Scholarly article on topic 'Two weeks of moderate intensity continuous training, but not high intensity interval training increases insulin-stimulated intestinal glucose uptake'

Two weeks of moderate intensity continuous training, but not high intensity interval training increases insulin-stimulated intestinal glucose uptake Academic research paper on "Medical engineering"

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Academic research paper on topic "Two weeks of moderate intensity continuous training, but not high intensity interval training increases insulin-stimulated intestinal glucose uptake"

Articles in PresS. J Appl Physiol (February 9, 2017). doi:10.1152/japplphysiol.00431.2016

1 Two weeks of moderate intensity continuous training, but not high

2 intensity interval training increases insulin-stimulated intestinal glucose

3 uptake

1 1 1 1 123 1

4 Kumail K. Motiani , Anna M. Savolainen ,Jari-Joonas Eskelinen , Jussi Toivanen , Tamiko Ishizu ' ' , Minna Yli-Karjanmaa ,

5 Kirsi A. Virtanen4, Riitta Parkkola5, Jukka Kapanen6, Tove J. Gronroos1,2, Merja Haaparanta-Solin1, Olof Solin7, Nina

6 Savisto1, 8Markku Ahotupa, Eliisa Loyttyniemi9, Juhani Knuuti1, Pirjo Nuutila1,10, Kari K. Kalliokoski1, Jarna C.

7 Hannukainen1

8 1Turku PET Centre, University of Turku, Turku, Finland

9 2Medicity Research Laboratory, University of Turku, Turku, Finland

10 3Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland

11 4Turku PET Centre, Turku University Hospital, Turku, Finland

12 5Department of Radiology, Turku University Hospital, Turku, Finland

13 6Paavo Nurmi Centre, Turku, Finland

14 7Turku PET Centre, Abo Akademi University, Turku, Finland

15 8Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland

16 9Department of Biostatistics, University of Turku, Finland

17 10Department of Endocrinology, Turku University Hospital, Turku, Finland

19 Short Title: Exercise training and intestinal metabolism

20 Financial support:

21 This study was conducted within the Centre of Excellence in Cardiovascular and Metabolic Diseases and supported by

22 the Academy of Finland, the University of Turku, Turku University Hospital, and Abo Akademi University. The study was

23 financially supported by the European Foundation for the Study of Diabetes, the Emil Aaltonen Foundation, the Hospital

24 District of Southwest Finland, the Orion Research Foundation, the Finnish Diabetes Foundation, the Ministry of

25 Education of the State of Finland, the Academy of Finland (grants 251399, 251572, 256470, 281440, and 283319), the

26 Paavo Nurmi Foundation, the Novo Nordisk Foundation and the Centre of Excellence funding.

27 Corresponding author and person to whom reprint requests should be addressed:

Copyright © 2017 by the American Physiological Society.

28 Jarna C. Hannukainen, PhD

29 Turku PET Centre

30 University of Turku

31 Turku P.O. Box 52, FIN-20521, Finland.

32 Tel: +35823131878

33 Fax: +35822318191

34 Email: jarna.hannukainen@tyks.fi

36 Keywords: Intestine, intestinal metabolism, high intensity interval training, moderate intensity continuous training,

37 exercise, positron emission tomography.

38 Conflict of interest: No conflict of interest

ABSTRACT

41 Similar to muscles, the intestine is also insulin resistant in obese subjects and subjects with impaired glucose tolerance.

