Subjects The Genetics of Obesity Study (GOOS) is a cohort of 5,000 individuals with severe early-onset obesity; age of obesity onset is less than 10 years. Severe obesity is defined as a body mass index (weight in kilograms divided by the square of the height in meters) standard deviation score greater than 3 (standard deviation scores calculated according to the United Kingdom reference population). 1770 individuals from the cohort of European descent were randomly selected for KSR2 screening by Sanger sequencing. In addition, we reviewed KSR2 sequence data obtained from whole-exome sequencing of 331 unrelated individuals from the GOOS cohort. The Ely study is a prospective population based cohort study of the etiology and pathogenesis of type 2 diabetes and associated conditions in UK Caucasians. 1536 individuals from the cohort were randomly selected for KSR2 screening.

Genetic Screening The coding region and intron/exon boundaries of the human KSR2 gene (ENSG00000171435) were screened in genomic DNA isolated from whole-blood lymphocytes (primers and conditions available on request). Genetic screening was undertaken by PCR, followed by direct sequencing using BigDye terminator chemistry (Applied Biosystems, UK) and analyzed on an ABI 3730 automated sequencer (Applied Biosystems, UK). Exome capture was performed using the Agilent Sure-Select Human All Exon 50 Mb array, followed by high-throughput sequencing using the Illumina HiSeq sequencing system. High-quality sequence data were obtained (mean target coverage of approximately 50× was achieved with 90% of the samples reaching 10× coverage for >80% of target bases). Raw-sequencing reads were mapped to the GRCh37 reference human genome and variants called using Burrows-Wheeler Alignment and Sequence Alignment/Map (SAM) tools. SNPs and insertion/deletions (indels) were identified and realigned using the Genome Analysis Toolkit (GATK).

Statistical Analysis of Rare Variants in Cases versus Controls We used Fisher’s exact test to compare the number of rare (MAF < 0.5%) variants (both SNVs and indels) between cases and controls. Reported results are using the dominant genetic model. Results were robust to using an additive model. Various MAF thresholds were used to define rare variants (0.005, 0.001, 0.0005) with the smallest p value chosen. This process was repeated over 10,000 permutations in order to assess the p value correcting for multiple testing using various MAF thresholds. Finally, the entire process was repeated excluding population controls with a BMI > 30. The analysis was repeated within a replication sample of 238 cases and 1,117 controls and a combined analysis using the original and replication data for a total of 2,339 cases and 2,653 controls was completed. Variants within each MAF category were defined based on the sample for which the test is run (i.e., original, replication, combined).

Generation of KSR2 Expression Constructs A human cDNA clone encoding the 921 amino acid isoform of KSR2 (ENSEMBL transcript ID: ENST00000425217) was obtained from Origene (SC318393). Standard PCR methods were used to create a clone identical in sequence to the longest 950 amino acid isoform of KSR2 (ENSEMBL transcript ID: ENST00000339824). These were subcloned into the pCR-Blunt vector (Invitrogen) and further subloned as a KpnI/XbaI fragment into pEGFPC1 (N-terminal GFP tag) and pCDNA3.1(+) (N-terminal Flag tag) vectors. Mutagenesis of Flag-KSR2 950 was performed using QuikChange site-directed mutagenesis kit (Stratagene).

Cell Culture and Transfection HEK293 and Cos7 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1 mM Pen/Strep. HEK293 cells were transiently transfected with either PEI or Lipofectamine LTX (Invitrogen) for immunoblotting or flow cytometry, respectively. Cos7 cells were transiently transfected with Lipofectamine 2000 (Invitrogen). C2C12 myotubes were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1 mM Pen/Strep and transfected using Lipofectamine LTX Plus (Invitrogen).

