A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity Rikke Krogh-Madsen, John P. Thyfault, Christa Broholm, Ole Hartvig Mortensen, Rasmus H. Olsen, Remi Mounier, Peter Plomgaard, Gerrit van Hall, Frank W. Booth and Bente K. Pedersen J Appl Physiol 108:1034-1040, 2010. First published 31 December 2009; doi:10.1152/japplphysiol.00977.2009 You might find this additional info useful... Supplemental material for this article can be found at: http://jap.physiology.org/content/suppl/2010/01/27/00977.2009.DC1.html This article cites 29 articles, 25 of which can be accessed free at: http://jap.physiology.org/content/108/5/1034.full.html#ref-list-1 Physical Activity, Sedentary Behavior, and Health: Paradigm Paralysis or Paradigm Shift? Peter T. Katzmarzyk Diabetes, November , 2010; 59 (11): 2717-2725. [Full Text] [PDF] Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle Søren Nielsen, Camilla Scheele, Christina Yfanti, Thorbjörn Åkerström, Anders R. Nielsen, Bente K. Pedersen and Matthew Laye J Physiol, October 15, 2010; 588 (20): 4029-4037. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://jap.physiology.org/content/108/5/1034.full.html Additional material and information about Journal of Applied Physiology can be found at: http://www.the-aps.org/publications/jappl This infomation is current as of March 25, 2011. Journal of Applied Physiology publishes original papers that deal with diverse areas of research in applied physiology, especially those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2010 by the American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/. Downloaded from jap.physiology.org on March 25, 2011 This article has been cited by 3 other HighWire hosted articles Variability in training-induced skeletal muscle adaptation James A. Timmons J Appl Physiol, March , 2011; 110 (3): 846-853. [Abstract] [Full Text] [PDF] J Appl Physiol 108: 1034–1040, 2010. First published December 31, 2009; doi:10.1152/japplphysiol.00977.2009. A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity Rikke Krogh-Madsen,1 John P. Thyfault,2 Christa Broholm,1 Ole Hartvig Mortensen,1 Rasmus H. Olsen,1 Remi Mounier,1 Peter Plomgaard,1 Gerrit van Hall,1 Frank W. Booth,3 and Bente K. Pedersen1 1 Centre of Inflammation and Metabolism at Department of Infectious Diseases and Copenhagen Muscle Research Centre, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 2Harry S. Truman Memorial Veterans Hospital, Health Activity Center, Departments of Nutrition and Exercise Physiology and Internal Medicine, University of Missouri, Columbia, Missouri; and 3Health Activity Center, Departments of Biomedical Sciences and of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri Submitted 30 August 2009; accepted in final form 29 December 2009 insulin resistance; clamp; insulin signaling and at the low range may increase risk for developing chronic metabolic disease(s) (4). We postulated that healthy, nonexercising subjects who transitioned from a high to low level of ambulatory activity (from ⬎10,000 to ⬍2,000) would quickly display metabolic alterations. Our initial findings showed that healthy young men who reduced their daily steps from an average of 10,501 ⫾ 808 to 1,344 ⫾ 33 for a 2-wk period displayed a clustering of metabolic alterations including increased insulin response to an oral glucose tolerance test (OGTT), increased plasma triglyceride response to an oral fat tolerance test, and a 7% increase in visceral fat (21). These findings led us to question if the loss of insulin sensitivity induced by reduced ambulatory activity was due to peripheral or hepatic decreases in insulin sensitivity. Moreover, if there was a decline in peripheral insulin sensitivity we questioned if it was associated with reduced activation of the insulin signaling pathway in skeletal muscle as has been shown in rodent models in which daily physical activity was ceased for acute periods of time (16). We additionally wished to determine if reduced ambulatory activity would negatively impact lean body mass or cardiorespiratory fitness [maximal oxygen consumption (V̇O2 max)], two factors strongly linked to metabolic health and mortality risk. Herein we provide evidence that 2 wk of reduced daily ambulatory activity in healthy young, nonexercising subjects decreases peripheral insulin sensitivity and insulin stimulation of Akt in skeletal muscle without change in rate of glucose appearance during a hyperinsulinemic-euglycemic clamp. In addition, we show evidence that reduced ambulatory activity significantly lowers both lean body mass and cardiorespiratory fitness. METHODS EPIDEMIOLOGICAL EVIDENCE indicates that low physical activity plays a causal role in the development of Type 2 diabetes (11, 12, 17). Our longer-term hypothesis is that reductions in daily ambulatory activity levels (reduced steps taken throughout the day) initiate attenuation in skeletal muscle insulin sensitivity that precedes a later development of overt insulin resistance. It is estimated that most free-living, nonexercising US adults take between ⬃2,000 and ⬃12,000 steps per day, a wide range of ambulatory activity that at the high range may decrease risk Address for reprint requests and other correspondence: R. Krogh-Madsen, Centre of Inflammation and Metabolism, Rigshospitalet-Section 7641, Blegdamsvej 9, DK-2100 Copenhagen, Denmark (e-mail: krogh-madsen@inflammationmetabolism.dk). 