42 Exercise training improves muscle insulin sensitivity, but its effects on intestinal metabolism are not known. We studied

43 the effects of high intensity interval training (HIIT) and moderate intensity continuous training (MICT) on intestinal

44 glucose and free fatty acid uptake from circulation in humans. Twenty-eight healthy middle-aged sedentary men were

45 randomized for two weeks of HIIT or MICT. Intestinal insulin-stimulated glucose uptake and fasting free fatty acid uptake

46 from circulation were measured using positron emission tomography and [18F]FDG and [18F]FTHA. In addition, effects of

47 HIIT and MICT on intestinal Glut2 and CD36 protein expression were studied in rats. Training improved aerobic capacity

48 (p=0.001) and whole-body insulin sensitivity (p=0.04), but not differently between HIIT and MICT. Insulin-stimulated

49 glucose uptake increased only after the MICT in the colon [HIIT=0%; MICT=37%] (p=0.02 for time*training) and tended

50 to increase in the jejunum [HIIT=-4%; MICT=13%] (p=0.08 for time*training). Fasting free fatty acid uptake decreased in

51 the duodenum in both groups [HIIT=-6%; MICT=-48%] (p=0.001 time) and tended to decrease in the colon in the MICT

52 group [HIIT=0%; MICT=-38%] (p=0.08 for time*training). In rats, both training groups had higher Glut2 and CD36

53 expression compared to control animals. This study shows that already two weeks of MICT enhances insulin-stimulated

54 glucose uptake while both training modes reduce fasting free fatty acid uptake in the intestine in healthy middle-aged

55 men, providing an additional mechanism by which exercise training can improve whole body metabolism.

56 New & Noteworthy

57 This is the first study where the effects of exercise training on the intestinal substrate uptake have been investigated

58 using the most advanced techniques available. We also show the importance of exercise intensity in inducing these

59 changes.

INTRODUCTION

61 The intestine is a large organ and a major determinant of whole body energy homeostasis through its control

62 over nutrient absorption and release of gut hormones during digestion (6). Evidence demonstrating the potential role of

63 the intestine in the pathogenesis of obesity and insulin resistance is rapidly increasing. In type 2 diabetes, there is a

64 continuous deterioration of intestinal endocrine function (16) and alterations in the intestinal microbiota content have

65 been shown to be associated with the development of insulin resistance in humans and animals (8; 9; 26). Splanchnic

66 glucose uptake (SGU) accounts up to 60 % of total glucose metabolism after an oral glucose load. In insulin resistance

67 splanchnic glucose uptake is impaired and plays a role in the pathogenesis of hyperglycaemia in type 2 diabetes.(10;

68 27)We have previously shown that tissue-specific intestinal glucose uptake from circulation into enterocytes is impaired

69 in insulin stimulated state, i.e. intestinal insulin resistance exists, in obese and type 2 diabetic subjects (29). The role of

70 intestinal insulin resistance in the pathology of type 2 diabetes is unclear, however, it has been suggested that intestinal

71 insulin resistance leads to abnormalities in the signalling mechanism responsible for the Glut2 mediated glucose uptake

72 in the small intestine, particularly in the jejunum, leading to increased transepithelial or lumen to blood glucose

73 exchange, causing hyperglycemia (3).

74 Regular exercise training enhances skeletal muscle insulin sensitivity (11; 20; 23; 35) in working muscles.

75 Exercise training also enhances the regulation and utilization of lipids in the skeletal muscle (13; 19; 22; 42). The training-

76 induced adaptations in muscle substrate metabolism and oxidative capacity lead to improvements in the whole body

77 metabolism and insulin sensitivity. Although muscle is widely studied, previous data about the effects of exercise on

78 abdominal organs concerns mainly on liver and pancreas and data is limited about the effects of exercise on intestine

79 (28; 33; 33; 36). Thus, it is not known whether exercise training could enhance intestinal substrate metabolism, and

80 whether any possible changes would be reflected in the insulin sensitivity of the whole body.