Immunoprecipitation and Immunoblotting 24 hr posttransfection, HEK293 cells were serum starved overnight. Cells were then stimulated with 50 ng/ml Human recombinant EGF (Invitrogen) for 10 min. For metformin treatment, transfected HEK293 cells were incubated with 5 mM metformin overnight. Cells were harvested in NP-40 lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM Sodium orthovanadate, 50 mM Sodium fluoride, 10 mM Sodium pyrophosphate, 10 mM Sodium glycerophosphate, 1% (v/v) NP-40 and protease inhibitors) and lysates were clarified by centrifugation at 12,000 rpm for 15 min at 4°C. Lysates (0.5–1 mg protein) were precleared by incubation with Protein A/G agarose (Santa Cruz Biotechnology) at 4°C for 30 min before incubation with anti-FLAG M2 affinity gel (Sigma) at 4°C for 2 hr. Immunoprecipitates were washed twice with lysis buffer, twice with buffer A (50 mM Tris [pH 7.5], 0.1 mM EGTA), resuspended in SDS sample buffer and filtered through a Spin-X filter to remove the resin. NuPAGE reducing agent (1X; Invitrogen) was added to the eluted samples, which were subjected to electrophoresis and immunoblot analysis. Immunoblotting was performed at 4°C overnight using the following antibodies: anti-Flag (Sigma, #F1804), anti-B-Raf (Santa Cruz Biotechnology, #sc-9002), anti-ACC [pS79] (#3661], anti-AMPK (#2603), anti-pAMPK [pT172] (#2535), anti-ERK1/2 (#9102), anti-pERK1/2 (#9101), anti-MEK1/2 (#9122), anti-pMEK1/2 [pS217/pS221] (#9121), anti-pRaptor [pS792] (#2083) or anti-pTSC2 [S1387] (Cell Signaling Technology].

Immunofluorescence and Confocal Microscopy 40,000 Cos7 cells were seeded onto glass coverslips in 12-well plates and cultured for 24 hr before transfection using Lipofectamine 2000. After 6 hr, the cells were serum starved overnight. Cells were stimulated with 100 ng/ml EGF for 5 min and then fixed in 4% paraformaldehyde in PBS and subsequently permeabilised with 0.01% Triton X-100 in PBS for 5 min. Coverslips were blocked for 1 hr in 3% BSA in PBS then incubated with the indicated primary antibody. In addition to the antibodies listed above, anti-MEK1 (Santa Cruz Biotechnology, #sc-219) was specifically used for immunostaining. After 1 hr, coverslips were washed in PBS and incubated for 45 min with the appropriate secondary antibody (Dylight488 anti-mouse IgG [Vector Laboratories, DI-2488] or Alexa Fluor568 anti-Rabbit IgG ]Molecular probes, A-11036]) before washing in PBS. Coverslips were mounted in Vectashield mounting medium (Vector Laboratories) containing DAPI and examined using a Zeiss LSM510 confocal microscope.

Flow Cytometry HEK293 cells were seeded into 6-well plates for 24 hr before transfection with 6 μg of Flag-tagged constructs using Lipofectamine LTX. 6 hr posttransfection the cell culture medium was replaced with serum free DMEM. After overnight serum starvation cells were stimulated with 100 ng/ml EGF for 5 min and then harvested using trypsin. Harvested cells were centrifuged and the cell pellet resuspended in 4% formaldehyde in PBS and incubated for 10 min. The following steps were performed in 15 ml falcon tubes: one million cells were permeabilised in staining buffer (3% BSA, 2 mM EDTA and 1% saponin in PBS) for 60 min before immunostaining with primary antibodies (mouse anti-Flag (Sigma, #F1804), and rabbit anti-p-ERK (Cell Signaling Technology, 4370) diluted in staining buffer for 45 min. Cells were rinsed once in washing buffer (1% BSA, 2 mM EDTA and 0.1% saponin in PBS) then stained with secondary antibodies (anti-mouse dylight 488 [Vector Laboratories, DI-2488] and anti-rabbit PE [Molecular probes,A-10542]) diluted in staining buffer for 45 min. Cells were washed once more with washing buffer before resuspension in 700 μl of PBS. Cells were immediately analyzed using a BD FACS Calibur flow cytometer using a 488 nm laser and 530/30 nm filter to detect the anti-mouse daylight 488 secondary antibody signal and 585/42 nm filter to detect the anti-rabbit PE secondary antibody signal. A total of 50,000 events were acquired. Data were interpreted using flowing software 2.