1034 Ethical approval. All subjects gave oral and written consent, and the study was approved by the Scientific-Ethics Committee of Copenhagen and Frederiksberg Municipalities (file no. KF01268925) in accordance with the Helsinki Declaration. Subjects. Ten healthy human males (mean age 23.8 ⫾ 1.5; body mass index of 22.1 ⫾ 0.7 kg/m2) participated in the study [reported in a preliminary report (21)]. Before the study all 10 subjects underwent a thorough clinical examination. All subjects were nonsmokers, asymptomatic, with no family history of diabetes, did not take medications, and revealed no physical abnormalities during examination. All subjects had normal resting values of blood pressure, normal plasma levels of glucose, insulin, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TG), normal hematological parameters including leukocyte counts, and normal hepatic, thyroid, and renal http://www.jap.org Downloaded from jap.physiology.org on March 25, 2011 Krogh-Madsen R, Thyfault JP, Broholm C, Mortensen OH, Olsen RH, Mounier R, Plomgaard P, van Hall G, Booth FW, Pedersen BK. A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity. J Appl Physiol 108: 1034 –1040, 2010. First published December 31, 2009; doi:10.1152/japplphysiol.00977.2009.—US adults take between ⬃2,000 and ⬃12,000 steps per day, a wide range of ambulatory activity that at the low range could increase risk for developing chronic metabolic diseases. Dramatic reductions in physical activity induce insulin resistance; however, it is uncertain if and how low ambulatory activity would influence peripheral insulin sensitivity. We aimed to explore if healthy, nonexercising subjects who went from a normal to a low level of ambulatory activity for 2 wk would display metabolic alterations including reduced peripheral insulin sensitivity. To do this, ten healthy young men decreased their daily activity level from a mean of 10,501 ⫾ 808 to 1,344 ⫾ 33 steps/day for 2 wk. Hyperinsulinemic-euglycemic clamps with stable isotopes and muscle biopsies, maximal oxygen consumption (V̇O2 max) tests, and blood samples were performed pre- and postintervention. A reduced number of daily steps induced a significant reduction of 17% in the glucose infusion rate (GIR) during the clamp. This reduction was due to a decline in peripheral insulin sensitivity with no effect on hepatic endogenous glucose production. The insulinstimulated ratio of pAktthr308/total Akt decreased after step reduction, with a post hoc analysis revealing the most pronounced effect after 4 h of insulin infusion. In addition, the 2-wk period induced a 7% decline in V̇O2 max (ml/min; cardiovascular fitness). Lean mass of legs, but not arms and trunk, decreased concurrently. Taken together, one possible biological cause for the public health problem of Type 2 diabetes has been identified. Reduced ambulatory activity for 2 wk in healthy, nonexercising young men significantly reduced peripheral insulin sensitivity, cardiovascular fitness, and lean leg mass. AMBULATORY ACTIVITY AND INSULIN SENSITIVITY HR variability, and ECG magnitude for epoch settings of 15, 30, and 60 s and can be recorded for a set time. Data on interbeat intervals (IBI logging) and ECG waveforms can also be recorded. Acceleration is measured by a piezoelectric element within the Actiheart with a frequency range of 1–7 Hz (3 dB). For every participant, the Actiheart monitor was tested for adequate HR pickup by recording ECG waveforms for ⬃30 s before the rest test. If pickup was adequate, the Actiheart was set up to record HR and movement continuously for 1 wk, after which it was reloaded for the rest of the study period. Fat and fat-free tissue masses for the whole body, trunk, and extremities were measured using a dual-energy X-ray absorptiometry (DXA) scanner, Lunar Prodigy Advance (GE Healthcare, Madison, WI) (21). Hyperinsulinemic-euglycemic clamps were performed after an overnight fast (Fig. 1). Peripheral catheters were placed in an antecubital vein for blood sampling, in the contralateral antecubital vein for infusion of glucose, insulin, and stable isotopes, and in a dorsal hand vein for blood sampling; the catheterized hand was wrapped in a heating blanket to obtain arterialized venous blood for measurement of glucose and potassium during the clamp. The