81 We have previously shown that two weeks of low volume high intensity interval training (HIIT) and moderate

82 intensity training (MICT) increases both aerobic capacity and whole body and main working skeletal muscle insulin-

83 stimulated glucose uptake (GU) in sedentary middle-aged men (7). In the present study, using the intestine data from

84 this same clinical trial (NCT01344928) our aim was to quantify the effects of exercise on tissue-specific insulin-stimulated

85 glucose and fasting free fatty acid uptake (FAU) from circulation into the intestine (duodenum, jejunum and colon) using

86 positron emission tomography (PET) and radiotracers 2-[18F]fluoro- 2-deoxy-D-glucose (FDG) and 14(R,S)-[18F]fluoro-6-

87 thia-heptadecanoic acid (FTHA) before and after HIIT and MICT. We hypothesized that the higher training volume

88 instead of the intensity would strain the intestinal metabolism more and thus lead to the increased intestinal insulin-

89 stimulated GU and decreased FAU after MICT compared to HIIT. Additionally, to explore possible mechanisms behind

90 the changes in intestinal GU and FFAU, we also studied healthy Wistar rats, which underwent corresponding HIIT and

91 MICT interventions and analysed the intestinal protein expression of Glut2 and CD36. We hypothesized that training

92 would increase the expression of Glut2 and CD 36 in enterocytes more after MICT than HIIT.

94 MATERIALS AND METHODS

95 Subjects

96 Twenty-eight, middle-aged sedentary individuals were recruited and randomized into two groups; one with two weeks

97 of high intensity interval training (HIIT) and the other with two weeks of moderate intensity continuous training (MICT).

98 The subjects were non-obese (aged 40-55 years, VO2peak < 40 ml-kg-1'min-1) and had no previous experience of active

99 exercise training. The inclusion and exclusion criteria of the recruitment process have been described in detail previously

100 (24). Two of the subjects withdrew during the intervention, one from the HIIT group due to exercise-induced hip pain

101 and one from MICT group due to personal reasons; this left thirteen subjects in each group. The purpose, nature, and

102 potential risks involved in participating in the study were explained in detail and informed consent was obtained before

103 any measurements were performed. The study was approved (NCT01344928) by the local ethical committee of the

104 Hospital District of South-Western Finland (decision 95/180/2010 §228) and carried out in compliance with the

105 Declaration of Helsinki.

107 Study design

108 Initial screening included a physical examination, an oral glucose tolerance test (OGTT), and a VO2peak test to assess the

109 participant's health, glycemic status, and aerobic capacity. The participants then underwent two PET imaging sessions on

110 two different days. On the first day 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA) and PET was used to

111 measure, under a fasting state, the free fatty acid uptake in different intestinal regions (duodenum, jejunum, and colon)

112 and the quadriceps femoris (QF) and deltoid muscles (the muscle results were taken from our previous publication (10)).

113 On the second day 2-[18F] fluoro-2-deoxy-D-glucose ([18F]FDG) and PET was used to measure the insulin-stimulated

114 glucose uptake in the intestine and the muscles during hyperinsulinemia. Once again the muscle results used were from

115 our previous publication (10). An overnight fast of at least 10 hours was required before the OGTT and PET

116 measurements. Participants were also asked to abstain from any caffeinated and alcoholic beverages, and to avoid

117 strenuous exercise 48 hours prior to these studies. After the two weeks exercise training intervention, all measurements

118 were repeated starting with [18F]FTHA PET 48 hours after the last exercise session and continuing with a [18F]FDG PET

119 post 72 hours and finally an OGTT and VO2peak test were done post 96 hours (Fig. 1).

121 Exercise interventions

122 Participants were randomized into HIIT and MICT exercise groups and both training groups had six supervised training

123 sessions within two weeks. Each HIIT session consisted of 4-6 x 30 s exercise bouts of all out cycling efforts (Wingate

124 protocol, load 7.5 % of the whole body weight, Monark Ergomedic 828E, Monark, Vansbro, Sweden) with 4 mins of

125 recovery in between the exercise bouts (5). All the participants were familiarized with the HIIT training protocol (2 x 30 s

126 bouts) before they were randomized into training groups. MICT training consisted of 40-60 min of cycling at a moderate

127 intensity (60 % of VO2peak intensity). In both groups, the training was progressive and in the HIIT group the number of

128 cycling bouts increased from 4 to 5 and finally to 6, and in the MICT group the training time increased from 40 to 50 and

129 then to 60 min in every second training session.

