Summary of Study ST002361

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 PR001516. The data can be accessed directly via it's Project DOI: 10.21228/M8R12T 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	ST002361
Study Title	UCP2-dependent redox-sensing in POMC neurons regulates feeding
Study Summary	Paradoxically, glucose, the primary driver of satiety, activates a small population of anorexigenic POMC neurons. Here we show that lactate levels in the circulation and in the cerebrospinal fluid are elevated in fed state and addition of lactate to glucose activates the majority of POMC neurons while increasing cytosolic NADH generation, mitochondrial respiration and extracellular pyruvate levels. Inhibition of lactate dehydrogenases diminishes mitochondrial respiration, NADH production, and POMC neuronal activity. However, inhibition of the mitochondrial pyruvate carrier has no effect. POMC-specific downregulation of Ucp2 (Ucp2PomcKO), a molecule regulated by fatty acid metabolism and shown to play a role as transporter in the malate-aspartate shuttle, abolishes lactate- and glucose-sensing of POMC neurons. Ucp2PomcKO mice have impaired glucose metabolism and are prone to obesity on a high fat diet. Altogether, our data show that lactate through redox signaling and blocking mitochondrial glucose utilization activates POMC neurons to regulate feeding and glucose metabolism.
Institute	Columbia University
Last Name	Diano
First Name	Sabrina
Address	1150 St. Nicholas Avenue Russ Berrie Medical Science Pavilion Rm 405 New York, NY, 10032
Email	sd3449@cumc.columbia.edu
Phone	212 8514554
Submit Date	2022-11-28
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-12-15
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001516
Project DOI:	doi: 10.21228/M8R12T
Project Title:	UCP2-dependent redox-sensing in POMC neurons regulates feeding
Project Summary:	Paradoxically, glucose, the primary driver of satiety, activates a small population of anorexigenic POMC neurons. Here we show that lactate levels in the circulation and in the cerebrospinal fluid are elevated in fed state and addition of lactate to glucose activates the majority of POMC neurons while increasing cytosolic NADH generation, mitochondrial respiration and extracellular pyruvate levels. Inhibition of lactate dehydrogenases diminishes mitochondrial respiration, NADH production, and POMC neuronal activity. However, inhibition of the mitochondrial pyruvate carrier has no effect. POMC-specific downregulation of Ucp2 (Ucp2PomcKO), a molecule regulated by fatty acid metabolism and shown to play a role as transporter in the malate-aspartate shuttle, abolishes lactate- and glucose-sensing of POMC neurons. Ucp2PomcKO mice have impaired glucose metabolism and are prone to obesity on a high fat diet. Altogether, our data show that lactate through redox signaling and blocking mitochondrial glucose utilization activates POMC neurons to regulate feeding and glucose metabolism.
Institute:	Columbia University
Last Name:	Diano
First Name:	Sabrina
Address:	1150 St. Nicholas Avenue Russ Berrie Medical Science Pavilion Rm 405 New York, NY, 10032
Email:	sd3449@cumc.columbia.edu
Phone:	212 8514554

Subject:

Subject ID:	SU002450
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammals

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype
SA237014	Ucp2PomcKO_2_HC_Neg	UCP2 POMC-knockout
SA237015	Ucp2PomcKO_2_RP_Pos	UCP2 POMC-knockout
SA237016	Ucp2PomcKO_1_RP_Pos	UCP2 POMC-knockout
SA237017	Ucp2PomcKO_1_HC_Neg	UCP2 POMC-knockout
SA237018	Ctrl_1_HC_Neg	wild type
SA237019	Ctrl_2_RP_Pos	wild type
SA237020	Ctrl_1_RP_Pos	wild type
SA237021	Ctrl_2_HC_Neg	wild type

Showing results 1 to 8 of 8

Showing results 1 to 8 of 8

Collection:

Collection ID:	CO002443
Collection Summary:	Hypothalamic primary neuronal cell culture Eight to ten neonatal (0 days old) pubs from either control or Ucp2PomcKO mice derived from homozygous Cre-positive parents were used for hypothalamic primary neuronal cell culture. In brief, we carefully removed the brain's hypothalamus and placed it onto a small culture dish containing a small volume of Hibernate-A Medium (Cat# A1247501, Thermo Fisher Scientific). After digestion, the tissues dissociated to single cells with 6 mL of Hibernate-A Medium containing 2.5 % of Trypsin-EDTA for 15 minutes at 37℃. Suspended cells were filtered (40 μm) and centrifuged for 5 min at 1000 rpm. The pellet was re-suspended and plated on XF96 cell culture microplates (Cat# 101085-004, Agilent Technologies) coated with poly-D-lysine (Cat# P6407, Sigma-Aldrich) at a density of 0.2 x104 cells per well. Cells were cultured in Neurobasal-A medium (Cat# 10888022, Thermo Fisher Scientific) supplemented with 1 % penicillin-streptomycin, 2 % B-27 Supplement (Cat# 17504044, Thermo Fisher Scientific), and GlutaMAX-I (Cat# 35050061, Thermo Fisher Scientific), CultureOne supplement (Cat# A3320201, Thermo Fisher Scientific). For control culture, we used either Ucp2fl/fl; Pomc-CreERT2; tdTomato mice which neuronal cultures were treated with vehicle (0.03 % ethanol diluted in medium) or Ucp2+/+; Pomc-CreERT2; tdTomato mice which neuronal cultures were treated with 2 μM 4-hydroxytamoxifen (Cat# H7904, Sigma-Aldrich). After 10 days in culture, primary neuronal cells isolated from control and Ucp2PomcKO mice were treated with 2 μM 4-hydroxytamoxifen for expression of a CreER recombinase. Primary neuronal cells were used for the measurement of mitochondria oxidation two days later.
Sample Type:	Neurons

Treatment:

Treatment ID:	TR002462
Treatment Summary:	We used the inducible Cre/loxP technology to generate mice in which UCP2 was selectively ablated in POMC neurons (Ucp2PomcKO mice). First, mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in cells expressing POMC (Pomc-CreERT2) were crossed with Rosa26-lox-stop-lox-tdTomato (Ai14; cre-recombinase-dependent expression) mice (Ai14 reporter mice; stock #007914; The Jackson Laboratory) to label POMC-expressing cells. Pomc-CreERT2; Rosa26-lox-stop-lox-tdTomato (Pomc-CreERT2; tdTomato) mice have POMC-expressing cells with the expression of tdTomato by tamoxifen administration. No observation of POMC-tdTomato expression was found in the absence of tamoxifen administration, indicating that recombination was strictly dependent upon tamoxifen-induced Cre recombinase activation. The mice with Pomc-CreERT2; tdTomato were then crossed with mice harboring conditional alleles Ucp2 floxed (Ucp2fl/fl; B6;129S-Ucp2tm2.1Lowl/J, Stock# 022394; The Jackson Laboratory) to generated mice with inducible deletion of Ucp2 specifically in POMC neurons (Ucp2PomcKO mice). All animal experiments were conducted with the mice expressing POMC-CreERT2. As control groups, Ucp2fl/fl; Pomc-CreERT2 mice were injected with tamoxifen, and Ucp2fl/fl; Pomc-CreERT2; tdTomato were mice injected with corn oil (vehicle), and Ucp2fl/fl; Pomc-CreERT2; tdTomato mice were injected intraperitoneally (IP) with tamoxifen (0.1 mg/g BW for 5 consecutive days) starting at 5 weeks of age to induce mature-onset deletion of Ucp2 in POMC neurons of Ucp2PomcKO mice. However, because there were no differences between these two control groups, most of the experiments were performed using Ucp2+/+; Pomc-CreERT2; tdTomato mice injected with tamoxifen (to label POMC neurons with tdTomato expression) as a control group, unless otherwise stated. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX) monthly at 10:00 AM. We performed transcriptomic profiling by using a ribosomal tagging strategy to analyze POMC neuron-specific mRNA expression in vivo. We crossed Pomc-CreERT2 mice30 with Rpl22 floxed (RiboTag, Stock# 029977, The Jackson Laboratories) mice to generate Pomc-CreERT2; RiboTag mice, expressing a hemagglutinin A (HA)-tagged ribosomal protein in the POMC neurons upon tamoxifen injection.