experiment was commenced by priming the glucose pool by administrating a bolus of 17.5 mol/kg of [6,6-2H2]glucose (Cambridge Isotopes Laboratories). This was followed by a continuous infusion (rate 0.4 mol·kg⫺1·min⫺1) for 2 h before the start of the clamp. Insulin (Actrapid, Novo Nordisk Insulin, 100 IE/ml) was infused continuously at a rate of 40.0 mU·min⫺1·m⫺2, and the plasma glucose concentration was kept at 5.0 mM by a coinfusion of glucose (200 g/1,000 ml, enriched with [6,6-2H2]glucose to 2.5%) at a variable rate. During the 4-h clamps the infusion rate of [6,6-2H2]glucose was reduced to 0.08 mol·kg⫺1·min⫺1. Arterialized blood was analyzed Fig. 1. Experimental design. Time lines of the study (A) and of the hyperinsulinemiceuglycemic clamp (B). V̇O2 max, maximal oxygen consumption; DXA, dual-energy X-ray absorptiometry. J Appl Physiol • VOL 108 • MAY 2010 • www.jap.org Downloaded from jap.physiology.org on March 25, 2011 parameters. None of the participants performed planned exercise sessions of ⬎2 h/wk or walked ⬍3,500 steps/day. Study design. In a free-living environment, participants were instructed to nightly record their daily steps (cycling not included) for 1 inclusion-week and then reduce steps to 1,500 per day for 14 days (cycling not allowed). Step number was measured using a simple pedometer (Yamax Digi-Walker SW-200, London, UK); daily physical activity was also monitored by an Actiheart monitor (Cambridge, UK). Subjects did not exercise more than 2 h/wk on a recreational level. Subjects who walked less than 3,500 steps per day or performed regular exercise were excluded. All subjects agreed to refrain from vigorous physical activity and only performed physical activity corresponding to their normal routines, for 2 days preceding the first test day. The number of steps was registered every night at bedtime. Dietary records were taken during the inclusion-week. The subjects carefully noticed what they consumed day by day, and they maintained their usual dietary habits during the whole study. Subjects were not allowed to consume alcohol 2 days preceding the first test and during the study period. Participants agreed to weigh and register consumed food the day before pre- and post-insulin clamp and the V̇O2 max tests. Figure 1 illustrates testing times. Determinations. An incremental exercise test was performed on a cycle ergometer (Monark 839E, Monark Ltd, Varberg, Sweden) to obtain V̇O2 max for each participant with indirect calorimetry system (Moxus modular V̇O2 system, AEI Technologies, Pittsburgh, PA) as previously described (1). Preceding the study, subjects performed a familiarization test. The Actiheart monitor [used to measure daily energy expenditure (7)] is attached to the chest with two standard electrocardiogram (ECG) electrodes and is able to measure acceleration, heart rate (HR), 1035 1036 AMBULATORY ACTIVITY AND INSULIN SENSITIVITY for glucose and potassium concentrations at intervals of 5 min during the first hour, and every 10 min during the last 3 h of the clamp. Blood samples were collected in heparin-containing tubes and immediately centrifuged for 15 min at 3,500 rpm, and the plasma was stored at ⫺20°C until further analysis. The plasma glucose enrichment was measured as previously described (23). The glucose turnover rate of appearance (Ra) and rate of disappearance (Rd) were calculated assuming steady state: baseline: Ra⫽Rd⫽ Finfusion Eglucose clamp: Ra共endogenous兲⫽ Ftotal Eglucose ⫺ GIR, Rd⫽ Ftotal Eglucose RESULTS Pedometer and Actiheart data. For a 2-wk period, participants reduced the number of daily steps by more than 85% J Appl Physiol • VOL Table 1. Correlation coefficients ⌬Daily Steps ⌬Physiological Variables r P value ⌬Daily energy expenditure, kJ/day ⌬V̇O2max, ml·kg⫺1·min⫺1 ⌬V̇O2max, ml/min ⌬Leg lean mass. kg ⌬Glucose infusion rate, mg·kg⫺1·min⫺1 0.922 0.921 0.960 0.262 ⫺0.225 0.001 ⬍0.01 0.001 NS NS Pearson correlation coefficients (r) are given between ⌬ in daily steps and ⌬ in physiological variables, where ⌬ represents a decrease from pre- to postintervention. No other but the mentioned factors correlated. V̇O2max, maximal oxygen consumption; NS, nonsignificant; n ⫽ 10. 