1  

Supplemental Material

Submission to Genes and Development

Dietary obesity-associated Hif1α activation in adipocytes restricts

fatty acid oxidation and energy expenditure via suppression of the

Sirt2-NAD+ system

Jaya Krishnan, Carsten Danzer, Tatiana Simka, Josef Ukropec, Katharina Manuela

Walter, Susann Kumpf, Peter Mirtschink, Barbara Ukropcova, Daniela Gasperikova,

Thierry Pedrazzini, and Wilhelm Krek

Supplemental Materials and Methods

Mouse physiology

ERT2-Cre recombinase activity was induced by intra-peritoneal (ip) injections of

tamoxifen citrate (in 40% ethanol/0.9% NaCl) for 5 consecutive days at 20mg/kg

body weight. GTT and ITT were performed as previously described (Zehetner et al.,

2008). Echocardiography was performed as described (Barrick et al., 2007). Basal

body temperature was determined with a microprobe rectal thermometer (Stoelting).

Lean body mass was determined by carcass analysis and calculated by subtracting fat

mass from the total carcass weight (Brommage, 2003).

Mouse and metabolic studies

Mice were maintained on a 12-hour:12-hour light–dark. Mice were subjected to HFD

(D12331, Research Diets Inc. from postnatal week 4. Food and water intake, O2

consumption, and CO2 expiration and RER were simultaneously determined for four

mice in separate cages per experiment during a 72-hour period in an Oxymax

  2  

metabolic chamber system (Columbus Instruments), as previously described (Silva et

al., 2009). REE was calculated using the formula ((3.9x VO2) + (1.1x(VCO2)) X 1.44

(van der Kuip et al., 2004). overall food consumption was determined over a period of

3 weeks between weeks 12-18 post-tamoxifen induction.

Metabolic assays

Mitochondrial activity was assessed as described (Guzy et al., 2005). β-oxidation

capacity was measured with [9,10-3H(N)] palmitate as previously described (Djouadi

et al., 2003). Cellular oxygen consumption was measured using a Seahorse bioscience

XF24 analyzer in 24 well plates at 37°C, with correction for positional temperature

variations adjusted from four empty wells evenly distributed within the plate. 3T3-L1

cells were seeded at 20000 cells/well 8-10 days prior to the analysis. Once confluence

was reached, differentiation was induced and viral infection performed on day 4 of

differentiation. Each experimental condition was performed on 10 biological

replicates, and performed as recommended by the manufacturer (Seahorse

Bioscience).

Adipocyte isolation and 3T3-L1 culture and differentiation.

Primary adipocytes were isolated as described (Tozzo et al., 1995). 3T3-L1

preadipocytes (American Type Culture Collection, ATCC) were cultured in DMEM

(Sigma) with 10% fetal bovine serum at 37°C and 5% CO2. Primary preadipocytes

were isolated from dissected epididymal and subcutaneous fat pads. Tissues were

minced and then incubated with 2mg/ml collagenase (Sigma) at 37°C for one hour

while shaking. Cell suspensions were passed through a 70µm cell strainer and then

centrifuged at 1000 rpm for 5 minutes. After resuspension and brief incubation in

erythrocyte lysis buffer (154mM NH4Cl, 10mM KHCO3, 0.1mM EDTA), cells were

centrifuged again, resuspended in DMEM with 10% FCS and plated. Cells were

  3  

grown to confluence and differentiation was induced by adding 100nM insulin, 1 µM

dexamethasone and 0.5 mM IBMX (all Sigma) to the serum-containing medium.

After 2 days, medium was replaced with fresh serum containing medium including

100nM insulin and the appropriate lentiviruses. After additional 48 hours, medium

was replaced with tamoxifen containing medium to induce Cre activation. Adipocytes

were ready and used for further assays 7 days after differentiation induction. For 3T3-

L1 differentiation, 3T3-L1 cells were grown to confluence. Two days past reaching

confluence differentiation was induced by adding 100nM insulin, 1 µM

dexamethasone and 0.5 mM IBMX (all Sigma) to the serum-containing medium.

After 2 additional days, medium was replaced by DMEM, 10% serum and 100nM

insulin, and then media were changed every 2 days until cells were ready for

harvesting (6 to 8 days) (Elks and Manganiello, 1985). Transient transfections of

differentiated cells were performed on day 4 post-differentiation as described

(Shigematsu et al., 2001). All experiments were performed with three biological

replicates and repeated three times.