131 PET scans

132 Participants underwent four PET sessions: one [18F]FTHA PET and one [18F]FDG PET before and after the training

133 intervention. Antecubital veins of both arms were cannulated for the PET studies. One catheter was used to inject the

134 radiotracers [18F]FTHA and [18F]FDG while the other one was for blood sampling. To arterialize the venous blood samples

135 the arm was heated using an electronically powered cushion. On the first PET scan session, intestinal free fatty acid

136 uptake was measured using [18F]FTHA PET in a fasting state. [18F]FTHA radiotracer (155 [SEM 0.4] MBq) was injected and

137 dynamic imaging of the abdominal region (frames 3x300 sec) was acquired starting on average at 46 minutes after the

138 tracer injection. This was followed by a femoral region scanning (quadriceps femoris) (frames 3x300sec), starting

139 approximately 65 min after the tracer injection. Finally, the shoulder region (deltoid) (frames 3x300 sec) was scanned

140 starting approximately 90 min after the tracer injection. On the second day intestinal glucose uptake was measured

141 using [18F]FDG under euglycemic hyperinsulinemic clamp. On average 87 [SEM 1] minutes after the start of the clamp

142 [18F]FDG (156[SEM 0.5] MBq) was injected and similar time frames were acquired as described earlier for [18F]FTHA

143 scans, starting at 49, 70, and 90 minutes after the tracer injection. Arterialized blood samples were obtained at regular

144 intervals during both the [18F]FTHA and [18F]FDG scans to measure the plasma radioactivity in order to calculate the

145 tracer input function. . An automatic gamma counter (Wizard 1480, Wallac, Turku, Finland) was used to measure the

146 plasma radioactivity. A GE Discovery TM ST system (General Electric Medical Systems, Milwaukee, WI, USA) was used to

147 acquire the PET/CT images. CT images were acquired for anatomical references.

149 Image analysis

150 The imaging data obtained from the PET scanner was corrected for dead time, decay, and photon attenuation and the

151 images were reconstructed using the 3D-OSEM method. Carimas 2.7 (www.pet.fi/carimas) was used to manually draw

152 the regional tubular three-dimensional regions of interest (ROIs) on sections of the descending duodenum, the jejunum,

153 and the transverse colon, using CT images as anatomical reference. The tubular ROIs were carefully drawn to outline the

154 intestinal wall while avoiding the intestinal contents and external metabolically active tissues (17). From these regional

155 (duodenum, jejunum, and colon) ROI's time activity curves (TAC) were extracted.

156 The rate constant (Ki) for the uptake of the radiotracer ([18F]FTHA, [18F]FDG) into the cells was calculated using tissue

157 TACs obtained from the duodenum, jejunum, and colon and a tracer input function using a fractional uptake rate (FUR)

158 method as previously described (17). Regional glucose and free fatty acid uptakes were calculated by multiplying region

159 specific Ki by the corresponding plasma glucose or free fatty acid concentration respectively. For glucose uptake the

160 products were further divided by a lumped constant (LC) of 1.15 (17) and a recovery coefficient of 2.5 (17) was applied

161 for the colonic glucose uptake to take into account the partial volume effect (4; 25). For the duodenal and jejunal

162 glucose uptake, no recovery coefficient was needed. The ROI's for the deltoid and quadriceps femoris muscles were

163 drawn as explained previously (7).