Treatment ID:

TR002462

Treatment Summary:

We used the inducible Cre/loxP technology to generate mice in which UCP2 was selectively ablated in POMC neurons (Ucp2PomcKO mice). First, mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in cells expressing POMC (Pomc-CreERT2) were crossed with Rosa26-lox-stop-lox-tdTomato (Ai14; cre-recombinase-dependent expression) mice (Ai14 reporter mice; stock #007914; The Jackson Laboratory) to label POMC-expressing cells. Pomc-CreERT2; Rosa26-lox-stop-lox-tdTomato (Pomc-CreERT2; tdTomato) mice have POMC-expressing cells with the expression of tdTomato by tamoxifen administration. No observation of POMC-tdTomato expression was found in the absence of tamoxifen administration, indicating that recombination was strictly dependent upon tamoxifen-induced Cre recombinase activation. The mice with Pomc-CreERT2; tdTomato were then crossed with mice harboring conditional alleles Ucp2 floxed (Ucp2fl/fl; B6;129S-Ucp2tm2.1Lowl/J, Stock# 022394; The Jackson Laboratory) to generated mice with inducible deletion of Ucp2 specifically in POMC neurons (Ucp2PomcKO mice). All animal experiments were conducted with the mice expressing POMC-CreERT2. As control groups, Ucp2fl/fl; Pomc-CreERT2 mice were injected with tamoxifen, and Ucp2fl/fl; Pomc-CreERT2; tdTomato were mice injected with corn oil (vehicle), and Ucp2fl/fl; Pomc-CreERT2; tdTomato mice were injected intraperitoneally (IP) with tamoxifen (0.1 mg/g BW for 5 consecutive days) starting at 5 weeks of age to induce mature-onset deletion of Ucp2 in POMC neurons of Ucp2PomcKO mice. However, because there were no differences between these two control groups, most of the experiments were performed using Ucp2+/+; Pomc-CreERT2; tdTomato mice injected with tamoxifen (to label POMC neurons with tdTomato expression) as a control group, unless otherwise stated. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX) monthly at 10:00 AM. We performed transcriptomic profiling by using a ribosomal tagging strategy to analyze POMC neuron-specific mRNA expression in vivo. We crossed Pomc-CreERT2 mice30 with Rpl22 floxed (RiboTag, Stock# 029977, The Jackson Laboratories) mice to generate Pomc-CreERT2; RiboTag mice, expressing a hemagglutinin A (HA)-tagged ribosomal protein in the POMC neurons upon tamoxifen injection.

Sample Preparation:

Sampleprep ID:	SP002456
Sampleprep Summary:	Cells cultured in 6-well plates were quickly washed once with ice cold 5 mM HEPES, then immediately quenched in 150ul/well ice cold quench buffer (20% methanol, 3 mM sodium fluoride, 0.1% formic acid, 1mM phenylalanine and 100uM EDTA). Each well was harvested using a cell lifter and immediately transferred to a pre-chilled 96-well plate on dry ice. Once completely frozen, cell lysates were lyophilized and stored in the -80oC freezer until the sample run. Lyophilized samples were prepared by resuspending in 50 µL water with D4-taurine (50 µM) as an internal standard and 5 µL of the supernatant was injected for each analysis mode using high performance liquid chromatography (HPLC) into the 6600 Triple TOF LC-MS/MS mass spectrometer (Sciex).

Sampleprep ID:

SP002456

Sampleprep Summary:

Cells cultured in 6-well plates were quickly washed once with ice cold 5 mM HEPES, then immediately quenched in 150ul/well ice cold quench buffer (20% methanol, 3 mM sodium fluoride, 0.1% formic acid, 1mM phenylalanine and 100uM EDTA). Each well was harvested using a cell lifter and immediately transferred to a pre-chilled 96-well plate on dry ice. Once completely frozen, cell lysates were lyophilized and stored in the -80oC freezer until the sample run. Lyophilized samples were prepared by resuspending in 50 µL water with D4-taurine (50 µM) as an internal standard and 5 µL of the supernatant was injected for each analysis mode using high performance liquid chromatography (HPLC) into the 6600 Triple TOF LC-MS/MS mass spectrometer (Sciex).

