171208 Nrf1 in BAT Supplementary Figures nb 0.0 0.5 1.0 1.5 ASGR secretion * * * Supplementary Fig. 2 Proteasomal activity is required for mitochondrial respiration. (a) ER functional

Hspa5 Ddit3 Xbp1s Atf4 Cd680

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* *

*

* *

* * *

* *

* *

**

**

**

Caspase- like

Chemotrypsin- like

Trypsin- like

c e

Supplementary Fig. 1 Role of UPR and UPS in BAT for ER homeostasis. (a,b) Whole body energy expenditure during light phase, dark phase or during CL316,243 (CL) stimulation in mice lacking Ire1α or Xbp1 in adipocytes (Biol. replicates n=4, *P<0.05 by T-Test). (c) Proteasome activity in livers from wild-type (WT) mice after 7 days of adaptation to 4 °C or 30 °C (biol. replicates n=6, *P<0.05 by T-Test). (d) Representative H&E histology as well as tissue weights of interscapular brown adipose tissue (iBAT) from WT mice housed at 22 °C or 30 °C 16 h after Bortezomib (2.5 mg/kg) or control DMSO treatment (Biol. replicates n=8, *P<0.05 by 2-way ANOVA, Scale bar: 100 µm) (e) mRNA levels of stress markers in the livers of WT mice housed at 22 °C or 30 °C 16 h after Bortezomib (2.5 mg/kg) or control DMSO treatment (Biol. replicates n=8, normalized to ΔCT, *P<0.05 by 2-way ANOVA). (f) Body core temperature after 4 h carfilzomib (4 mg/kg) or control DMSO treatment in WT mice housed at 22 °C or 30 °C (Biol. replicates n=8, *P<0.05 by 2-way ANOVA). (g) mRNA levels of stress markers in BAT of WT mice housed at 22 °C or 30 °C after 6 h carfilzomib (4 mg/kg) or control DMSO treatment in (Biol. replicates n=8, normalized by ΔCT, *P<0.05 by 2-way ANOVA).

Nature Medicine: doi:10.1038/nm.4481

State 3 State 4 FCCP GDP-sensitive0

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OC

R

(pm

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e f g

h i

* IB: Nrf1

IB: β-Tubulin

BAT 22° DMSO

BAT 22° Carfilzomib

120 – 95 –

50 –

DMSO Epox DMSO Epox

Control Nrf1

IB: β-Tubulin

IB: Nrf1 (short exposure)

120 – 95 –

120 – 95 –

IB: Nrf1 (long exposure)

50 –

DMSO

100 n

m Epo

xomici

n

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m Bort

ezom

ib

50 nm

Thaps

igargi

n0.0

0.5

1.0

1.5

ASG

R s

ecre

tion

* *

*

Supplementary Fig. 2 Proteasomal activity is required for mitochondrial respiration. (a) ER functional reporter assay measuring asialoglycoprotein receptor (ASGR) secretion in control, epoxomicin, bortezomib or thapsigargin treated 3T3-L1 adipocytes (Tech. replicates n=4, *P<0.05 by 1-way ANOVA). (b) Oxygen consumption rate (OCR) in mitochondria isolated from BAT of WT mice 16 h after bortezomib (2.5 mg/kg) or DMSO at 22 °C (representative traces) (c) State 3, state 4, FCCP-induced and GDP-sensitive OCR in mitochondria isolated from BAT of WT mice 16 h after bortezomib (2.5 mg/kg) or DMSO at 22 °C (Biol. replicates n=4 for state 3 and state 4 and n=3 for FCCP and GDP, *P<0.05 by T-Test). (d-f) OCR in brown adipocytes 16 h after epoxomicin (100 nM) or DMSO treatment (representative traces in (d), tech. replicates n=4 in (e,f), *P<0.05 by T-Test). (g) Nrf1 protein levels in BAT after 6 h carfilzomib (4 mg/kg) or DMSO treatment (22 °C, lanes represent biological replicates, cropped images). (h) Nrf1 protein levels in control brown adipocytes or cells overexpressing Nrf1. Cells were treated with 250 nM epoxomicin or DMSO for 6 h (biol. Replicates n=3, cropped images). (i) Tissue mRNA levels of Nfe2l1 in WT mice after adaptation to 30 °C or 4 °C for 7 days (Biol. replicates n=4, *P<0.05 by T-Test with Holm-Sidak correction for multiple testing, SkeMus: Skeletal muscle).