Immunofluorescence staining of cryosections

Tissue preparation and Immunofluorescence staining was performed as described

(Hirschy et al., 2006). Oil Red O staining was performed and quantified as previously

described (Krishnan et al., 2009). BODIPY staining was performed with the LipidTox

Lipid Stain (Invitrogen) as recommended by the manufacturer.

Antibodies, fluorescent reagents and chemicals

Cpt1 and Peroxiredoxin 6 antibodies were provided by Rene Lerch (University of

Geneva, Switzerland) and Sabine Werner (ETH-Zurich, Switzerland). Sirt1, Sirt2 and

acetylated-Lysine antibodies were from Cell Signaling. Antibodies against pan-

  4  

cadherin, b-actin and acetylated-tubulin were from Sigma. Hif1α, Hif1β, Pgc1α,

tubulin and lamin antibodies were from Santa Cruz Biotechnology. A second Pgc1α

antibody from Upstate/Millipore was utilized ti assess the specificity of Pgc1α

immunoprecipitation. Phalloidin, and DAPI were used as recommended by the

manufacturer (Invitrogen). DMOG (Cayman Chemical) and DFX were used 3mM

and 150µM, respectively. All chemical were purchased from Sigma unless indicated

otherwise.

Lentiviruses and plasmids

Lentiviral shRNAs were purchased from OpenBioSystems. The Hif1α ΔODD

expression construct was generated as described (Huang et al., 1998).

SDS-PAGE, immunoblotting and immunoprecipitation

Adipose tissue lysates were prepared and immunoblotting performed as described

(Hirschy et al., 2006). Immunoprecipitation for Pgc1α was performed as previously

described (Rodgers et al., 2005). The in vitro Sirt2 deacetylation assay as previously

described with bacterial purified Sirt2 (Ellis et al., 2008).

Luciferase assays

1kb upstream of the Sirt2 TSS was amplified from mouse BAC genomic DNA

(Imagenes) and cloned into the pGL3 luciferase reporter vector (Stratagene). The

Sirt2 ΔHRE-mutant was generated by recombinant PCR (Casonato et al., 2004; Elion

et al., 2007). Sequence integrity of the respective wildtype and mutant promoters were

verified by sequencing (Microsynth) and BLAST alignment

(http://www.ncbi.nlm.nih.gov/blast). The reporter assay was performed by transient

cotransfection of the appropriate luciferase reporter, the normalization construct pSV-

  5  

β-galactosidase (Promega), in the presence of Hif1α shRNA-encoding or of Hif1α

overexpressing viruses. The PEPCK-luciferase reporter and the E-box reporter

constructs were kindly provided by Pere Puigserver (Dana Farber Cancer Institute,

Boston) and Javier León (University of Cantabria, Spain), respectively. Luciferase

and β-galactosidase activity was measured using the Luciferase Assay System kit

(Promega) as recommended by the manufacturer and analyzed on the

MicroLumatPlus LB 96V (luciferase activity: Berthold Technologies) and Spectra

MAX 190 plate reader (β-galactosidase activity: Molecular Devices). All experiments

were performed with 3-5 biological replicates and repeated three times, unless

indicated otherwise.

Quantitative RT-PCR and ChIP assays

RNA was isolated with Trizol (Invitrogen) and cDNA generated using Superscript II

(Invitrogen), and performed as recommended by the manufacturer (Roche) and

analyzed on the Roche LightCycler 480. The ChIP assay was performed with

material from visceral adipose tissue and the assay performed using the ChIP-IT kit

(Active Motif) as recommended by the manufacturer. In silico promoter analyses and

alignments were performed using MatInspector and DiAlignTF (Genomatix).

Sequences of primers used in the various assays are listed in Supplemental Material

(Supplemental Tables 2 and 3).

Statistical analysis

All statistical analyses and tests were carried out using Excel software (Microsoft).

Data are given as mean +/- standard error of mean (SEM). Bars in graphs represent

standard errors and significance was assessed by two-tailed t-test.

  6  

Supplemental Figure Legends

Supplemental Fig. 1 Adipocyte Hif1α inactivation alleviates nutrient overload

induced pathologies

A-B) Various organs of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the

NCD/NCD (A) or HFD/HFD (B) protocol were assayed for Hif1α mRNA expression

at week 10-12 post-tamoxifen injection. All values were normalized internally to 18S

RNA expression and to the Hif1α iC(T) control, respectively. Data are mean +/- SEM

of values from 5 mice/group.