Combined analysis:

Analysis ID	AN003855	AN003856
Analysis type	MS	MS
Chromatography type	Reversed phase	Unspecified
Chromatography system	SCIEX ExionLC	SCIEX ExionLC
Column	Phenomenex Kinetex F5 Core-shell LC (100 x 2.1 mm,2.6um)	Thermo Scientific Hypercarb (100 x 4.6 mm,3um)
MS Type	ESI	ESI
MS instrument type	Triple TOF	Triple TOF
MS instrument name	ABI Sciex 6600 TripleTOF	ABI Sciex 6600 TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH002853
Chromatography Summary:	Reverse phase column with positive MS mode
Instrument Name:	SCIEX ExionLC
Column Name:	Phenomenex Kinetex F5 Core-shell LC (100 x 2.1 mm,2.6um)
Column Temperature:	30
Flow Rate:	0.3ml/min
Solvent A:	95% water/5% acetonitrile; 0.1% formic acid
Solvent B:	95% acetonitrile/5% water; 0.1% formic acid
Analytical Time:	20min
Washing Buffer:	50%Methanol, 50% water
Chromatography Type:	Reversed phase

Chromatography ID:	CH002854
Chromatography Summary:	Hypercarb column with negative MS mode
Instrument Name:	SCIEX ExionLC
Column Name:	Thermo Scientific Hypercarb (100 x 4.6 mm,3um)
Column Temperature:	50
Flow Rate:	1ml/min
Solvent A:	100% water; 15 mM ammonium formate; 0.03% acetyl acetone; 0.1% formic acid
Solvent B:	60% acetonitrile/35% isopropanol; 0.1% formic acid; 15 mM ammonium formate
Analytical Time:	18min
Washing Buffer:	50%Methanol, 50% water
Chromatography Type:	Unspecified

MS:

MS ID:	MS003596
Analysis ID:	AN003855
Instrument Name:	ABI Sciex 6600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	Data were collected using an information-dependent analysis (IDA) workflow consisting of a TOF MS scan (200 msec) and a high-resolution IDA experiment (70 msec each) monitoring 10 candidate ions per cycle. Former target ions were excluded after 2 occurrences for 5 seconds and dynamic background subtraction was employed. The mass range for both TOF MS and IDA MS/MS scans was 60-1000. Ion spray voltage = 5000 V; ion source gas 1 (GS1) = 50 psi, ion source gas 2 (GS2) = 50 psi, curtain gas (CUR) = 30 psi, temperature (TEM) = 400 oC. declustering potential (DP) = 35 V, collision energy (CE) = 30 V, collision energy spread (CES) = 20 V. El-MAVEN software (Elucidata.io) was used for peak picking and curation from house built targeted and untargeted libraries. Targeted libraries were developed using a commercial standard kit of ~600 metabolites (IROA Technologies) in each mode, yielding reference data (molecular ions and retention times) with wide coverage of the endogenous metabolome. Untargeted libraries contained ~2,700 metabolites drawn from the KEGG database where the top 5 candidates with the highest intensity throughout a sample run for each metabolite were automatically curated.
Ion Mode:	POSITIVE

MS ID:	MS003597
Analysis ID:	AN003856
Instrument Name:	ABI Sciex 6600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	Data were collected using an information-dependent analysis (IDA) workflow consisting of a TOF MS scan (200 msec) and a high-resolution IDA experiment (70 msec each) monitoring 10 candidate ions per cycle. Former target ions were excluded after 2 occurrences for 5 seconds and dynamic background subtraction was employed. The mass range for both TOF MS and IDA MS/MS scans was 70-1000. Ion spray voltage = -4500V, ion source gas 1 (GS1) = 50 psi, ion source gas 2 (GS2) = 50 psi, curtain gas (CUR) = 30 psi, temperature (TEM) = 500 oC.declustering potential (DP) = 35 V, collision energy (CE) = 30 V, collision energy spread (CES) = 20 V. El-MAVEN software (Elucidata.io) was used for peak picking and curation from house built targeted and untargeted libraries. Targeted libraries were developed using a commercial standard kit of ~600 metabolites (IROA Technologies) in each mode, yielding reference data (molecular ions and retention times) with wide coverage of the endogenous metabolome. Untargeted libraries contained ~2,700 metabolites drawn from the KEGG database where the top 5 candidates with the highest intensity throughout a sample run for each metabolite were automatically curated.
Ion Mode:	NEGATIVE