165 Maximal exercise test

166 As previously described (24) the maximal oxygen uptake (VO2peak) was determined by performing an incremental bicycle

167 ergometer test (Ergoline 800s, VIASYS Healthcare, USA) with direct respiratory measurements using a ventilation and

168 gas exchange (Jaeger Oxycon Pro, VIASYS Helthcare, Germany) at the Paavo Nurmi Centre (Turku, Finland). Initial

169 exercise intensity was 50 W and after every two minutes the exercise intensity was increased by 30 W until volitional

170 exhaustion. VO2peak was expressed as the highest 1 min mean oxygen consumption. The workload at the last two

171 minutes of the test was averaged and used as a measure for maximal performance. The peak respiratory exchange ratio

172 was >1.15 and peak blood lactate concentration, measured from capillary samples obtained immediately and 1 min after

173 exhaustion (YSI 2300 Stat Plus, YSI Incorporated Life Sciences, USA), was >8.0 mmol-L-1 for all the tests. A peak heart

174 rate (HR) (RS800CX, Polar Electro Ltd., Kempele, Finland) within 10 beats of the age-appropriate reference value (220 -

175 age) was true in all except one participant in the both groups and in both pre- and post-training tests. Therefore, the

176 highest value of oxygen consumption was expressed as VO2peak and not VO2max.

178 The euglycemic hyperinsulinemic clamp

179 The euglycemic hyperinsulinemic clamp technique was used as previously described (7; 39). Insulin was infused at a rate

180 of lmU/kg/min (Actrapid; Novo Nordisk, Copenhagen, Denmark) and blood samples were taken every 5-10 min to adjust

181 the exogenous glucose infusion and to maintain the plasma glucose concentration as closely as possible to the level of 5

182 mmol/l. Euglycemic hyperinsulinemic clamp was performed after the subjects had fasted at least for 10 h. Insulin

183 (Actrapid, 100 U/ml, Novo Nordisk, Bagsvaerd, Denmark) infusion was started with the rate of 40 mU/min/m2 during the

184 first 4 min. After 4 min and up to 7 min, infusion rate was reduced to 20 mU/min/m2, and, after 7 min to the end of the

185 clamp, it was kept constant at 10 mU/min/m2. Glucose infusion was started 4 min after the start of the insulin infusion

186 with a rate of subject's weight (kg)-0.1-1-g-1-h-1. At 10 min, glucose infusion was doubled, and after that further adjusted

187 according to plasma glucose levels to maintain the steady state level of 5 mmol/l. Arterialized venous blood samples

188 were collected before the clamp and every 5-10 min to measure the plasma glucose concentration for adjusting the

189 glucose infusion rate. Arterialized plasma glucose was determined in duplicate by the glucose oxidase method (Analox

190 GM9 Analyzer; Analox Instruments LTD, London, United Kingdom). Whole body insulin-stimulated glucose uptake rate

191 (M-value) was calculated from the measured glucose values collected when the subjects had reached the the steady

192 state during the PET scan that was started 87 min (SEM 1) after the start of the clamp. FDG-PET study was performed

193 when the subject had reached the stable glucose concentrations at the level of 5 mmol/l (within 5 % range for at least 15

194 min) after positioning into the PET scanner.

196 MRI

197 Adipose tissue depot masses were measured with MRI. MRI scans were performed using Philips Gyroscan Intera 1.5 T CV

198 Nova Dual scanner (Philips Medical Systems, the Netherlands). Abdominal area axial T1 weighted dual fast field echo

199 images (TE 2.3 and 4.7 ms, TR 120ms, slice thickness 10mm without gap) were obtained. To measure different adipose

200 tissue masses the images were analyzed using SliceOmatic software v. 4.3

201 (http://www.tomovision.com/products/sliceomatic.htm). To obtain the mass the pixel surface area was multiplied with

202 the slice thickness and the density of adipose tissue 0.9196 kg/l (1).

204 Other measurements

205 A two hour 75 g oral glucose tolerance test (OGTT) was conducted after the subjects had fasted for 12-hours. Blood

206 samples were collected at 0, 15, 30, 60, 90, and 120 minutes after the glucose ingestion to determine the glucose and

207 insulin levels. Measurements of oxidized LDL and oxidized HDL were based on spectrophotometric analyses of oxidized

208 lipids in lipoproteins isolated by precipitation methods (2). Whole body fat percentage was measured at the Paavo

209 Nurmi Centre using a bioimpedance monitor (InBody 720, Mega Electronics, Kuopio, Finland).

211 Animal study design

212 Twenty-four male Wistar rats were randomly divided into three groups: HIIT (n=8), MICT (n=8) and control (CON) (n=8).