a b

c d

BAT IngWAT EpiWAT Liver Heart SkeMus Kidney Pancreas0

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T B

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Nrf1 120 kDa Nrf1 95 kDa Ucp1

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22 °C Nfe2l1ΔBAT

*

* *

* *

*

*

* * * *

*

* *

* *

* *

* *

50 –

30 –

Supplementary Fig. 3 Validation of the Nfe2l1ΔBAT model. (a) PCR detection of cre recombinase-mediated recombination of the floxed Nfe2l1 locus (upper panel) as well as cre genotyping (lower panel) in selected tissues (cropped images). (b,c) Nrf1 and Ucp1 protein levels determined by immunoblot analysis (cropped images) in BAT (b) and quantified (c) from WT and Nfe2l1ΔBAT mice born and raised at 30 °C or 22 °C (n=3 biol. replicates). Lysates from Nrf1-deficient mouse embryonic fibroblasts (MEFs) or BAT from bortezomib-treated mice (20 % protein amount loaded on the gel) served as controls. (d) mRNA levels of proteasome subunits in the livers of WT or Nfe2l1ΔBAT mice after 16 h bortezomib (2.5 mg/kg) or DMSO treatment (22 °C; biol. replicates n=4, normalized to ΔCT, *P<0.05 by 2-way ANOVA). (e) mRNA levels of stress markers in BAT of WT or Nfe2l1ΔBAT mice born at 22 °C and then placed at 30 °C for 7 days (Biol. replicates n=6, normalized to 18s, *P<0.05 by 2-way ANOVA).


Hspa1

bHsp

a2Hsp

a4

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l

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Macroautophagy Autophagy

Lysosomal membrane

Pre-autophagosomal structure

0% 10% 20% 30% 40% 50%

Vacuole

Lysosomal membrane

Autophagy

Macroautophagy

Pre-autophagosomal

Increased pathways in Nfe2l1ΔBAT based on protein amount (GO term coverage)

P = 7.7 × 10-21

P = 9.8 × 10-9

P = 2.2 × 10-7

P = 5.1 × 10-7

P = 2.9 × 10-5

Nfe2l1ΔBAT

*

* * * * *

* *

* *

* * * * *

*

*

Supplementary Fig. 4 Broad survey of protein homeostasis in BAT from Nfe2l1ΔBAT mice. (a) Representative electron microscopy (EM) of BAT from 4 week-old WT or (b) Nfe2l1ΔBAT mice born and raised at 22 °C (White arrows pointing to ER, biol. replicates n=3; Scale bar in (a) 2 µm and in (b) 1 µm) (c) Protein levels of heat-shock proteins detected in the proteomic analysis of cold-adapted BAT (biol. replicates n=3 for WT and n=4 for Nfe2l1ΔBAT, *P<0.05 by T-Test with Holm-Sidak correction for multiple testing). (d) Pathway analysis of autophagy-related processes in BAT of Nfe2l1ΔBAT mice using ConsensusPathwayDB interaction database. (e) EM of BAT from 4 week-old Nfe2l1ΔBAT mice born and raised at 22 °C (White arrows pointing to vacuole/autophagosome formation). Scale bar: 1µm.


