Participants. A full description of our study population and general procedures has been published previously (9; Table 1). The sample size was calculated to reveal any significant difference ≥1.5-fold larger than the coefficient of variation (which was <10% in most cases) between the mean values for any individual variable, with a significance level of P < 0.05 and statistical power of 0.8. Ethical approval was obtained from the Regional Ethical Review Board of Umeå University (Umeå, Sweden), as well as the ethical committee of the University of Las Palmas de Gran Canaria (Canary Islands, Spain). All subjects were informed about potential risks and benefits before providing their written consent. Table 1. Baseline characteristics of our subjects Diet Sucrose

(n = 7) Whey Protein

(n = 8) Age, yr 38.7 ± 8.2 43.0 ± 8.0 Height, cm 181 ± 5.5 180 ± 4.2 Weight, kg 98 ± 12.0 100 ± 14.9 BMI, kg/m2 29.9 ± 3.1 30.9 ± 4.2 Lean mass, kg 63.1 ± 3.1 65.4 ± 6.0 Fat mass, kg 31.5 ± 9.1 31.4 ± 9.2 Body fat, % 31.6 ± 5.3 30.9 ± 4.1 V̇ o 2max , l/min 3.8 ± 0.3 3.9 ± 0.3 Daily energy intake, kcal 2,256 ± 513 2,086 ± 489 Physical activity (IPAQ), kcal/day 612 ± 315 601 ± 289

Experimental protocol. The experimental protocol consisted of a three consecutive phases: pretest (PRE), caloric restriction with exercise for 4 days (CRE), and a controlled diet with reduced exercise for 3 days (CD; Fig. 2). The aim of the CD phase was to allow the reestablishment of water shifts due to changes in glycogen stores and to reveal changes in protein expression involved in leptin signaling, which may require 2–3 days to develop. During the PRE and at the end of the CRE and CD phases, body composition was assessed by dual-energy X-ray absorptiometry (Lunar iDXA; GE Healthcare, Madison, WI; 9); 30-ml blood samples were drawn (in the supine position) following a 12-h overnight fast; and three muscle biopsies were obtained, one from each deltoid muscle (posterior portion) and one from the middle portion of the vastus lateralis (Fig. 2). All muscle biopsies were obtained early in the morning, immediately after drawing the blood samples. The PRE biopsies were obtained 7 days before the start of the CRE phase. The second round of biopsies was taken the starting day of the CD phase, i.e., in the morning of the first day after the end of the CRE phase. The last biopsies were performed the day after the end of the CD phase. During PRE and after CRE and CD, maximal fat oxidation (MFO) and peak oxygen uptake (V̇o 2peak ) were determined separately during arm cranking (in each arm separately) and two-legged pedaling by indirect calorimetry. Fig. 2.Experimental protocol. DXA, dual-energy X-ray absorptiometry; PRE, before the intervention; CRE, caloric restriction in combination with prolonged exercise; CD, isocaloric control diet. Download figureDownload PowerPoint

During CRE, the subjects were assigned randomly to ingest a very low calorie diet (0.8 kcal·kg body wt−1·day−1) consisting of either sucrose (n = 7) or whey protein (n = 8; Syntrax Nectar; Syntrax Innovations, Scott City, MO). This experimental approach allowed testing of the metabolic and molecular effects of the recommended dietary allowance for protein compared with ingestion of the same amount of energy as carbohydrates. With this experimental design, we avoided potential confusing effects due to other constituents of the diet, had mixed low-calorie diets been used. The CRE phase began with a 12-h overnight fast, after which a blood sample was taken between 6:30 and 8:00 AM. Each day, the subjects performed 45 min of one-arm cranking (at 15% of maximal intensity), followed by 8 h of walking at 4.5 km/h (35 km/day; at environmental temperatures ranging from 2.9 to 10.2°C). In a double-blind fashion they ingested sucrose (0.8 g/kg body wt) or whey protein (0.8 g/kg body wt), both dissolved in 1.5 liters of distilled water. The whey protein solution also contained Na+ (308 mg/l) and K+ (370 mg/l), as did the sucrose solution (160 and 100 mg/l, respectively). The subjects drank 0.5 liters of the assigned solution in the morning (just before arm cranking) and again at midday and 8 PM. In addition, both groups were allowed to drink a hypotonic rehydrating solution containing Na+ (160 mg/l), Cl− (200 mg/l), K+ (100 mg/l), citrate (700 mg/l), and sucrose (3 g/l) ad libitum. Each day during the CD phase, each participant ate three standardized meals containing his normal daily intake of energy (as assessed by weighing all food ingested during 7 days of the pretest period) and was not allowed to walk >10,000 steps.