213 At the central animal laboratory of the University of Turku, the animals (aged between 8 to 12 weeks) were housed

214 under standard conditions (temperature 21°C, humidity 55±5%, lights on from 6:00 a.m. to 6:00 p.m.) with free access

215 to food and tap water. Before the exercise intervention rodents' body weight, body fat mass, and lean tissue mass were

216 measured using EchoMRI-700 (Echo Medical Systems LLC, Houston, TX, USA), and OGTT and VO2max test were

217 performed, and free living energy consumption measured. Animals in the HIIT and MICT groups had 10 exercise sessions

218 within two weeks. Each HIIT exercise session comprised of 8-10 x about 30 sec swimming bouts with 1 min resting

219 period after each bout. Animals in the HIIT group had extra weights of 30 - 50 grams tied to the waist to force them to

220 make all-out efforts. Animals in the MICT group started with 40 min swimming exercise and thereafter the exercise

221 duration was increased by 10 minutes every second session until 80 min was reached in the last two sessions. In the

222 MICT group, the rats did not bear any additional weights. One day after the last training session OGTT was performed

223 which followed VO2max tests on the second and third day after the last exercise session. Thereafter the animals were

224 kept in the metabolic gages for two days. Animals were sacrificed five days after from the last exercise session and

225 intestinal samples from duodenum were collected for protein expression analyses. All animal procedures were approved

226 by the National Animal Experimental Board (ESAVI/5053/04.10.03/2011) and performed in accordance with the

227 guidelines of the European Community Council Directives 86/609/EEC.

229 Western blot

230 The frozen duodenal tissue pieces were homogenized on ice in a lysis buffer (150 mM NaCl, 1% NP-40, 0,5% Na-

231 deoxycholate, 0,1% SDS, 50 mM Tris-HCl pH 8,0), supplemented with a protease inhibitor cocktail with an Ultra-Turrax

232 T25 (Ika® -Werke GmbH & Co. KG). The protein concentration was then quantified with the Thermo Scientific Pierce™

233 BCA protein assay kit (Thermo Fisher Scientific) prior to the sample denaturation with SDS loading buffer containing P-

234 mercaptoethanol (Sigma-Aldrich) in +95°C for 5 min. Samples were run on a 10% SDS-polyacrylamide gel and, after

235 electrophoresis, transferred onto a nitrocellulose membrane (Santa Cruz Biotechnology, Inc.). An incubation with 5%

236 (w/v) milk diluted in TBS-T (0,02 M Tris-buffered saline, 0,1% Tween-20) was used to block the unspecific binding sites

237 prior to the overnight incubation in +4°C with the following primary antibodies: Glut2 (#07-1402, Millipore), CD36 (#sc-

238 9154, Santa Cruz Biotechnology, Inc.), vascular endothelial growth factor 2 (VEGFR2) (#NB-100-530, Novus Biologicals)

239 and P-actin (#sc-8432, Santa Cruz Biotechnology, Inc.). The fluorescent signal from the secondary antibodies IRDye®

240 800CW Donkey anti-Rabbit lgG (H+L) and IRDye® 800CW Donkey anti-Mouse lgG (H+L) (LI-COR Biosciences) was

241 detected by using the LI-COR Odyssey® CLx Imager (LI-COR,Inc.). The intensities were normalized to a reference band in

242 each membrane and the relative values were used for fold-change calculations.