Nfe2l1 Ucp1 Ppara Pparg Ppargc1a Ppargc1b Ddit30

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Supplementary Fig. 5 The phenotype of Nfe2l1ΔBAT mice is progressive and occurs only below thermoneutrality. (a) Representative H&E histology of BAT from WT and Nfe2l1ΔBAT mice born and raised for the indicated times at 22 °C (Biol. replicates n=4; Scale bar: 100 µm). (b) Representative H&E histology and (c) Ucp1 IHC staining of BAT from 4 week-old WT and Nfe2l1ΔBAT mice born and raised at 30 °C or 22 °C (Biol. replicates n=4; Scale bar: 100 µm). (d) Body weight of 4 week-old WT and Nfe2l1ΔBAT mice born and raised at 30°C or 22 °C (Biol. replicates n=4). (e) mRNA levels of critical BAT regulators in BAT of 4 week-old WT and Nfe2l1ΔBAT mice born and raised at 30 °C or 22 °C (Biol. replicates n=4 for 30 °C, n=3 22 °C WT, and n=5 for Nfe2l1ΔBAT, normalized to Tbp, *P<0.05 by 2-way ANOVA). (f) CL-stimulated whole body energy expenditure of 4 week-old WT and Nfe2l1ΔBAT mice born and raised at thermoneutrality or room temperature (Biol. replicates n=4, *P<0.05 by 2-way ANOVA). (g) Representative traces from Seahorse measurements comparing mitochondria isolated from BAT of WT and Nfe2l1ΔBAT mice (Biol. replicates n=4, BSA: fatty acid-free bovine serum albumin, GDP: guanosine diphosphate).


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Supplementary Fig. 6 Inducible Nrf1 deletion results in stress pathway activation and BAT dysfunction. (a) Immunoblot analysis of OXPHOS complexes (CI-V) in BAT or isolated mitochondrial from WT and Nfe2l1ΔBAT mice (KO) as well as DMSO or bortezomib (bort)-treated animals, respectively (cropped images). (b,c) Ex vivo lipolysis assay measuring isoproterenol-stimulated non-esterified fatty acid (NEFA) release from (b) BAT and (c) WAT explants from 6 week-old WT and Nfe2l1ΔBAT mice born at 22 °C (Biol. replicates n=3). (d) Measurement of body core, BAT and skin temperature in WT and Nfe2l1ΔBAT mice adapted to cold for 7 days. (biol. replicates, WT n=4, Nfe2l1ΔBAT n=5, *P<0.05 by T-Test). (e,f) BAT mRNA levels in WT and mice, 7 days after the Nfe2l1 gene was disrupted in adult mice conditionally by tamoxifen (Ind.Nfe2l1−/−) at room temperature (biol. replicates n=4, normalized to Rn18s, *P<0.05 by (e) T-Test or (f) Mann-Whitney U-Test). (g) Representative photographs (Scale bar: 5 mm), H&E histology and Ucp1 IHC (Scale bar: 100 µm) of iBAT from WT and Ind.Nfe2l1−/− mice (h) CL-stimulated whole body energy expenditure in WT and Ind.Nfe2l1−/− (biol. replicates n=3 for WT and n=5 for Ind.Nfe2l1−/− mice, *P<0.05 by T-Test).