C) Lean body mass of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the

NCD/NCD or HFD/HFD protocol was assessed by carcass analysis.

D) Cardiac contractile function, expressed as percentage (%) fractional shortening, of

Hif1α iC(T) (n=5 NCD/NCD; n=5 HFD/HFD) and Hif1α icKO(T) (n=8 NCD/NCD;

n=7 HFD/HFD) mice maintained on the HFD/HFD protocol at the indicated time-

points both prior- (week 16), and at 10 and 22 weeks post-tamoxifen-mediated Hif1α

excision (*,% p<0.05 vs. Hif1α iC(T) NCD/NCD). Data are mean +/- SEM of values

from each group.

E) Representative M-mode echocardiographic tracing of Hif1α iC(T) and Hif1α

icKO(T) mice maintained on the HFD/HFD protocol at week 13 post-tamoxifen-

mediated Hif1α excision. Closed arrows indicates ventricular lumen internal diameter.

F) Representative images depicting the gross morphology of hearts derived from

Hif1α iC(T) and Hif1α icKO(T) mice maintained on the HFD/HFD protocol. Region

corresponding to the lung (lu) and epicardiac fat (epi) are indicated.

G) Schematic representation of the NCD/NCD and HFD/NCD protocol. Littermate

Hif1α iC and Hif1α icKO mice were placed on a NCD or HFD diet from 4 weeks

postnatal, for 14 weeks. Thereafter, at week 18, tamoxifen was administered to Hif1α

  7  

iC and Hif1α icKO mice of both the NCD and HFD groups. Mice from both groups

were maintained for an additional 20 weeks on the NCD diet. GTT measurements

were taken prior to Hif1α excision (at week 17), and at 10 and 20 weeks post-Hif1α

excision (at weeks 28 and 38).

H) Body weight measurements of Hif1α iC/Hif1α iC(T) (n=9 NCD/NCD; n=10

HFD/NCD) and Hif1α icKO/ Hif1α icKO(T) (n=8 NCD/NCD; n=12 HFD/NCD)

mice subjected to the HFD/NCD protocol were performed at the indicated time points

throughout the course of the protocol (* p<0.05 vs. Hif1α icKO(T) HFD/HFD, %

p<0.05 vs. Hif1α iC HFD/HFD and Hif1α icKO HFD/HFD). Data are mean +/- SEM

of values from each group.

I) GTT and area under curve (AUC) measurements of HFD/NCD maintained Hif1α

iC (n=10) and Hif1α icKO (n=12) mice prior to Hif1α excision (at week 17). Data are

mean +/- SEM of values from each group.

J) GTT and AUC measurements of HFD/NCD maintained Hif1α iC(T) (n=10) and

Hif1α icKO(T) (n=12) mice at 10 weeks post-Hif1α excision (* p<0.05). Data are

mean +/- SEM of values from each group.

K) GTT and AUC measurements of HFD/NCD maintained Hif1α iC(T) (n=10) and

Hif1α icKO(T) (n=12) mice at 20 weeks post-Hif1α excision (* p<0.05). Data are

mean +/- SEM of values from each group.

L-M) Interscapular BAT (L) and retroperitoneal visceral WAT (M) resected from

Hif1α f/f (n=9) and Hif1α BATcKO (n=11) mice were weighed. Data are mean +/-

SEM of values from each group.

Supplemental Fig. 2 Adipose Hif1α inactivation promotes the expression of fatty acid

oxidation genes

  8  

A) Individually housed Hif1α iC(T) (n=10) and Hif1α icKO(T) (n=12) mice

maintained on a HFD/HFD protocol were assessed for food intake over a period of 1

weeks (between weeks 12-13 post-tamoxifen administration). Data are mean +/- SEM

of values from each group.

B) Primary subcutaneous white adipocytes isolated from Hif1α iC(T) and Hif1α

icKO(T) mice maintained on the HFD/HFD protocol were assessed for palmitate

oxidation. Data are means ± SEM of values from 4 mice/group.

C) Primary visceral white adipocytes isolated from Hif1α iC(T) and Hif1α icKO(T)

mice maintained on the NCD/NCD protocol were assessed for palmitate oxidation.

Data are means ± SEM of values from 4 mice/group.