MFO and V̇ o 2peak . Oxygen uptake and CO 2 production were assessed by employing a metabolic cart (Jaeger Oxycon Pro; Viasys Healthcare, Hoechberg, Germany), calibrated with 16.0% O 2 and 4.0% CO 2 (Air Liquid, Kungsängen, Sweden), at low, medium, and high flow rates with a 3-liter air syringe (Hans Rudolph, Kansas City, MO), in accordance with the recommendations of the manufacturer. MFO was determined using three separate incremental tests: one-arm cranking with the control or the exercised arm alone in random order, followed by two-legged pedaling (1, 42). The arm-cranking MFO test began at 10 W for 5 min followed by a 10-W increase every 3 min. The leg MFO test started at 30 W for 5 min, followed by a 30-W increment every 3 min. At the end of the 3-min period during which the subjects exhibited a respiratory exchange ratio >1.0, the exercise was stopped. After 5 min of recovery, an incremental test (10 and 30 W/min for the arm and leg protocols, respectively) beginning at the highest load reached during the MFO test was performed to determine the V̇o 2peak . During the tests they were instructed to maintain cranking and pedaling rates at 80 rpm. Between the incremental exercise tests, the subjects rested for 30 min or until the earlobe blood lactate concentration fell below 3.0 mmol/l (Biosen C-line; EKF Diagnostics, Barleben, Germany). Fat and carbohydrate oxidation were calculated from the oxygen uptake (V̇o 2 ) and carbon dioxide production (V̇co 2 ) values observed during the last 60 s of the 3-min steps of the MFO tests (16), assuming that the rate of protein oxidation was small and remained relatively constant. We also assumed that nearly steady-state conditions were reached during the last minutes of a 3-min constant-intensity load, as the load increments were small and the intensities were low (1). The highest V̇o 2 value during any 20-s interval of an incremental test on the cycle ergometer (Monark Ergomedic 839E; Monark Exercise, Vansbro, Sweden) was designated as the V̇o 2peak .

Assessment of physical activity and nutrition. Physical activity was assessed with the International Physical Activity Questionnaire (13, 45), and nutrition was assessed with a 7-day dietary record (Dietist XP; Kost & Näringsdata, Bromma, Sweden). During CD, each subject received a diet with the same energy content as that recorded during PRE containing 17% protein, 30% fat, and 53% carbohydrate. This food was provided and eaten in our facilities, so the weight of the food consumed could be measured and its composition determined with the Dietist XP software. During this phase, the sucrose and whey protein groups ingested 2,135 ± 390 and 2,129 ± 404 kcal/day (means ± SD), respectively.

Biochemical and hormonal analyses. After a 12-h overnight fast, 30-ml blood samples were drawn from a forearm vein directly into Vacutainer tubes (no. 368499 and no. 368498; BD Vacutainer, Stockholm, Sweden), and the serum concentration of glucose, insulin, and free fatty acids was quantified as previously reported (9). The Homeostasis Model Assessment index (HOMA) was calculated as fasting plasma concentration of insulin (µU/ml) × corresponding concentration of glucose (mmol/l)/22.5 (35). Cortisol and total testosterone were measured with chemiluminescence enzyme immunoassays (Immulite 2000 Cortisol, no. L2KCO2, and Immulite 2000 Total Testosterone, no. L2KTW2; Siemens) exhibiting sensitivities of 5.5 and 0.5 nmol/l and intra-assay and interassay coefficients of variation of 6.2 and 7.3% and 8.2 and 9.1%, respectively. Free testosterone was determined by a radioimmunoassay (Coat-A-Count Free Testosterone, no. TKTF1; Siemens) with a sensitivity of 0.5 pmol/l and intra-assay and interassay coefficients of variation of <8%. Sex hormone-binding globulin (SHBG) was assessed with a chemiluminescence enzyme immunoassay (Immulite SHBG, no. L2KSH2; Siemens) having a sensitivity of 0.02 nmol/l and intra-assay and interassay coefficients of variation of 2.7 and 5.2%, respectively. The free androgen index was calculated as [TT (nmol/l)/SHBG (nmol/l)] × 100, where TT is total testosterone. Following automated precolumn derivatization of plasma amino acids with o-phthalaldehyde, the resulting derivatives were separated by reversed-phase HPLC (on a 5-μm Resolve C18 column; Waters) and quantified by fluorescence detection.