Measurement of Glucose Oxidation 48 hr after transfection, C2C12 cells were subjected to experimental treatment in Seahorse XF24 V7 assay plates coated with poly-L-lysine (20,000 cells/well). Oxygen Consumption Rate (OCR) was then determined using an XF24 extracellular Immuflux analyzer (Seahorse Bioscience) as follows: cells were washed twice in Seahorse assay medium (4.15 g DMEM base, 1.85 g/l NaCl, 1x glutamax, 1 mM sodium pyruvate, 5 mM D-glucose, 15 mg/l phenol red, 20 mM HEPES [pH 7.4]) and incubated in 630 μl of assay medium at 37°C for 1 hr in a non-CO 2 incubator. To investigate cellular response to the presence of respiratory inhibitors and uncouplers 70 μl of oligomycin, 70 μl of FCCP and 70 μl of antimycin A/rotenone were loaded into Seahorse injection ports to achieve a final concentration of 1 μg/ ml oligomycin, 2 μM FCCP, 5 μM antimycin A and 4 μg/ml rotenone. The experimental set-up involved an initial 20 min equilibration step, two cycles of 3 min mix–2 min delay, 3 min measure steps to obtain basal OCR and sequential injections of oligomycin, FCCP and antimycin A/rotenone, each of which was followed by two cycles of mix-delay-measure steps as above. Oxygen consumption rates (OCR) were normalized to cell number as measured using a sulphorhodamine B (SRB) assay. Cells from Seahorse XF24 extracellular flux experiments were fixed with 200 μl 50% trichloroacetic acid at 4°C for 1 hr. Cells were then washed three times in water and allowed to air-dry. Cells were stained for 20 min at room temperature with 50 μl 0.4% (v/v) SRB in 1% (v/v) acetic acid, and then washed three times in 1% (v/v) acetic acid. The incorporated sulphorhodamine B dye (Sigma) was next solubilised in 100 μl 10 mM unbuffered Tris base at room temperature for 5 min, after which 20 μl of each sample was transferred to fresh wells in duplicates in a 96-well plate and further mixed with 80 μl 10 mM unbuffered Tris base. The absorbance at 565 nm was then measured. To obtain the cell count for each well, the absorbance was compared to a standard curve that was constructed by seeding known number of cells in quadruplicate wells and fixing and staining them in parallel with sample cells.

Measurement of Fatty Acid Oxidation 48 hr posttransfection, C2C12 cells were seeded in Seahorse XF24 V7 assay plates coated with poly-L-lysine (20,000 cells/well) and in order to initiate differentiation, cells were grown for 96 hr in medium with 1% FBS. Cells were either pretreated for 1 hr with 1 mM metformin or left untreated and the Oxygen Consumption Rate (OCR) was then determined using an XF24 extracellular flux analyzer (Seahorse Bioscience) as follows: cells were washed twice in KHB buffer (110 mM NaCl, 4.7 mM KCl, 2 mM MgSO 4 , 1.2 mM Na 2 HPO 4 , 2.5 mM glucose adjusted to pH 7.4 and supplemented with 0.5 mM carnitine) and incubated in 630 μl of KHB buffer at 37°C for 1 hr in a non-CO 2 incubator. To investigate FAO to the presence of BSA-conjugated palmitate (Seahorse Bioscience), 70 μl of BSA vehicle, 70 μl of BSA-Palmitate and 70 μl of FAO inhibitor etomoxir (Sigma) were loaded into Seahorse injection ports to achieve final concentration of 33 μM BSA, 100 μM palmitate, 50 μM etomoxir. The experimental set-up involved an initial 20 min equilibration step, two cycles of 3 min mix–2 min delay, 3 min measurement steps to obtain basal OCR and sequential injections of BSA vehicle or BSA-palmitate and etomoxir, each of which was followed by two cycles of mix-delay-measure steps as above. Oxygen consumption rates (OCR) were normalized to cell number as described above.

KSR1 and KSR2 Gene Expression in Human Tissues A human tissue cDNA library was prepared using 1 μl RNA (Clontech), which was reverse transcribed to cDNA using a Retroscript kit (Ambion). 1 μl cDNA was then used as a template in a PCR reaction using the following primers: KSR1 forward primer, 5′-AAGAGACTGGCCCTTGAAGAAC-3′; KSR1 reverse primer, 5′ - AAGTTTCTCCAGCATGTCCATC-3′; KSR2 forward primer, 5′-GGAGCAAATCCCACGAGTTCCAGC-3′; KSR2 reverse primer, 5′-GGTGCGTGTCCCACAAAGAAGG-3′.