a b c

0

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GENERATION_OF_PRECURSOR_METABOLITES_AND_ENERGY

MITOCHONDRIAL_MEMBRANE

MITOCHONDRIAL_ENVELOPE

ENVELOPE

ORGANELLE_ENVELOPE

CELLULAR_CATABOLIC_PROCESS

CATABOLIC_PROCESS

MITOCHONDRIAL_PART

ORGANELLE_MEMBRANE

OXIDOREDUCTASE_ACTIVITY

MITOCHONDRION

CYTOPLASMIC_PART

CYTOPLASM

MEMBRANE

TRANSPORT

ORGANELLE_PART

INTRACELLULAR_ORGANELLE_PART

CELLULAR_LIPID_METABOLIC_PROCESS

LIPID_METABOLIC_PROCESS

MEMBRANE_PART

ESTABLISHMENT_OF_LOCALIZATION

CARBOXYLIC_ACID_METABOLIC_PROCESS

ORGANIC_ACID_METABOLIC_PROCESS

MACROMOLECULAR_COMPLEX

PROTEIN_COMPLEX

INTRINSIC_TO_MEMBRANE

INTEGRAL_TO_MEMBRANE

Normalized enrichment score

Mitochondrion

Mitochondrial matrix Mitochondrial envelope

Inner mitochondrial membrane protein complex

Mitochondrial inner membrane

Cellular respiration

Respiratory electron transport chain

Respiratory chain complex

Oxidative phosphorylation

Ribose phosphate metabolic process

Purine-containing compound metabolic process

Nucleoside phosphate metabolic process

Nucleoside metabolic process

Acyl-CoA metabolic process

Coenzyme metabolic process

Pyridine-containing compound metabolic

process

Fatty acid metabolic process

Oxoacid metabolic process

Lipid modification

Cellular lipid catabolic process

Lipid oxidation

Organic acid catabolic process

Lipid catabolic process

Small molecular metabolic process

Neutral lipid metabolic process

Monosaccharide metabolic process

Microbody Extracellular exosome

Supplementary Fig. 7 Analysis of the BAT ubiquitome. (a) Proteomic quantification of ubiquitin modifications. (b) Gene set enrichment analysis (GSEA) of hyperubiquitinated proteins in BAT of Nfe2l1ΔBAT mice (c) Pathway analysis of hyperubiquitinated proteins in BAT of Nfe2l1ΔBAT mice using ConsensusPathwayDB interaction database.


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* *

* *

* *

* *

* *

Supplementary Fig. 8 Metabolic phenotype of lean Nfe2l1ΔBAT mice. (a,b) Body weight (a) and glucose tolerance (b, 1 g/kg) in lean WT and Nfe2l1ΔBAT mice born and raised at room temperature (Biol. replicates n=8). (c) Representative gating strategy for macrophage flow cytometry analysis in BAT. (d) Gene expression analysis of cultured brown adipocytes after 8h treatment with DMSO or 20 nM bortezomib. (Biol. replicates n=3, normalized to Tbp, *P<0.05 by T-Test). (e) mRNA levels of inflammatory markers in iBAT of WT or Nfe2l1ΔBAT mice after 16 h bortezomib (2.5 mg/kg) or DMSO control treatment (22 °C; biol. replicates n=4, normalized to Rn18s, *P<0.05 by 2-way ANOVA).


IB: Ucp1

IB: β-tubulin

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ob/ob AV-LacZ ob/ob AV-Nrf1

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Supplementary Fig. 9 Adenoviral manipulation of BAT. (a) Nrf1 and Ucp1 protein levels determined by immunoblot analysis in BAT of WT and Nfe2l1ΔBAT mice contralateral BAT-injected with AV-LacZ or AV-Nrf1 (Biol. replicates n=2, same interscapular BAT depot is shown for one mouse, cropped images). (b) Nrf1 and Ucp1 protein levels determined by immunoblot analysis in BAT of ob/ob mice BAT-injected with either AV-LacZ or AV-Nrf1 (Biol. replicates n=7, *P<0.05 by Mann-Whitney U-Test, cropped images). (c) mRNA levels of Nfe2l1 and proteasome subunits in BAT of ob/ob mice BAT-injected with either AV-LacZ or AV-Nrf1 (Biol. replicates AV-LacZ n=8, AV-Nrf1 n=9, normalized to Tbp, *P<0.05 by T-Test). (d) Insulin tolerance test in ob/ob mice BAT-injected with AV-LacZ or AV-Nrf1 (Biol. replicates AV-LacZ n=8, AV-Nrf1 n=9, *P<0.05 by T-Test on AUC).


NRF1 Proteasome

subunits

Cold-induced proteometabolic activity

Global cellular quality control

Brown fat adaptation & systemic metabolic health

Ub Ub Ub Ub Ub

ER Mitochondria Cytosol Lipid droplets Cytoskeleton

Nucleus

Protein ubiquitination

Protein degradation

Proteasome

Supplementary Fig. 10 Schematic model summarizing the main findings of this study. Cold adaptation introduces an unusual and extreme metabolic burden on brown fat cells. The mechanism by which brown fat cells adapt to increased turnover of proteins, metabolites as well as synthesis and breakdown of cellular components is unknown. Here we show that proteasomal degradation of ubiquitinated proteins is a fundamental adaptive mechanism by which cellular integrity and quality control is achieved and identify the cold-inducible transcription factor NRF1 as the critical driver of proteasomal activity, brown fat metabolic adaptation and systemic homeostasis. Nrf1 loss and gain-of-function in preclincal models of obesity-induced insulin resistance demonstrate that Nrf1 is a guardian of metabolic health and a promising future drug target.