108 • MAY 2010 • www.jap.org Downloaded from jap.physiology.org on March 25, 2011 where Finfusion is the infusion rate of glucose tracer (mol·kg⫺1·min⫺1) in terms of lean body mass; Eglucose is the enrichment of glucose in plasma; Ftotal is the sum of Finfusion; and the [6,6-2H2]glucose infused by the clamp; GIR is the glucose infusion rate; and kilograms refers to lean body mass. Baseline blood samples were obtained before initiation of the stable isotope infusion (time point ⫺2 h, Fig. 1). Glucose, triglyceride (TG), and free fatty acids (FFA) were measured with an automatic analyzer (Cobas Fara, Roche, Basel, Switzerland); insulin and C-peptide by ELISA (DAKO, Glostrup, Denmark); tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-6, IL-15, and leptin by ELISA (R&D Systems); and adiponectin by a RIA kit (LINCO Research). Vastus lateralis muscle biopsies were taken before and 1 and 4 h after the starting of the clamp. Muscle was quickly frozen in liquid nitrogen and stored at ⫺80°C until later analysis. Muscle tissue was homogenized in ice-cold buffer containing protease inhibitors and phosphatase inhibitors as previously referenced (13, 29). Insulin receptor  (IR), phosphorylation of Akt at threonine 308 (pAktthr308), total Akt, phosphorylation of AS160 (pAS160), and AS160 total content were performed as referenced previously (29). Regarding tyrosine phosphorylation of IR (pTyIR), muscle homogenate was incubated overnight with anti-phosphotyrosine agarose beads (Sigma). Both immunoprecipitates and muscle homogenates were separated by SDSPAGE and transferred to PVDF membranes before probing with appropriate antibodies. The protein bands were detected using Supersignal West femto (Pierce, Rockford, IL, USA) or enhanced chemiluminescence (Amersham Biosciences, UK) and quantified using a CCD image sensor and software (Quantity One, Bio-Rad, CA). Total RNA was extracted and analyzed from skeletal muscle using previously referenced methods (15). The primers and probes for TNF-␣ and 18S rRNA were predeveloped TaqMan probes and primer sets from Applied Biosystems (Foster City, CA). The relative expression of TNF-␣ was normalized to endogenous 18S. Statistical analyses. Data were tested for normal distribution by the Kolmogorov-Smirnov analysis. Variables were analyzed using parametric methods (paired t-tests; pre vs. post) on the absolute data, and reported values are means ⫾ SE. Correlations were determined with a Pearson correlation test. All Western blots were normally distributed when log-transformed before analysis and then analyzed using parametric methods and expressed as geometric mean (95% confidence interval). Within-subject variation over time and variation between trials were analyzed using repeated-measures (2-way ANOVA, timeby-trial). If a significant interaction (time ⫻ trial) was found, Bonferroni-corrected P values were obtained as appropriate to identify significant differences between trials and to identify significant differences from baseline values. P ⬍ 0.05 was considered statistically significant. Analysis was performed using a statistical software package (SAS version 9.1.3 SAS Institute). from a baseline of ⬃10,000 per day, as published previously (21), resulting in reduced energy expenditure (kJ/day; estimated by Actiheart) from a mean of 5,020 ⫾ 515 to 3,010 ⫾ 294 kJ/day. The decline in number of steps from baseline to 2 wk significantly correlated with the decline in daily energy expenditure (kJ/day) (r ⫽ 0.922; P ⫽ 0.001), and decline in V̇O2 max (ml/min) (r ⫽ 0.960; P ⫽ 0.001) or V̇O2 max (ml·min⫺1·kg⫺1) (r ⫽ 0.921; P ⬍ 0.01) (Table. 1). Cardiovascular fitness and body composition (DXA). A significant 7.2% and 6.6% decline was observed in V̇O2 max (ml/min and ml·min⫺1·kg⫺1, respectively) following reduced stepping (Table 2). Lean mass of legs, but not arms or trunk, also decreased significantly after reduced ambulatory activity (Table 2). The decline in number of steps from baseline to 2 wk did not correlate with the decline in lean mass of the legs (Table 1). There was no change in total fat mass (21). Metabolism. GIR during the 240-min clamp significantly declined 17% after 2 wk of reduced stepping (pretesting ⫽ 6.29 ⫾ 0.62 mg·kg⫺1·min⫺1; posttesting ⫽ 5.22 ⫾ 0.54 mg·kg⫺1·min⫺1) (Fig. 2A). Plasma glucose was kept at ⬃5.0 mM during the clamps (pretesting ⫽ 4.96 ⫾ 0.05 mM; posttesting 4.92 ⫾ 0.08 mM) (Fig. 2B). Reduced stepping also resulted in reduced insulin-stimulated glucose disappearance rate (Rd) (an index of decreased peripheral insulin sensitivity), but did not change endogenous hepatic glucose production rate (Ra) (Fig. 2C) or basal glucose turnover (data not shown). Calculations for glucose metabolism were performed during steady-state euglycemia (120 –240 min of clamp). There was no significant difference in baseline values preand postintervention with regard to plasma levels of TG, FFA, glucose, insulin, and C-peptide (Table 2). The decline in number of steps from baseline to 2 wk did not correlate with the decline in GIR (Table 1). Insulin signaling. Insulin stimulation increased ratios of phosphorylated to total protein for both Akt protein (pAktthr308/ total Akt) and AS160 protein (pAS160/total AS160) in the vastus lateralis muscle at 1 and 4 h of the clamp compared with baseline (time point 0 h) (P ⬍ 0.05 for all comparisons), but not for IR protein (pTyIR/total IR) (Fig. 3). Reduced stepping led to a significant reduction in insulin-stimulated pAktthr308/total Akt (pre- vs. postintervention) at the 