Muscle biopsies, extraction of total protein, Western blotting, PCR, and myosin heavy chain composition. Muscle biopsies were taken under local anesthesia. After disinfection, the skin and subcutaneous adipose tissue were infiltrated with 1–2 ml of lidocaine 2%, avoiding the injection of anesthetic below the superficial fascia. Ten minutes later, a 6–7-mm incision was made, and a Bergstrom-type biopsy needle was inserted 2 cm into the muscle belly to obtain ~100 mg of tissue. The muscle sample was dissected free of any debris and adipose tissue, immediately frozen in liquid nitrogen, and stored at −80°C for later analysis. Extracts of muscle protein were prepared as described previously (37), and total protein content was quantified using the bicinchoninic acid assay (46). In brief, 30 mg of muscle were homogenized in urea lysis buffer (6 M urea, 1% SDS, 1× Complete protease inhibitor cocktail, and 1× PhosSTOP phosphatases inhibitor cocktail), and the lysate then was centrifuged for 15 min at 20,000 rpm at 4°C. The resulting supernatant was diluted with electrophoresis loading buffer (62.50 mM Tris·HCl, pH 6.8; 2.3% SDS; 10% glycerol; 5% β-mercaptoethanol; and bromophenol blue), and 35 μg of protein were then loaded onto each gel. The nine samples from each subject and four control samples (prepared from healthy human skeletal muscle) were loaded onto the same gel; that is, the four control samples always had the same composition. The sample protein bands were normalized to the mean value of the control band densities to account for the variability between gels (4). Proteins were labeled with the specific antibodies diluted in 4% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T; BSA-blocking buffer). The protein bands were revealed by incubation with a horseradish peroxidase-conjugated anti-rabbit antibody (diluted 1:20,000 in Blotto blocking buffer) and visualized using Immmuno Western CTM-Star (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom) using a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Finally, the bands were quantified with the Quantity One image analyzer (Bio-Rad Laboratories, Hercules, CA). To control for differences in loading and transfer efficiency, the membranes were subsequently stained with Reactive Brown 10 (56), and the bands were quantified with the ChemiDoc XRS. Since loading was homogeneous in all membranes, no further corrections were performed. The levels of OBR mRNA in the muscle biopsies were determined by real-time PCR. Total RNA was isolated using a RNeasy tissue fibrous kit (Qiagen, Valencia, CA), and its concentration was measured with spectrophotometry (Nanodrop; Thermo Scientific, Wilmington, DE); all RNA measurements had a ratio of absorbance at 260 nm (A260) to A280 of 2.0 ± 0.2. An aliquot of each RNA sample was used to determine the RNA quality indicator (RQI; Experion; Bio-Rad Laboratories), which was comprised between 5 and 8. For synthesis of first-strand cDNA, 100 ng of total RNA were reverse transcribed using a mix of oligo(dT) and random hexamer primers (Transcriptor; Roche Diagnostics, Mannheim, Germany). The transcript levels of OBR gene were quantified by real-time PCR (Light Cycler 480; Roche Diagnostics) using primers and Taqman probes designed with the Universal ProbeLibrary (UPL) assay system (Roche Diagnostics). Each cDNA was amplified using LC 480 Probes Master Mix (Roche Diagnostics) under the following conditions: 95°C for 10 min, followed by 35 cycles of 10 s at 95°C, 30 s at 60°C, and 15 s at 72°C. All the results were normalized to the levels of two housekeeping transcripts (cyclophilin gene and GAPDH), and relative quantification was calculated by the Livak method with an “all-to-mean” analysis including an interassay normalization (calibrator). All samples were run in duplicate, the average values were calculated, and the relative mRNA levels were reported as fold expression over the calibrator. Myosin heavy chain composition was determined as previously reported (20).

Materials. The Complete protease inhibitor cocktail and PhosSTOP phosphatases inhibitor cocktail were obtained from Roche Diagnostics (no. 04693116001 and no. 04906845001, respectively). The horseradish peroxidase-conjugated secondary anti-rabbit antibody was from Jackson ImmunoResearch (no. 111-035-144 and no. 715-035-150, respectively; West Grove, PA). The Immun-Blot nitrocellulose membranes and the Inmmun-Star WesternC were from Bio-Rad Laboratories (Hemel Hempstead, United Kingdom). The ChemiDoc XRS System and the image analysis software Quantity One were obtained from Bio-Rad Laboratories. The corresponding catalog numbers of the antibodies from Cell Signaling were as follows: anti-phospho-STAT3 (Tyr705), no. 9145; anti-STAT3 no. 9139; anti-phospho-JAK2 (Tyr1007/1008), no. 3771; and anti- JAK2, no. 3229. The polyclonal rabbit anti-human SOCS3 (no. sc-9023), anti-Tyr1141OBR (no. sc-16420), and anti-Tyr985OBR (no. sc-16419) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal mouse anti-PTP1B antibody was obtained from Calbiochem (no. FG6-1G; Darmstadt, Germany). The polyclonal rabbit anti-human leptin receptor antibody was obtained from Linco Research (no. 4781-L; St. Charles, MO). The anti-α-tubulin antibody was obtained from Biosigma (no. T5168, Madrid, Spain). The primers and UPL probes used for PCR were as follows: OBR gene (NM_002303.5*), forward (5′-CCTGGGCACAAGGACTTAAT-3′) and reverse (5′-TGTCACTGATGCTGTATGCTTG-3′), UPL no. 129; cyclophilin gene (NM_000942.4), forward (5′-TGTGGTGTTTGGCAAAGTTC-3′) and reverse (5′-GTTTATCCCGGCTGTCTGTC-3′), UPL no. 10; and GAPDH gene (NM_002046.3), forward (5′-AGCCACATCGCTCAGACAG-3′) and reverse (5′-GCCCAATACGACCAAATCC-3′), UPL no. 60.