Figure 2g

Nrf1

β-Tubulin

55

100

Figure 2d

Nrf1

β-Tubulin

50

100

Figure 4a

Ubiquitin

Supplementary Fig. 2g

Nrf1

β-Tubulin

120 100

50

Supplementary Fig. 2h

Nrf1 (short exposure)

β-Tubulin

120 100

50

120 100

Nrf1 (long exposure)

Supplementary Fig. 11 Uncropped immunoblot pictures #1.


Supplementary Fig. 3c

Nrf1

Ucp1

50

30

120 100

β-Tubulin

Supplementary Fig. 3b

Supplementary Fig. 6a

Mitochondrial complexes CI-CV

500

500

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40

Supplementary Fig. 12 Uncropped gel and immunoblot pictures #2.


Supplementary Fig. 9a

120

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Supplementary Fig. 9b

120 95

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Nrf1

Ucp1

β-Tubulin

Nrf1

Ucp1

β-Tubulin

Supplementary Fig. 13 Uncropped immunoblot pictures #3.


Supplementary Methods

Immunohistochemistry. We performed immunohistochemistry on paraffin slides. We

mounted sections on adhesion slides, deparaffinized and rehydrated using a standard

protocol (2 x Xylene, 2 x 100 % Ethanol, 95 % v/v Ethanol, 70 % v/v Ethanol, 50 % v/v

ethanol, water, 5 min each). To block endogenous peroxidase activity, we incubated

sections in 3 % v/v H2O2 in PBS for 10 min. We performed antigen retrieval by boiling

the slides in Citrate buffer (10 mM sodium citrate, 0.05 % v/v Tween 20, pH 6) in a

microwave oven for 10 min. We cooled slides down to room temperature for 20 min,

rinsed these with PBS and then placed them in a humidity chamber for blocking and

permeabilization in blocking buffer (2 % v/v goat serum, 1 % w/v BSA, 0.1 % v/v Triton

X-100, 0.05 % v/v Tween 20 in PBS) for 1.5 h at room temperature. We incubated the

slides with the primary antibody (anti-UCP1, abcam # 10981), diluted in 1 % w/v BSA,

0.05 % v/v sodium azide in PBS over night at 4 °C. After the primary antibody

incubation, we washed the slides with PBS for 3 x 10 min and then incubated them with

goat anti-rabbit horseradish peroxidase secondary antibody (Santa Cruz #2030) at a 1:500

dilution for 1 h at room temperature. After the secondary antibody incubation, sections

were washed with PBS for 3 x 10 min. We performed staining using a 3,3’-

diaminobenzidine (DAB) substrate kit (abcam) following the manufacturer’s instructions.

After DAB staining, we rinsed the slides with PBS to stop the chromogenic reaction and

counterstained with hematoxylin QS (Vector Laboratories) for 2 min. We incubated the

slides under running tap water for 10 min, after which the slides were dehydrated in 50 %

v/v Ethanol, 70 % v/v Ethanol, 95 % v/v Ethanol, 2 x 100 % Ethanol and 2 x Xylene for

5 min each. Mounting was performed using Permount (Electron Microscopy Sciences).

Macrophage isolation and flow cytometry. We performed the isolation of stromal

vascular fraction (SVF) of BAT containing the immune cells as previously described71

with some modifications in 1 representative cohort of HFD-fed mice. Briefly, we minced


the interscapular BAT depot from individual mice and incubated the tissue in DMEM

containing collagenase I and II (Calbiochem, 2 mg/ml each) for 40 min at 37 °C with

120 rpm shaking. We stopped the reaction with FACS buffer (PBS containing 5% FBS)

and filtered the suspensions through 100 µm cell strainers (BD Falcon). The cell

suspensions were centrifuged at 2500 rpm for 5 min. We re-suspended the pellet in FACS

buffer and filtered it through a 40 µm cell strainer (BD Falcon). We centrifuged the cell

suspension at 3500 rpm for 5 min and the pellet was incubated for 5 min in erythrocyte-

lysing buffer (Sigma). Finally, we suspended the cells in FACS buffer. The antibodies

(1:100) used for the analysis of the SVF of BAT were anti-CD11b (eBioscience M1/70),

anti-F4/80 (Biolegend BM8), anti-CD11c (Biolegend N418). A representative gating

strategy can be found in Supplementary Fig. 7d. We analyzed the data using FACS

Calibur (BD Falcon) and FlowJo software (FlowJo). We expressed the total number of

macrophages per interscapular BAT depot per mouse and the M1/2 polarization within

these fractions was expressed as the percentage of total macrophages.