4th hour of clamp (P ⫽ 0.03) (Fig. 3) and although pAS160/total AS160 and pTyIR/total IR signaling tended to decrease from prevs. postintervention, the changes were not significant (Fig. 3). Inflammation. Baseline (time point ⫺2 h) plasma levels of TNF, IL-6, IL-15, leptin, and adiponectin (Table 2) and skel- AMBULATORY ACTIVITY AND INSULIN SENSITIVITY Table 2. Pre- and post-2-wk intervention results Measurement Postintervention Mean SE Mean 3,435 47.7 259 194 149 1.3 11 3 3,194 45.1 243 193 70.9 26.3 6.7 18.6 1.9 0.8 0.3 0.5 69.7 25.6 6.7 18.1 SE 130 1 11 4 2 0.6 0.3 0.5 5.39 0.11 5.26 0.11 31.9 3.7 31.8 3.6 337.6 27.5 335.4 22.6 1,001 199 1,266 430 617 56 647 47 1.31 0.13 1.38 0.16 0.79 0.22 1.02 0.19 1.29 0.1 1.27 0.2 5,056 803 4,841 812 2,334 389 2,676 410 P Value ⬍0.01 ⬍0.01 ⬍0.01 NS ⬍0.001 NS NS ⬍0.001 NS NS NS NS NS NS NS NS NS NS V̇O2max test results, dual-energy X-ray absorptiometry (DXA) scans, and plasma concentrations on lipid and glucose metabolism as well as inflammation are shown. Total lean mass decreased significantly post-2 wk intervention vs. preintervention (see Ref. 21). HR, heart rate, FFA, free fatty acids; TG, triglycerides. n ⫽ 10. etal muscle TNF mRNA and protein expression (data not shown) were not affected by 2 wk of reduced daily stepping. DISCUSSION Major novel findings of this study were that 2 wk of reduced daily ambulatory activity in a free-living condition resulted in decreases in insulin-stimulated muscle Akt phosphorylation, peripheral insulin sensitivity, leg lean mass, and V̇O2 max in young healthy men who had not been previously exercise trained. As shown by others in previous studies, the later three aforementioned decreases are primary decrements that can lead to chronic metabolic disorders and premature mortality. Importantly, the experimental design, used here, models the extremes [⬃2,000 to ⬃12,000 (4)] in numbers of daily steps in the majority of free-living adults in developed countries, most of whom do not partake in structured exercise programs (4). Therefore, the present study is distinctly different from previous studies in which nontypical and extreme changes in activity levels were deployed including continuous bed rest in which subjects are not allowed to stand or walk (8) and detraining studies in which intense structured-exercise programs ceased (2, 18). Furthermore, subjects served as their own control, eliminating potential gene differences that might occur by comparing separate groups of active and less active subjects. The attenuation of peripheral insulin sensitivity after 2 wk of the decreased ambulatory activity occurred without any detectable alteration in endogenous hepatic glucose production, implying that a peripheral decrement in insulin sensitivity is primary to hepatic insulin resistance on reductions in daily stepping. These findings further reinforce the dogma that glucose entry is exquisitely matched with substrate usage in skeletal muscle, in part by modulating muscle insulin sensitivJ Appl Physiol • VOL ity. A remaining question to be answered is whether reduced ambulatory activity either increases susceptibility to or is a necessary component of insulin resistance. There is strong support for the latter suggestion as high-fat diet-induced insulin resistance or obesity-associated insulin resistance does not normally occur in skeletal muscle when a sufficient level of increased physical activity or acute exercise is imposed (19, 22, 26, 29), and epidemiological evidence clearly shows that reduced physical activity is a major risk for Type 2 diabetes while high activity levels is protective (11, 12, 17). Previous studies have shown that acute, 7-day exercise protocols dramatically improve insulin sensitivity (10, 27). However, the ability of increased physical activity (7 days of exercise) to improve insulin sensitivity is blocked if subjects are maintained in an energy balance by consuming postworkout calories that match the amount of calories expended in each daily exercise bout (3). This evidence strongly suggests that the ability of increased activity to improve insulin action is linked to acutely induced energy deficits. Because the subjects in the present study maintained the same caloric intake but dramatically lowered their daily activity it is likely that they were in a state of positive energy balance. This concept is contradicted by the evidence that subjects had a 1.6% reduction in total body mass. That being said, if the subjects were in a positive energy balance, it is likely that this excess energy played a role in the loss of peripheral insulin sensitivity after reduced stepping. A followup study could examine this link by having one group of subjects consume a reduced caloric intake that matches the reduction in energy expenditure that occurs after reducing daily stepping from 12,000 to 2,000 