less effective.
| S-EPMC6199571 | biostudies-other
Project description:ObjectiveCold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue.MethodsWe investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT.ResultsCold exposure or treatment with a β3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or β3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity.ConclusionsOur study implicates NNAT in the regulation of adipocyte thermogenesis.
| S-EPMC9932177 | biostudies-literature
Project description:Objective: Energy expenditure (EE) increases in response to cold exposure, which is called cold induced thermogenesis (CIT). Brown adipose tissue (BAT) has been shown to contribute significantly to CIT in human adults. BAT activity and CIT are acutely influenced by ambient temperature. In the present study, we investigated the long-term effect of seasonal temperature variation on human CIT. Materials and Methods: We measured CIT in 56 healthy volunteers by indirect calorimetry. CIT was determined as difference between EE during warm conditions (EEwarm) and after a defined cold stimulus (EEcold). We recorded skin temperatures at eleven anatomically predefined locations, including the supraclavicular region, which is adjacent to the main human BAT depot. We analyzed the relation of EE, CIT and skin temperatures to the daily minimum, maximum and mean outdoor temperature averaged over 7 or 30 days, respectively, prior to the corresponding study visit by linear regression. Results: We observed a significant inverse correlation between outdoor temperatures and EEcold and CIT, respectively, while EEwarm was not influenced. The daily maximum temperature averaged over 7 days correlated best with EEcold (R2 = 0.123, p = 0.008) and CIT (R2 = 0.200, p = 0.0005). The mean skin temperatures before and after cold exposure were not related to outdoor temperatures. However, the difference between supraclavicular and parasternal skin temperature after cold exposure was inversely related to the average maximum temperature during the preceding 7 days (R2 = 0.07575, p = 0.0221). Conclusion: CIT is significantly related to outdoor temperatures indicating dynamic adaption of thermogenesis and BAT activity to environmental stimuli in adult humans. Clinical Trial Registration: www.ClinicalTrials.gov, Identifier NCT02682706.
| S-EPMC6115528 | biostudies-literature
Project description:Regulation of cold-induced thermogenesis by the stress granule protein FAM195A
| PRJNA692630 | ENA
Project description:BACKGROUND:Hypothyroidism is a frequent endocrine disorder with common symptoms of increased cold sensitivity and unintended weight gain, indicating changes in energy expenditure (EE) and response to cold exposure. Thyroid hormones (TH) play an important role for proper function of brown adipose tissue (BAT) and cold-induced thermogenesis (CIT) in rodents, but the role of hypothyroidism on CIT in humans is uncertain. METHODS:This was a prospective observational study. Forty-two patients presenting with subclinical or overt hypothyroidism in whom TH replacement was planned were recruited. Thirty-three patients completed the study. Thermogenesis was measured by indirect calorimetry during warm conditions and after a mild cold stimulus of 90 minutes, both during the hypothyroid state and after at least three months of sufficient TH replacement. CIT was determined as the difference between EE during mildly cold and warm conditions. The primary endpoint was the change of CIT between the hypothyroid and euthyroid state. RESULTS:EE during warm conditions increased from a median of 1330 (interquartile range [IQR] 1251-1433) kcal/24 hours in the hypothyroid state to a median of 1442 (IQR 1294-1579) kcal/24 hours in the euthyroid state (+8.5%; p?=?0.0002). EE during mild cold exposure increased from 1399 (IQR 1346-1571) kcal/24 hours to 1610 (IQR 1455-1674) kcal/24 hours (+15%; p?<?0.0001). The median CIT was 55 (IQR 1-128) kcal/24 hours at the baseline visit, after restoration of euthyroidism CIT increased by 102% to a median of 111 (IQR 15.5-200) kcal/24 hours (p?=?0.011). Serum levels of free thyroxine at the respective visit and mean outdoor temperature during the preceeding 30 days were significantly associated with CIT (p?=?0.021 and p?=?0.001, respectively). CONCLUSION:Restoring euthyroidism significantly increases CIT in hypothyroid humans.
| S-EPMC6482913 | biostudies-literature
Project description:Recent studies indicate that a substantial amount of metabolically active brown adipose tissue (BAT) exists in adult humans. Given the unique ability of BAT to convert calories to heat, there is intense interest in understanding the regulation of BAT metabolism in hopes that its manipulation might be an effective way of expending excess calories. Because of the established role of AMP-activated protein kinase (AMPK) as a "metabolic master switch" and its extremely high levels of activity in BAT, it was hypothesized that AMPK might play a central role in regulating BAT metabolism. To test this hypothesis, whole body ?(1)-AMPK(-/-) (knockout) and wild-type mice were studied 1) under control (room temperature) conditions, 2) during chronic cold exposure (14 days at 4°C), and 3) during acute nonshivering thermogenesis (injection of a ?(3)-adrenergic agonist). Under control conditions, loss of ?(1)-AMPK resulted in downregulation of two important prothermogenic genes in BAT, thyrotropin-releasing hormone (-9.2-fold) and ciliary neurotrophic factor (-8.7-fold). Additionally, it caused significant upregulation of ?(2)-AMPK activity in BAT, white adipose tissue, and liver, but not cardiac or skeletal muscle. During acute nonshivering thermogenesis and chronic cold exposure, body temperature was indistinguishable in the ?(1)-AMPK(-/-) and wild-type mice. Similarly, the degree of cold-induced hyperphagia was identical in the two groups. We conclude that ?(1)-AMPK does not play an obligatory role in these processes and that adaptations to chronic loss of ?(1)-AMPK are able to compensate for its loss via several mechanisms.
| S-EPMC3154713 | biostudies-literature
Project description:Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP(-/-)) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP(-/-) mice. Brain TNF-? mRNA and protein expressions along with IL-1? protein levels were significantly increased in WT mice, which was not seen in the CIRP(-/-) mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-? and IL-1? were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.
| S-EPMC3815202 | biostudies-literature
Project description:Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate-forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate-forming protein. Here, using the arginine-glycine/arginine-glycine-glycine (RG/RGG)-rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD+ , NADH, NADP+ , and NADPH directly interact with CIRBP, involving both the folded RNA-recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA-driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid-liquid phase separation studies of RG/RGG and other condensate-forming proteins in order to better mimic the cellular environment in the future.
| S-EPMC8197425 | biostudies-literature