Sample preparation and liquid chromatography-MS3 spectrometry for proteomics.

BAT samples from 1 representative cohort of mice for multiplexed quantitative mass

spectrometry analysis were processed and analyzed through the Thermo Fisher Scientific

Center for Multiplexed Proteomics at Harvard Medical School. Immunoprecipitation of

diGly-containing peptides was performed as described previously72. Samples for

proteomic analysis were prepared as previously described73 with the following

modification. All solutions are reported as final concentrations. Lysis buffer (8 M Urea,

1 % SDS, 50 mM Tris pH 8.5, Protease and Phosphatase inhibitors from Roche) was

added to the tissue to achieve a cell lysate with a protein concentration between 2-

8 mg/mL. In order to facilitate tissue lysis a mechanical tissue grinder was used to

homogenize the tissue. A micro-BCA assay (Pierce) was used to determine the final

protein concentration in the cell lysate. Proteins were reduced and alkylated as previously


described. Proteins were precipitated using methanol-chloroform. In brief, 4 volumes of

methanol were added to the cell lysate, followed by 1 volume of chloroform, and finally

3 volumes of water. The mixture was vortexed and centrifuged to separate the chloroform

phase from the aqueous phase. The precipitated protein was washed with one volume of

ice-cold methanol. The washed precipitated protein was allowed to air dry. Precipitated

protein was resuspended in 4 M Urea, 50 mM Tris pH 8.5. Proteins were first digested

with LysC (1:50; enzyme:protein) for 12 h at 25 °C. The LysC digestion was diluted

down to 1 M Urea, 50 mM Tris pH8.5 and then digested with trypsin (1:100;

enzyme:protein) for another 8 h at 25 °C. Peptides were desalted using a C18 solid phase

extraction cartridges as previously described. Dried peptides were resuspended in

200 mM EPPS, pH 8.0. Peptide quantification was performed using the micro-BCA assay

(Pierce). The same amount of peptide from each condition was labeled with tandem mass

tag (TMT) reagent (1:4; peptide:TMT label) (Pierce). The 10-plex labeling reactions were

performed for 2 h at 25 °C. Modification of tyrosine residue with TMT was reversed by

the addition of 5 % hydroxyl amine for 15 min at 25 °C. The reaction was quenched with

0.5 % TFA and samples were combined at a 1:1:1:1:1:1:1:1:1:1 ratio. Combined samples

were desalted and offline fractionated into 24 fractions as previously described. 12 of the

24 peptide fraction from the basic reverse phase step (every other fraction) were analyzed

with an LC-MS3 data collection strategy74 on an Orbitrap Fusion mass spectrometer

(Thermo Fisher Scientific) equipped with a Proxeon Easy nLC 1000 for online sample

handling and peptide separations. Approximately 5 µg of peptide resuspended in 5 %

formic acid + 5 % acetonitrile was loaded onto a 100 µm inner diameter fused-silica

micro capillary with a needle tip pulled to an internal diameter less than 5 µm. The

column was packed in-house to a length of 35 cm with a C18 reverse phase resin (GP118

resin 1.8 µm, 120 Å, Sepax Technologies). The peptides were separated using a 180 min

linear gradient from 3 % to 25 % buffer B (100 % ACN + 0.125 % formic acid)

equilibrated with buffer A (3 % ACN + 0.125 % formic acid) at a flow rate of


400 nL/min across the column. The scan sequence for the Fusion Orbitrap began with an