steps. This type of design would not only help to define the role of energy balance on insulin resistance induced by reduced stepping but would also help to determine the impact of positive energy balance on the loss of total body mass and lean body mass that was witnessed. Although we provide novel evidence that acutely reducing ambulatory activity negatively impacts insulin sensitivity, previous examinations have found that exercise cessation in endurance athletes has a similar impact. Heath et al. (9) showed that 10 days of exercise cessation in endurance athletes dramatically increased insulin responses to an OGTT. A similar result was witnessed in master endurance athletes (⬃61 yr old) who ceased exercise training for 10 days (25). King et al. (14) showed that endurance athletes who cease training for 10 days have a reduction in peripheral insulin sensitivity measured by hyperinsulinemic-euglycemic clamp. They further determined that this was due to a loss of insulin sensitivity and not a total loss of insulin responsiveness as a much larger infusion of insulin actually sustained insulin sensitivity after 10 days of exercise cessation. In a study from another group, the cessation of exercise in endurance athletes for only 60 h lowered insulin sensitivity to what was measured in sedentary age-matched controls (6). Similar results have been found in rodents. Kump and Booth (16) have shown that rats allowed daily access to voluntary running wheels for 3 wk (average daily run distance equaled 5.7 km) display rapid losses of insulin-stimulated glucose uptake and insulin signaling in skeletal muscle after their daily running was inhibited by wheel lock for only ⬃2 days (53 h). Although the evidence that exercise cessation or reduced ambulatory activity leads to a rapid decline in skeletal muscle 108 • MAY 2010 • www.jap.org Downloaded from jap.physiology.org on March 25, 2011 V̇O2max test V̇O2max, ml/min V̇O2max, ml·min⫺1·kg⫺1 Maximal power, W Maximal HR, beats/min DXA scan Total body mass, kg Trunk lean mass, kg Arm lean mass, kg Leg lean mass, kg Plasma Glucose, mmol/l Insulin, pmol/l C-peptide, pmol/l FFA, mmol/l TG, mol/l TNF, pg/ml IL-6, pg/ml IL-15, pg/ml Adiponectin, ng/ml Leptin, pg/ml Preintervention 1037 1038 AMBULATORY ACTIVITY AND INSULIN SENSITIVITY insulin sensitivity in human and animal models is very strong, it remains underappreciated as an initiator of metabolic disease and an underused model to study initial declines in insulin sensitivity that likely precede overt insulin resistance. The most common experimental model for studying insulin resistance is acute and long-term high-fat diets, which, as we have already stated, appear to be effective in sedentary conditions only. A recent study by Brons et al. (5) showed that a 5-day Fig. 3. Insulin signaling in skeletal muscle during hyperinsulinemic-euglycemic clamp. Akt, AS160, and insulin receptor (IR)- protein content and phosphorylation (p) were examined in biopsies taken from vastus lateralis muscle pre- and post-euglycemic clamp. Muscle biopsies were obtained at time points 0 h (before insulin clamp) and 1 h and 4 h (after initiation of insulin clamp); labels on x-axis indicate these times. Labels on y-axis indicate: phosphorylation of IR beta expressed relative to IR protein (A); phosphorylation of Akt at threonine308 expressed relative to Akt protein (B); phosphorylation of AS160 expressed relative to AS160 protein (C); and representative immunoblots of signaling proteins. pTYIR, tyrosine phosphorylated IR. Data are expressed as geometric means (95% confidence interval). *Post hoc analysis, P ⫽ 0.03 (difference between pre- and postintervention). **Post hoc analysis, P ⬍ 0.05 (different from 0 h). n ⫽ 10. J Appl Physiol • VOL 108 • MAY 2010 • www.jap.org Downloaded from jap.physiology.org on March 25, 2011 Fig. 2. Glucose values during hyperinsulinemic-euglycemic clamp. A: glucose infusion rate (GIR) during the euglycemic clamp. B: blood glucose concentration just before (time point 0) and during the euglycemic clamp. C: area under the curve (AUC) for glucose rate of appearance (Ra) and rate of disappearance (Rd) during the euglycemic clamp (120 –240 min). *Significant difference between preintervention (Pre) and postintervention (Post) (paired t-test, P ⫽ 0.01) Preintervention is before reduced daily steps; postintervention is final 3 days of 14-day period of reduced daily steps. , Preintervention determination; , postintervention determination; filled bars, preintervention determination; open bars, postintervention determination. Data are presented as mean ⫾ SEM. n ⫽ 10. AMBULATORY ACTIVITY AND INSULIN SENSITIVITY ACKNOWLEDGMENTS All authors have contributed to the conception and design, or the analysis and interpretation of data; the drafting of the article or revising of the article; and the final approval of