Human Metabolic Phenotyping Subjects were invited to participate in clinical studies at the Wellcome Trust Clinical Research Facility at Addenbrooke’s Hospital, Cambridge, UK. All studies were approved by the Cambridge regional ethics committee and conducted in accordance with the principles of the Declaration of Helsinki. Each subject, or his or her parent for those under 16, provided written informed consent, minors provided oral consent. Weight and height were measured barefoot in light clothing. Dual X-ray absorptiometry (DEXA) (DPX software; Lunar Corp) was used to determine body composition. Ad libitum energy intake was assessed using a 18MJ breakfast of known macronutrient content after an overnight fast and was expressed per kilogram of lean body mass as measured by DEXA to allow comparison between individuals of different body weights and compositions. Basal metabolic rate and respiratory quotient were determined by indirect calorimetry after an overnight fast using an open circuit, ventilated, canopy measurement system (Europa Gas Exchange Monitor; NutrEn Technology). After adjustment for body composition, basal metabolic rate was compared to predicted metabolic rate based on standard age and sex specific equations. Blood pressure was measured using automated brachial (DINAMAP, GE Healthcare) or wrist (OMRON Healthcare) monitors. Heart rate was recorded using a wearable sensor (Actiheart, CamNtech, Papworth, UK) from which heart rate variability parameters were derived from cleaned interbeat interval time-series. Heart rate was recorded using a wearable sensor (Actiheart, CamNtech, Papworth, UK). Measurements were made during two distinct states; asleep (overnight from 0030–0530 hr); baseline awake (0700 – 0730 hr). Heart rate variability parameters were derived in Kubios HRV software (version 2.1, Kuopio, Finland). Interbeat interval time series were manually cleaned and filtered using the low correction threshold (cleaning interbeat intervals that differ more than 0.35 s from the local mean interbeat interval). Groups were compared using a general linear model after log transformation of RMSSD, low- and high-frequency power (ms2) and LF/HF ratio. Interaction between physiological state (asleep/awake) and groups was also checked in a general linear model (SPSS 20).

Mouse Husbandry and Energy Balance Studies −/− mice, maintained on a mixed genetic background (129/SvEvBrd and C57BL/6J), have been described previously, as have the general methods for mouse husbandry ( Brommage et al., 2008 Brommage R.

Desai U.

Revelli J.P.

Donoviel D.B.

Fontenot G.K.

Dacosta C.M.

Smith D.D.

Kirkpatrick L.L.

Coker K.J.

Donoviel M.S.

et al. High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes. Revelli et al., 2011 Revelli J.P.

Smith D.

Allen J.

Jeter-Jones S.

Shadoan M.K.

Desai U.

Schneider M.

van Sligtenhorst I.

Kirkpatrick L.

Platt K.A.

et al. Profound obesity secondary to hyperphagia in mice lacking kinase suppressor of ras 2. Revelli et al., 2011 Revelli J.P.

Smith D.

Allen J.

Jeter-Jones S.

Shadoan M.K.

Desai U.

Schneider M.

van Sligtenhorst I.

Kirkpatrick L.

Platt K.A.

et al. Profound obesity secondary to hyperphagia in mice lacking kinase suppressor of ras 2. Brommage et al., 2008 Brommage R.

Desai U.

Revelli J.P.

Donoviel D.B.

Fontenot G.K.

Dacosta C.M.

Smith D.D.

Kirkpatrick L.L.

Coker K.J.

Donoviel M.S.

et al. High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes. Ksr2mice, maintained on a mixed genetic background (129/SvEvBrd and C57BL/6J), have been described previously, as have the general methods for mouse husbandry (). The Institutional Animal Care and Use Committee at Lexicon Pharmaceuticals approved all study protocols. All mice were fed a low-fat diet (10% kcal as fat; diet D12450B, Research Diets, New Brunswick, NJ). Pair-feeding studies were performed as described previously (). Mouse body composition was estimated by Quantitative Magnetic Resonance (QMR, ECHO Medical Systems, Houston, TX) as described previously (). Immediately after Ksr2−/− mice and WT littermates were housed individually at 4°C, they had their rectal temperature measured for 1 s using a Thermalert TH-8 thermometer (Physitemp Instruments, Clifton, NJ) and Physitemp RET-3 rectal probe. They also had their skin temperature measured by placing a Physitemp MT-4 skin probe over their interscapular space for 1 s. The mice then had their rectal and interscapular skin temperatures re-measured every 30 min over a span of 4 hr. Any mice that became lethargic at a low rectal temperature, or had their rectal temperature drop below 25°C, were removed from the cold room, placed in a warmed cage and their temperature data up to the time of rescue were included in the analyses.