MS1 spectrum (Orbitrap analysis, resolution 120,000, 400−1400 m/z scan range with

quadrapole isolation, AGC target 2 × 105, maximum injection time 100 ms, dynamic

exclusion of 90 seconds). ‘Top N” (the top 10 precursors) was selected for MS2 analysis,

which consisted of CID ion trap analysis, AGC 8 × 103, NCE 35, maximum injection

time 150 ms), and quadrapole isolation of 0.5 Da for the MS1 scan. The top ten fragment

ion precursors from each MS2 scan were selected for MS3 analysis (synchronous

precursor selection), in which precursors were fragmented by HCD prior to Orbitrap

analysis (NCE 55, max AGC 1 × 105, maximum injection time 150 ms, MS2 quadrapole

isolation was set to 2.5 Da, resolution 60,000. For diGly-enriched fractions the scan

sequence for the Fusion Orbitrap began with an MS1 spectrum (Orbitrap analysis,

resolution 120,000, 400−1250 m/z scan range with quadrapole isolation, AGC target 5 ×

105, maximum injection time 100 ms, dynamic exclusion of 60 seconds). ‘Top N” (the

top 10 precursors) was selected for MS2 analysis, which consisted of Orbitrap analysis

with quadrapole isolation of 0.5 Da for precursor selection, 1.5 × 104 resolution, AGC

1.5 × 105, NCE 35, maximum injection time 300 ms), and for the MS1 scan. The top

three fragment ion precursors from each MS2 scan were selected for MS3 analysis

(synchronous precursor selection), in which precursors were fragmented by HCD prior to

Orbitrap analysis (NCE 55, max AGC 1 × 105, maximum injection time 500 ms, MS2

quadrapole isolation was set to 2.5 Da, resolution 60,000. MS/MS data were searched

against a Uniprot mouse database (August 2013) with both the forward and reverse

sequences using the SEQUEST algorithm. Further data processing steps included

controlling peptide and protein level false discovery rates, assembling proteins from

peptides, and protein quantification from peptides. Statistical significance for proteins

and ubiquitin sites was calculated using the Benjamini-Hochberg method for multiple

hypothesis correction. For the differential BAT ubiquitome we divided the relative

intensities for ubiquitin-modified proteome by the relative intensities of the conventional


proteome. Several proteins, for which we found ubiquitination sites from the ubiquitin-

modified proteome were not found in the conventional proteome and therefore were

excluded from the differential ubiquitome analysis. We performed pathway analysis

using Gene Set Enrichment Analysis (GSEA, www.broadinstitute.org/gsea) with default

settings75,76. To increase signal-to-noise ratio we included only sites that were at least

significantly 2-fold higher in Nfe2l1ΔBAT BAT compared to WT and if multiple sites were

found per individual protein only the site displaying the highest ubiquitination relative to

the WT was included. For visualization of the pathway analysis we used the

ConsensusPathDB interaction database (CPDB; http://cpdb.molgen.mpg.de/MCPDB) and

included the proteins listed in the GSEA analysis with a significance cut-off of

6.92 × 10-15 and a relative overlap of 0.4 for GO term level 4.

SWIR quantum dots and SWIR in vivo imaging. For the synthesis of SWIR-emitting

quantum dots (QDs), 4 mmoles of indium(III) acetate and 16 mmoles of oleic acid were

mixed in 20 mL 1-octadecene (ODE) and degassed at RT for 30 minutes. The mixture

was heated to 115 °C under vacuum for another 60 minutes and then heated to 295 °C

under nitrogen. In a glovebox 0.2 mmoles (TMGe)3As77,78 were dissolved in 4 ml

trioctylphosphine (TOP) and subsequently injected into the solution. After 10 min a

0.17 M solution of (TMGe)3As in ODE was injected for 11 minutes at a speed of 8 mL

per h. The heat was removed and the growth solution was transferred to a glovebox and

purified by 2 precipitation/redispersion cycles. Purified InAs QDs (71 nmoles) in hexanes

were transferred to a mixture of 2 mL ODE and 2 mL oleylamine. The solution was

degassed for 30 min at RT and another 10 min at 110 °C. Subsequently, the solution was

heated to 280 °C for the shell growth. As soon as the mixture reached 240 °C, shell

precursor injection was started. 1 mL of a 0.05 M cadmium(II) oleate 0.3 M oleic acid

solution in ODE, and 1 mL of a 0.05 M solution of TOP-Se in TOP/ODE (1:19) were

injected over 40 minutes. InAsCdSe core-shell QDs were purified using the above


described procedure and the resulting QDs (ca. 60 nmoles) were redispersed in 2 mL