the version of the article to be published. Ruth Rousing and Hanne Villumsen are acknowledged for technical assistance. GRANTS The study was supported by the Commission of the European Communities (contract no. LSHM-CT-2004-005272 EXGENESIS) and by grants from the Augustinus Foundation, The Novo Nordisk Foundation, and from Unilever. R. H. Olsen received a scholarship from the Danish Research Council. The Centre of Inflammation and Metabolism is supported by a grant from the Danish National Research Foundation (DG 02-512-555). DISCLOSURES No conflicts of interest are declared by the authors. J Appl Physiol • VOL REFERENCES 1. Akerstrom T, Steensberg A, Keller P, Keller C, Penkowa M, Pedersen BK. Exercise induces interleukin-8 expression in human skeletal muscle. J Physiol 563: 507–516, 2005. 2. Arciero PJ, Smith DL, Calles-Escandon J. Effects of short-term inactivity on glucose tolerance, energy expenditure, and blood flow in trained subjects. J Appl Physiol 84: 1365–1373, 1998. 3. Black SE, Mitchell E, Freedson PS, Chipkin SR, Braun B. Improved insulin action following short-term exercise training: role of energy and carbohydrate balance. J Appl Physiol 99: 2285–2293, 2005. 4. Bravata DM, Smith-Spangler C, Sundaram V, Gienger AL, Lin N, Lewis R, Stave CD, Olkin I, Sirard JR. Using pedometers to increase physical activity and improve health: a systematic review. JAMA 298: 2296 –2304, 2007. 5. Brons C, Jensen CB, Storgaard H, Hiscock NJ, White A, Appel JS, Jacobsen S, Nilsson E, Larsen CM, Astrup A, Quistorff B, Vaag A. Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men. J Physiol 587: 2387–2397, 2009. 6. Burstein R, Polychronakos C, Toews CJ, MacDougall JD, Guyda HJ, Posner BI. Acute reversal of the enhanced insulin action in trained athletes. Association with insulin receptor changes. Diabetes 34: 756 –760, 1985. 7. Crouter SE, Churilla, Jr, Bassett DR Jr. Accuracy of the Actiheart for the assessment of energy expenditure in adults. Eur J Clin Nutr 62: 704 –711, 2008. 8. Greenleaf JE, Kozlowski S. Physiological consequences of reduced physical activity during bed rest. Exerc Sport Sci Rev 10: 84 –119.: 84 –119, 1982. 9. Heath GW, Gavin JR, III, Hinderliter JM, Hagberg JM, Bloomfield SA, Holloszy JO. Effects of exercise and lack of exercise on glucose tolerance and insulin sensitivity. J Appl Physiol 55: 512–517, 1983. 10. Houmard JA, Cox JH, MacLean PS, Barakat HA. Effect of short-term exercise training on leptin and insulin action. Metabolism 49: 858–861, 2000. 11. Hu FB, Leitzmann MF, Stampfer MJ, Colditz GA, Willett WC, Rimm EB. Physical activity and television watching in relation to risk for type 2 diabetes mellitus in men. Arch Intern Med 161: 1542–1548, 2001. 12. Hu G, Lindstrom J, Valle TT, Eriksson JG, Jousilahti P, Silventoinen K, Qiao Q, Tuomilehto J. Physical activity, body mass index, and risk of type 2 diabetes in patients with normal or impaired glucose regulation. Arch Intern Med 164: 892–896, 2004. 13. Iglay HB, Thyfault JP, Apolzan JW, Campbell WW. Resistance training and dietary protein: effects on glucose tolerance and contents of skeletal muscle insulin signaling proteins in older persons. Am J Clin Nutr 85: 1005–1013, 2007. 14. King DS, Dalsky GP, Clutter WE, Young DA, Staten MA, Cryer PE, Holloszy JO. Effects of exercise and lack of exercise on insulin sensitivity and responsiveness. J Appl Physiol 64: 1942–1946, 1988. 15. Krogh-Madsen R, Plomgaard P, Akerstrom T, Moller K, Schmitz O, Pedersen BK. Effect of short-term intralipid infusion on the immune response during low-dose endotoxemia in humans. Am J Physiol Endocrinol Metab 294: E371–E379, 2008. 16. Kump DS, Booth FW. Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise. J Physiol 562: 829 –838, 2005. 17. Laaksonen DE, Lindstrom J, Lakka TA, Eriksson JG, Niskanen L, Wikstrom K, Aunola S, Keinanen-Kiukaanniemi S, Laakso M, Valle TT, Ilanne-Parikka P, Louheranta A, Hamalainen H, Rastas M, Salminen V, Cepaitis Z, Hakumaki M, Kaikkonen H, Harkonen P, Sundvall J, Tuomilehto J, Uusitupa M. Physical activity in the prevention of type 2 diabetes: the Finnish diabetes prevention study. Diabetes 54: 158 –165, 2005. 18. Mikines KJ, Sonne B, Tronier B, Galbo H. Effects of acute exercise and detraining on insulin action in trained men. J Appl Physiol 66: 704–711, 1989. 19. Morris RT, Laye MJ, Lees SJ, Rector RS, Thyfault JP, Booth FW. Exercise-induced attenuation of obesity, hyperinsulinemia, and skeletal muscle lipid peroxidation in the OLETF rat. J Appl Physiol 104: 708–715, 2008. 20. Newman AB, Kupelian V, Visser M, Simonsick EM, Goodpaster BH, Kritchevsky SB, Tylavsky FA, Rubin SM, Harris TB. Strength, but not muscle mass, is associated with mortality in the health, aging and body composition study cohort. J Gerontol A Biol Sci Med Sci 61: 72–77, 2006. 