Summary of Study ST003199
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001993. The data can be accessed directly via it's Project DOI: 10.21228/M83T6C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003199 |
| Study Title | Metabolite profiling in the liver from fasted eIF4ES209A (S209A) mice compared to fasted wild type (WT) mice |
| Study Summary | Fasting is associated with a range of health benefits, including increased longevity, enhanced brain function, and improved metabolism. How fasting signals elicit changes in the proteome to establish metabolic programs that underlie lipid catabolism and the production of ketone bodies, an essential alternative fuel of energy, remain poorly understood. Here we show that paradoxically, while global translation is downregulated during fasting, hepatocytes selectively remodel the translatome to sustain lipid metabolism and ketogenesis. We discovered that phosphorylation of the major cap binding protein, eukaryotic translation initiation factor (P-eIF4E), is induced during fasting. By employing genome-wide unbiased polysome sequencing, we show that P-eIF4E is responsible for controlling the translation of the entire ketogenesis pathway, including the master regulator of fatty acid oxidation in the liver, peroxisome proliferator-activated receptor alpha (PPAR-alpha). Importantly, P-eIF4E regulates those mRNAs through a specific translation regulatory element within their 5’ untranslated regions. Genetic inhibition of P-eIF4E interrupts ketogenesis and fatty acid oxidation upon fasting. In addition, our findings reveal a new signaling property of fatty acids (FAs) derived from adipose tissue lipolysis, which are elevated during fasting. We uncovered that FAs bind and induce AMPK kinase activity that in turn enhances the phosphorylation of the kinase that phosphorylates eIF4E, the mitogen-activated protein kinase-interacting kinase (MNK). The AMPK-MNK axis controls ketogenesis revealing a new lipid-mediated kinase signaling pathway that links ketogenesis to translation control. We further show that genetically inhibiting P-eIF4E also impairs ketogenesis in response to a ketogenic diet. Certain types of cancers, such as pancreatic cancers use ketone bodies as an energy source which may rely on P-eIF4E suggesting a novel point of vulnerability. Our findings reveal that upon a ketogenic diet, treatment with eFT508 (a clinic P-eIF4E inhibitor) restrains pancreatic tumor growth in vivo. Importantly, restoring circulating β-hydroxybutyrate (BHB) or overexpression of PPAR-alpha in tumor cells ablate the effect of eFT508, demonstrating a systemic and tumor intrinsic role of P-eIF4E during tumorigenesis in response to a ketogenic diet. Thus, our findings unveil a novel fatty acid-induced signaling pathway that activates selective translation, which underlies the rapid cellular response to fasting and ketogenesis and provides a tailored diet intervention therapy for cancer. |
| Institute | University of Chicago |
| Last Name | Shah |
| First Name | Hardik |
| Address | 900 E 57th street |
| hardikshah@uchicago.edu | |
| Phone | 7738348830 |
| Submit Date | 2024-05-10 |
| Num Groups | 2 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-08-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001993 |
| Project DOI: | doi: 10.21228/M83T6C |
| Project Title: | Selective remodeling of the translatome underlies ketogenesis and diet associated tumor growth |
| Project Summary: | Fasting is associated with a range of health benefits, including increased longevity, enhanced brain function, and improved metabolism. How fasting signals elicit changes in the proteome to establish metabolic programs that underlie lipid catabolism and the production of ketone bodies, an essential alternative fuel of energy, remain poorly understood. Here we show that paradoxically, while global translation is downregulated during fasting, hepatocytes selectively remodel the translatome to sustain lipid metabolism and ketogenesis. We discovered that phosphorylation of the major cap binding protein, eukaryotic translation initiation factor (P-eIF4E), is induced during fasting. By employing genome-wide unbiased polysome sequencing, we show that P-eIF4E is responsible for controlling the translation of the entire ketogenesis pathway, including the master regulator of fatty acid oxidation in the liver, peroxisome proliferator-activated receptor alpha (PPAR-alpha). Importantly, P-eIF4E regulates those mRNAs through a specific translation regulatory element within their 5’ untranslated regions. Genetic inhibition of P-eIF4E interrupts ketogenesis and fatty acid oxidation upon fasting. In addition, our findings reveal a new signaling property of fatty acids (FAs) derived from adipose tissue lipolysis, which are elevated during fasting. We uncovered that FAs bind and induce AMPK kinase activity that in turn enhances the phosphorylation of the kinase that phosphorylates eIF4E, the mitogen-activated protein kinase-interacting kinase (MNK). The AMPK-MNK axis controls ketogenesis revealing a new lipid-mediated kinase signaling pathway that links ketogenesis to translation control. We further show that genetically inhibiting P-eIF4E also impairs ketogenesis in response to a ketogenic diet. Certain types of cancers, such as pancreatic cancers use ketone bodies as an energy source which may rely on P-eIF4E suggesting a novel point of vulnerability. Our findings reveal that upon a ketogenic diet, treatment with eFT508 (a clinic P-eIF4E inhibitor) restrains pancreatic tumor growth in vivo. Importantly, restoring circulating β-hydroxybutyrate (BHB) or overexpression of PPAR-alpha in tumor cells ablate the effect of eFT508, demonstrating a systemic and tumor intrinsic role of P-eIF4E during tumorigenesis in response to a ketogenic diet. Thus, our findings unveil a novel fatty acid-induced signaling pathway that activates selective translation, which underlies the rapid cellular response to fasting and ketogenesis and provides a tailored diet intervention therapy for cancer. |