ODE and 2 mL Oleylamine. To that solution 1.2 mL of a 0.05 M cadmium(II) oleate

0.3 M oleic acid solution in ODE and 1.2 mL of a 0.045 M sulfur solution in ODE were

added within 48 min. InAsCdSeCdS core-shell-shell QDs were purified using the

procedure described above and exhibited a fluorescence peak at 1129 nm and a

fluorescence quantum yield of 37 %. QD-labelled recombinant triglyceride-rich

lipoproteins were generated by a previously reported procedure79. Briefly, for

incorporating the QDs into the recombinant lipoproteins, 20 mg of lipid extract

(approximately 80 % triglycerides, 10 % cholesterol and 10 % phospholipids) were

dissolved in chloroform and mixed with 1 mg (dry weight) InAsCdSeCdS core-shell-

shell QDs. The solvent was removed and the lipoproteins were formed in 2 mL PBS or

isotonic saline by sonication with a probe sonicator for 10 minutes. Potential aggregates

were removed by filtration using a 0.45 µm filter prior to injection. For the SWIR

imaging set-up, we coupled a 10 W 808 nm laser (Opto Engine; MLL-N-808) in a 910

µm-core metal-cladded multimode fiber (Thorlabs; MHP910L02). The output from the

fiber is passed through a ground-glass plate (Thorlabs; DG20-220-MD) to provide

uniform illumination over the working area. We used a 4 in square first-surface silver

mirror (Edmund Optics; Part No 84448) to direct the emitted light through two colored

glass 1000 nm long-pass filters (Thorlabs) to a Princeton Instruments Nirvana equipped

with a C-mount camera lenses (Navitar). The whole assembly was surrounded by a

partial enclosure to eliminate excess light while enabling manipulation of the field of

view during operation. Cold exposure (4 °C) was performed in single cages for 24 h

including a 4 h fasting period. For in vivo imaging to measure metabolic rates mice were

anesthetized (150 mg/kg ketamine and 15 mg/kg xylazine) mice, their fur removed (Nair)

and placed into the SWIR imaging setting. QD-labeled lipoproteins were injected via a

tail vein catheter with a syringe pump at a rate of 13.3 µL/mL (2 mg lipid per mouse) and

SWIR signal was recorded over time as well as after the injection from dissected iBAT.


Transmisson electron microscopy. 4-week-old mice from 3 independent cohorts were

anesthetized (300 mg/kg ketamine and 30 mg/kg xylazine) and transcardially perfused

first with 0.9 % w/v NaCl and then with electron microscopy grade fixative buffer

containing: 2.5 % w/v glutaraldehyde, 2.5 % w/v paraformaldehyde in 0.1 M sodium

cacodylate buffer, pH 7.4 (Electron Microscopy Science). 2 min after perfusion, small

pieces (1 to 2 mm cubes) of tissue were stored in the same fixative buffer described

above until further processing. The tissues were then washed in 0.1 M cacodylate buffer

and postfixed with 1 % w/v osmiumtetroxide (OsO4)/1.5 % w/v potassium ferrocyanide

(K2FeCN6) for 3 h, washed in water three times and incubated in 1 % w/v uranyl acetate

in maleate buffer (pH 5.2) for 1 h followed by 3 washes in maleate buffer and subsequent

dehydration in grades of ethanol (10 min each; 50 %, 70 %, 90 %, 2 × 10 min 100 %),

followed by 3 propylene oxide washes over one hour. Then the samples were placed in

50 % w/w propylene oxide with plastic mixture including catalyst overnight. The

following day, samples were embedded in TAAB 812 Resin mixture (Marivac Ltd., Nova

Scotia, Canada) and polymerized at 60 °C for 48 h. Ultrathin sections (80 nm) were

generated using a Leica Ultracut microtome, picked up on 100 µm mesh formvar/carbon

copper grids, stained with 0.2 % w/v lead citrate and examined in a TecnaiG2 Spirit

BioTWIN microscope. Images were recorded with an AMT 2k CCD camera.

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Documents

171208 Nrf1 in BAT Supplementary Figures nb 0.0 0.5 1.0 1.5 ASGR secretion * * * Supplementary Fig. 2 Proteasomal activity is required for mitochondrial respiration. (a) ER functional