21. Olsen RH, Krogh-Madsen R, Thomsen C, Booth FW, Pedersen BK. Metabolic responses to reduced daily steps in healthy nonexercising men. JAMA 299: 1261–1263, 2008. 108 • MAY 2010 • www.jap.org Downloaded from jap.physiology.org on March 25, 2011 high-fat/high-calorie diet in young healthy men (daily activity levels were not reported) induced hepatic but not peripheral insulin resistance measured by a hyperinsulinemic-euglycemic clamp. This suggests that diet-induced insulin resistance may initially be activated through hepatic disturbances while reductions in activity lead to declines in skeletal muscle insulin sensitivity. Type 2 diabetes has been linked to both increased consumption of dietary fats and a sedentary lifestyle, making it plausible that both of these lifestyle factors work in parallel to impair both hepatic and skeletal muscle insulin sensitivity and are an underlying feature of the dramatic increase in rates of Type 2 diabetes. The loss of 2.8% in leg lean mass with 2 wk of reduced daily ambulatory activity was unanticipated as most literature suggests that reduced muscle loading must be drastic, such as limb immobilization or bed rest, to observe muscle atrophy. Thus it is likely that decreases in leg lean mass and in the rate of glucose disappearance per kilogram of whole body lean mass (Fig. 2C) both contribute to decreased glucose infusion rate in a hyperinsulinemic-euglycemic clamp (Fig. 2A) after 2 wk of reduced ambulatory activity. A 7% suppression of cardiovascular fitness (V̇O2 max) after lowered ambulatory activity was also unanticipated based on our notion that the loss in ambulatory activity would be of insufficient magnitude and duration to produce a loss. Because of the previously established strong connection between reduced cardiorespiratory fitness and premature mortality, the rapid decline in V̇O2 max is clinically significant in these young, healthy subjects after only 2 wk of reduced stepping. The parallel losses of peripheral insulin sensitivity, leg lean mass, and V̇O2 max are clinically relevant as all three independently increase mortality. While low cardiovascular fitness is the best predictor of mortality (28), low skeletal muscle strength also increases mortality (20), and insulin resistance is believed to be a primary initiator of many chronic diseases (24), which also increase mortality. Taken together, we speculate that reduced ambulatory activity may be primary and predispose to other environmental-gene interactions, especially in at-risk populations such as overweight, elderly, or genetically predisposed humans, that lead to the early development of chronic diseases and/or premature mortality. Chronic diseases take years to become overtly clinical. Our observations suggest that reductions in daily steps or physical activity may initiate or contribute to progressive declines in metabolic function. 1039 1040 AMBULATORY ACTIVITY AND INSULIN SENSITIVITY 22. Pauli JR, Ropelle ER, Cintra DE, Carvalho-Filho MA, Moraes JC, DE Souza CT, Velloso LA, Carvalheira JB, Saad MJ. Acute physical exercise reverses S-nitrosation of the insulin receptor, insulin receptor substrate 1 and protein kinase B/Akt in diet-induced obese Wistar rats. J Physiol 586: 659 –671, 2008. 23. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, Pedersen BK. Tumor necrosis factor-␣ induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 54: 2939 –2945, 2005. 24. Reaven GM. The insulin resistance syndrome: definition and dietary approaches to treatment. Annu Rev Nutr 25: 391– 406.: 391– 406, 2005. 25. Rogers MA, King DS, Hagberg JM, Ehsani AA, Holloszy JO. Effect of 10 days of physical inactivity on glucose tolerance in master athletes. J Appl Physiol 68: 1833–1837, 1990. 26. Ropelle ER, Pauli JR, Prada PO, DE Souza CT, Picardi PK, Faria MC, Cintra DE, Fernandes MF, Flores MB, Velloso LA, Saad MJ, Carvalheira JB. Reversal of diet-induced insulin resistance with a single bout of exercise in the rat: the role of PTP1B and IRS-1 serine phosphorylation. J Physiol 577: 997–1007, 2006. 27. Solomon TP, Haus JM, Kelly KR, Cook MD, Riccardi M, Rocco M, Kashyap SR, Barkoukis H, Kirwan JP. Randomized trial on the effects of a 7-d low-glycemic diet and exercise intervention on insulin resistance in older obese humans. Am J Clin Nutr 90: 1222–1229, 2009. 28. Sui X, LaMonte MJ, Laditka JN, Hardin JW, Chase N, Hooker SP, Blair SN. Cardiorespiratory fitness and adiposity as mortality predictors in older adults. JAMA 298: 2507–2516, 2007. 29. Thyfault JP, Cree MG, Zheng D, Zwetsloot JJ, Tapscott EB, Koves TR, Ilkayeva O, Wolfe RR, Muoio DM, Dohm GL. Contraction of insulin-resistant muscle normalizes insulin action in association with increased mitochondrial activity and fatty acid catabolism. Am J Physiol Cell Physiol 292: C729 –C739, 2007. Downloaded from jap.physiology.org on March 25, 2011 J Appl Physiol • VOL 108 • MAY 2010 • www.jap.org