244 Other measurements in rats

245 Body composition was measured using EchoMRI-700 (Echo Medical Systems LLC, Houston, TX, USA). Each animal was

246 scanned before and after the exercise intervention and body fat mass and lean tissue mass was measured. The aerobic

247 capacity was studied by measuring the VO2max with rat single lane treadmill (Panlab- Harvard Apparatus, Spain). Animals

248 were familiarized to the rat single lane treadmill (Panlab- Harvard Apparatus) for three days before the VO2max test. The

249 test started after a warm up period. During the test the angle of the treadmill was 25° degrees and the speed was

250 increased by 3 cm/s after every other minute until exhaustion. Oral glucose tolerance test (OGTT) was performed after 6

251 hours fast. Glucose (20%, wt/vol, 1 ml /100g) was administered orally and tail vein glucose was measured at 0, 30, 60, 90

252 and 120 min with a Precision Xceed Glucose Monitoring Device (Abbott Diabetes Care Ltd, Abbot Park, IL, USA). Whole

253 body energy expenditure was measured with a metabolic gage (Oxylet system, Panlab, Harvard Apparatus, Spain) over

254 48 hours. The energy expenditure was calculated according to the measured carbon dioxide (CO2) production and

255 oxygen (O2) consumption and averaged over 24 hours.

257 Statistics

258 Descriptive statistics shown in the tables and the figures are based on model based means [95 % confidence intervals,

259 CI]. Association between the anthropometrics, glucose profile, and the lipid profile and the training groups, time points,

260 and time*training interaction were performed with hierarchical linear mixed model, using the compound symmetry

261 covariance structure for time. Transformations (logarithmic or square root) were done to (insulinfasting, HDL, colonic,

262 quadriceps femoris and deltoid glucose uptake; duodenal, jejunal, colonic and quadriceps femoris free fatty acid

263 uptake) to achieve the normal distribution assumption. All tests were performed as 2-sided, with a significance level set

264 at 0.05. Correlations were calculated using Pearson r. In the animal study, one-way analysis of variance was used. All the

265 analyses were performed using SAS System, version 9.3 for Windows (SAS Institute Inc., Cary, NC, US).

267 RESULTS

268 Characteristics

269 The effects of exercise on whole-body fat percentage, aerobic capacity (VO2peak), and whole body insulin sensitivity (M-

270 value) have been published in our previous study (5). Total, LDL, and HDL cholesterol levels decreased significantly after

271 training (Table 1). In the cholesterols the only difference between the training modes was the greater decrease in LDL

272 cholesterol in the HIIT group compared with the MICT group [p = 0.03, time*training].

274 Intestinal substrate uptake

275 Colonic insulin-stimulated glucose uptake improved in the MICT group (+ 37%) while no response was observed in the

276 HIIT group (+/- 0%) (p = 0.02 time*training) (Fig. 2). Jejunal glucose uptake tended to respond differently between the

277 training modes, with only MICT increasing the uptake (HIIT - 4%, MICT + 13 % p = 0.08 time*training) (Fig.2). Both

278 exercise modes decreased the free fatty acid uptake in the duodenum (p = 0.001 time, Fig. 2) and MICT tended to also

279 decrease the uptake in the colon (HIIT 0%, MICT -38%, p = 0.08 time*training, Fig. 2). The jejunal glucose uptake

280 associated positively with aerobic capacity (VO2peak) [Pre: r = 0.46, p = 0.03; Post: r = 0.45, p = 0.03] and negatively with

281 visceral fat mass [Pre: r = - 0.42, p = 0.05; Post: r = - 0.45, p = 0.03]. Glucose uptake both in the jejunum [Pre: r = - 0.31, p

282 = 0.15; Post: r = - 0.50, p = 0.02] and duodenum [Pre: r = - 0.12, p = 0.59; Post: r = - 0.53, p = 0.02] associated negatively

283 with HcA1c levels. In the MICT group, the glucose uptake in the colon associated positively [Pre: r = 0.17, p = 0.63; Post:

284 0.68, p = 0.03] (Fig. 3) and the duodenal free fatty acid uptake negatively [Pre: r = -0.38, p = 0.31; Post: r= -0.94, p = 0.01]

285 with the whole body glucose uptake after the training. Quadriceps femoris (QF) and deltoid muscle results in these

286 subjects have been published elsewhere (10). For comparison purposes those results have been added to Fig. 2.