| Institute: | University of Chicago |
| Last Name: | Shah |
| First Name: | Hardik |
| Address: | 900 E 57th street |
| Email: | hardikshah@uchicago.edu |
| Phone: | 7738348830 |
Subject:
| Subject ID: | SU003318 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype |
|---|---|---|---|
| SA348123 | S209A_01 | Liver | S209A |
| SA348124 | S209A_04 | Liver | S209A |
| SA348125 | S209A_02 | Liver | S209A |
| SA348126 | WT_05 | Liver | wild type |
| SA348127 | WT_01 | Liver | wild type |
| SA348128 | WT_03 | Liver | wild type |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO003311 |
| Collection Summary: | Food was removed from same-aged B6 wild-type mice or eIF4E_S209A/S209A mice for 24h. Then livers were isolated from 24h fasted mice. The largest lobe of each liver was snap-frozen using liquid nitrogen. |
| Sample Type: | Liver |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003327 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP003325 |
| Sampleprep Summary: | The polar metabolite profiling was performed on the snap-frozen liver tissue ground to powder using a mortar and pestle on dry ice. The metabolites were extracted using the ice-cold 4/4/2 acetonitrile/methanol/water (20µL solvent per mg of tissue, LC-MS grade solvents), homogenized (Omni International, TH115-PCR5H, stainless steel probe), vortexed and subjected 2 times- to sonication for 4 minutes in ice-cold water bath, freeze in liquid nitrogen for 1 minute, thaw on ice and subsequently vortex for 5 min at 2000 rpm and 4° C using Thermomixer. Samples were incubated on ice for 20 minutes, centrifuged at 20,000g for 20 minutes at 4°C and 500µL of supernatant from each sample was dry down using the Genevac EZ-2.4 elite evaporator. The dry-down samples were stored at -80°C until the analysis. On the day of the analysis, the samples were re-suspended in 140 µL of 60/40 acetonitrile/water. |
Combined analysis:
| Analysis ID | AN005249 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | HILICON iHILIC-(P) Classic (150 x 2.1 mm, 5 um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Scientific IQ-X tribrid |
| Ion Mode | UNSPECIFIED |
| Units | A.U. |
Chromatography:
| Chromatography ID: | CH003974 |
| Chromatography Summary: | The chromatography separation was performed using Thermo Scientific Vanquish Horizon UHPLC system and iHILIC-(P) Classic (2.1x150 mm, 5 µm; part # 160.152.0520; HILICON AB) column.The mobile phase A(MPA) was 20 mM ammonium bicarbonate at pH 9.6, adjusted by ammonium hydroxide addition and MPB was acetonitrile. The column temperature, injection volume, and the flow rate were 40°C, 2 µL, and 0.2mL/minute, respectively. The chromatographic gradient was 0 minutes: 85% B, 0.5 minutes: 85% B, 18 minutes: 20% B, 20 minutes: 20% B, 20.5 minutes: 85% B and 28 minutes: 85% B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | HILICON iHILIC-(P) Classic (150 x 2.1 mm, 5 um) |
| Column Temperature: | 40 |
| Flow Gradient: | The chromatographic gradient was 0 minutes: 85% B, 0.5 minutes: 85% B, 18 minutes: 20% B, 20 minutes: 20% B, 20.5 minutes: 85% B and 28 minutes: 85% B |
| Flow Rate: | 0.2mL/min |
| Solvent A: | 100% Water 20 mM ammonium bicarbonate; ammonium hydroxide 0.2% |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004982 |
| Analysis ID: | AN005249 |
| Instrument Name: | Thermo Scientific IQ-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The high-resolution Orbitrap IQ-X Tribrid mass spectrometer (Thermo Scientific) with a H-ESI probe operating in switch polarity was used to detect and quantify the metabolite levels.. MS parameters were as follows: Acquisition range of 70-1000 m/z at 60K resolution, spray voltage:3600V for positive ionization and 2800 for negative ionization modes, sheath gas: 35, auxiliary gas: 5, sweep gas: 1, ion transfer tube temperature: 250°C, vaporizer temperature: 350°C, AGC target: 100%, and a maximum injection time of 118 ms. AcquireX workflow was used to collect the MS/MS data in negative and positive separately using the assisted HCD collision energy 20,35,50,75,100 as well as targeted MS/MS with a defined retention time window for the in-house retention time database. Data acquisition was done using the Xcalibur software (Thermo Scientific) and data analysis was performed using Compound Discoverer 3.3 (± 5 ppm) & Tracefinder 5.1 software (Thermo Scientific). Metabolite identification was done by matching the retention time and MS/MS fragmentation to the in-house database generated using the commercially available reference standards. In the data table, the “RT+MS/MS” indicates the matching retention time & MS/MS, “RT”-indicates the only matching retention time and doesn’t have MS/MS while the MS/MS is for carnitine species identified based on the 85.0281 fragment. |
| Ion Mode: | UNSPECIFIED |