D) Gene expression profiling of visceral WAT of Hif1α iC(T) and Hif1α icKO(T)

mice subjected to the HFD/HFD protocol for expression of regulators of fatty acid

oxidation and uncoupling protein. All values were normalized internally to 18S RNA

expression and to the Hif1α iC(T) control, respectively (* p<0.01 compared to

control, set at 1.0). Data are mean +/- SEM of values from 5 mice/group.

E-F) Gene expression profiling of subcutaneous WAT of Hif1α iC(T) and Hif1α

icKO(T) mice subjected to the HFD/HFD protocol for expression of transcriptional

(D) and enzymatic (E) regulators of fatty acid oxidation. All values were normalized

internally to 18S RNA expression and to the Hif1α iC(T) control, respectively (*

p<0.01 compared to control, set at 1.0). Data are mean +/- SEM of values from 5

mice/group.

G-H) Preadipocytes isolated from the visceral (G) or subcutaneous (H) fat depots of

Hif1α iC(T) and Hif1α icKO(T) mice maintained on the HFD/HFD protocol were in

vitro differentiated to adipocytes and assessed for oleate-induced oxygen

consumption, as a measure of fatty acid β-oxidation, using the Seahorse Bioscience

  9  

24XF extracellular flux analyzer. Normalised oxygen consumption rate (OCR) is

shown (* p<0.01 compared to control Hif1α iC(T), set at 1.0). Data are mean +/- SEM

of values from 3 mice/group.

I) Average body weight of mice maintained on NCD (n=10) or HFD (n=10) for 20

weeks (* p<0.01 vs. NCD fed mice). Data are mean +/- SEM of values from each

group.

J) Visceral and subcutaneous white adipocytes isolated from wildtype C57/Bl6 mice

maintained on NCD or HFD for 20 weeks were assessed for palmitate oxidation (* p

< 0.05 vs. WT NCD). Data are means ± SEM of values from 4 mice/group.

K-M) Visceral WAT (K), subcutaneous (L) WAT and BAT (M) of mice maintained

on NCD or HFD for 20 weeks were assessed for Hif1α protein expression. β-actin

serves as a loading control.

N) Gene expression profiling of Hif1α-target genes in visceral WAT (vWAT),

subcutaneous WAT (scWAT) and BAT of wildtype C57/Black6 mice maintained on

NCD or HFD for 20 weeks. All values were normalized internally to 18S RNA

expression and to the NCD control, respectively (* p<0.01 compared to NCD, set at

1.0). Data are mean +/- SEM of values from 5 mice/group.

O-P) 3T3-L1-derived adipocytes  were subjected to ectopic Hif1α expression (O) or

hypoxia (at 3% O2) (P) and assessed for expression of mitochondrial biogenesis and

fatty acid oxidation regulators. Black bars indicate mock infectant (O) or normoxia

control (20% O2) (P) or, white bars indicate samples subjected to ectopic Hif1α

expression (O) or hypoxia (P). All values were normalized internally to 18S RNA

expression and to mock infectant (K) or normoxia (L) control, respectively (* p<0.01

compared to control, set at 1.0). Data are mean +/- SEM of values from each group.

  10  

Supplemental Fig. 3 Adipose Hif1α inactivation increases white adipocytes

mitochondrial density

A) Visceral WAT sections of Hif1α iC(T) and Hif1α icKO(T) mice maintained on the

HFD/HFD protocol were stained for the mitochondrial marker cytochrome oxidase

(COX, red), the neutral lipid marker (BODIPY, green), phalloidin (pink) and DAPI

(blue), and analyzed by immunofluorescence confocal microscopy.

Supplemental Fig. 4 Hif1α inactivation promotes Sirt2-mediated Pgc1α

transcriptional activation

A-C) COX3 (A), Cpt1 (B) and PEPCK (C) promoter activity in response to lentiviral

shHif1α-mediated Hif1α knockdown, was determined by transient cotransfection of

the respective promoters fused to luciferase in 3T3-L1 adipocytes. Adipocytes

infected with lentiviral non-silencing shRNAs (nsRNA) and transfected with the

respective reporters serve as controls. All transfections contained equal amounts of a

β-galactosidase expression vector for normalization of luciferase activity (* p<0.01

compared to control, set at 1.0). Data are mean +/- SEM of values from each group.

D) 3T3-L1-derived adipocytes  were subjected to normoxia (20% O2) or hypoxia (3%

O2) in the absence or presence of nsRNA or shHif1α. Cell lysates were

immunoprecipitated for Pgc1α and probed for acetylated lysine.

E) Hif1α was stabilized by addition of the prolyl hydroxylase (PHD) inhibitor

dimethyloxallyl glycine (DMOG), or by addition of the iron chelator desferrioxamine

(DFX) in 293 cells. Lysates were assessed for Hif1α protein levels by immunoblotting

(upper panel), and immunoprecipitated with the Pgc1α antibody and probed for lysine

acetylation and Pgc1α by immunoblotting (lower panels).

F) Visceral WAT of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the HFD/HFD

  11  

protocol were assayed for expression of Sirt3, Sirt4, Sirt5, Sirt6 and Sirt7. All values

were normalized internally to 18S RNA expression and to the Hif1α iC(T) control,

respectively. Data are mean +/- SEM of values from 3 mice/group.

G) Visceral WAT sections of HFD/HFD maintained Hif1α iC(T) and Hif1α icKO(T)

mice were stained for Sirt2 (red), DAPI (blue), the lipid marker BODIPY (green), and

phalloidin (pink) and analyzed by immunofluorescence confocal microscopy. Inlay

depicts co-localization of Sirt2 with DAPI predominantly in the nucleus.

H) Cell fractionation was performed on WAT biopsies of Hif1α icKO(T) HFD/HFD

mice and probed for Sirt2. Lamin serves as a nuclear marker, while peroxiredoxin 6

was used as a cytoplasmic marker (Kumin et al., 2007).

I) 3T3-L1-derived adipocytes  were subjected to normoxia (20% O2) or hypoxia (3%

O2) in the absence or presence of nsRNA or shHif1α, and assessed for Sirt2

expression. All values were normalized internally to 18S RNA expression and to the

normoxia control set (* p<0.01 compared to normoxia control, set at 1.0; % p<0.01

compared to hypoxia sample). Data are mean +/- SEM of values from each group.

Supplemental Fig. 5 Sirt2 is a Pgc1α deacetylase

A-B) 3T3-L1-derived adipocytes were infected with control or Sirt2-encoding (A)

lentivirus, or with nsRNA or shSirt2 (B), respectively, and assessed for expression of

Sirt2 protein. Loading was normalized to β-actin.

C) 3T3-L1-derived adipocytes were infected with control or Sirt1-encoding lentivirus

and assessed for expression of Sirt1 protein. Loading was normalized to β-actin.

D) 3T3-L1-derived adipocytes were infected with nsRNA or shPgc1α, and assessed

for Pgc1α protein by immunoblotting. Loading was normalized to β-actin.

E) Specificity of the Pgc1α antibody used for immunoprecipitation was determined by

  12  

infecting primary neonatal mouse cardiomyocytes (NMC) with nsRNA or shPgc1α

lentiviruses, and immunoprecipitating and immunoblotting Pgc1α. Lysates derived

from visceral WAT of Hif1α iC(T) and Hif1α icKO(T) mice maintained on

HFD/HFD were run in parallel. NMC express high basal levels of Pgc1α (Ventura-

Clapier et al., 2008).

Supplemental Fig. 6 Sirt2 is downregulated in a mouse model of dietary-induced

obesity

A) Sirt2 antibody specificity was verified by lentivirus-mediated knockdown of

SIRT2, and immunoblotting for SIRT2 in the human cell lines HeLa and A549.

B) Average fasting blood glucose index of mice maintained on NCD (n=10) or HFD

(n=10) for 20 weeks (* p<0.01 vs. NCD fed mice). Data are mean +/- SEM of values

from each group.

C) Visceral WAT biopsies of mice maintained on NCD or HFD for 20 weeks were

assessed for Sirt2 protein expression. β-actin serves as a loading control.

Supplemental Table Legends

Supplemental Table 1 Physiological characteristics of the human lean and obese

subjects

Adipose tissue area was determined by Magnetic Resonance Imaging (MRI) from a

single slice scan of abdomen in the umbilical region (between L4-5 vertebrae). None

of the individuals had fasting plasma glucose higher than 5.6 mmol/l. Oral glucose

tolerance test (oGTT); M-value, insulin sensitivity index was determined by

euglycemic hyperinsulinemic clamp. Data are presented as mean +/- SEM. The two

groups were compared by the student t-test and p < 0.05 (compared to lean subjects)

  13  

was considered statistically significant.

Supplemental Table 2 List of mouse qPCR and genotyping primers used in the study

Supplemental Table 3 List of human qPCR primers used in the study

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Supplemental  Table  1  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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