288 Animal results

289 There was a significant increase in the body weight and fat free mass of all the animal groups indicating to the age-

290 related growth during the study intervention. (Table 2) While the fat percentage increased in the CON group, it

291 significantly decreased in both HIIT and MICT groups after the training. There were no differences in glucose values at

292 time points 0' and 120' or in the glucose AUC in any of the group*s. The aerobic capacity (VO2 max) tended to improve

293 significantly in both HIIT and MICT groups compared to the CON group (Pre: HIIT: 70.07 [66.2, 74.0]; MICT: 71.2 [67.3,

294 75.1]; CON: 69.0 [65.1, 72.9] (ml/min/kgA0.75); Post: HIIT: 72.9 [69.0, 76.8]; MICT: 72.8 [68.9, 76.7]; CON: 68.9 [65.0,

295 72.8] (ml/min/kgA0.75) [95 % CI], p = 0.05). Glut2 protein expression in the rat intestine was significantly higher in the

296 HIIT and MICT groups compared to CON group (HIIT: 19090 [12930, 28190]; MICT: 11606 [7651, 17604]; CON: 4141

297 [2730, 2141] [95 % CI] (arbitrary units), p < 0.01). Also CD36 expression was higher in the HIIT and MICT groups

298 compared to CON group (HIIT: 635 [366, 1100]; MICT: 696 [387, 558]; CON: 79 [44, 63] [95 % CI] (arbitrary units), p <

299 0.05). While VEGFR2 was only higher in the HIIT group compared to MICT and CON group (HIIT: 704 [477, 976]; MICT:

300 345 [193, 541]; CON: 294 [147, 491] [95 % CI] (arbitrary units), p < 0.05). No significant differences were observed in

301 Glut2, CD36 or VEGFR2 expression between the HIIT and the MICT groups.

303 DISCUSSION

304 In the present study, the effects of two weeks of exercise training, HIIT and MICT, on intestinal substrate uptake

305 from circulation were studied in healthy, untrained, middle-aged men. The data shows that MICT increases insulin-

306 stimulated glucose uptake while both training modes decrease fasting free fatty acid uptake in the intestine and that

307 intestinal insulin-stimulated glucose uptake correlates positively with aerobic capacity and negatively with visceral fat

308 and HbA1c. In addition both training modes increased Glut2 and CD36 protein expressions in rat enterocytes. To our

309 knowledge, this is the first study that provides evidence about the beneficial effects of exercise training on the intestinal

310 substrate metabolism and an additional mechanism by which exercise improves whole body metabolism.

311 The intestinal glucose uptake values during hyperinsulinemia in the present study agree with our recent data in

312 healthy lean controls and obese subjects (17; 29). Studies by Honka et al. (2014) and Makinen et al (2015) show that

313 insulin increases the intestinal glucose uptake compared to fasting state in healthy lean controls but the increase is

314 blunted in obese subjects. This means that the intestine is an insulin sensitive organ and intestinal insulin resistance

315 exists in obesity. Furthermore, it was shown that in obese subject's intestinal insulin resistance is ameliorated after rapid

316 weight loss (17; 29). In enterocytes, glucose is transported from blood to lumen by Glut2 transporter proteins (40). In

317 obesity and intestinal insulin resistance there is an impairment in the insulin stimulated Glut2 internalisation in the

318 enterocyte; which has been suggested to restrain the normal glucose uptake in the intestine (41). In the present study,

319 the insulin-stimulated intestinal glucose uptake before the training intervention was at the same level as the healthy

320 controls in our previous study (29). Insulin-stimulated glucose uptake improved in the colon (+37%) and tended to

321 improve in the jejunum in the MICT group after the training, while it remained essentially unchanged in the HIIT group.

322 To study the mechanisms behind the exercise-induced improvements in intestinal glucose uptake in our human data, we

323 performed corresponding short HIIT and MICT training interventions in healthy rats. As Glut2 is responsible for the

324 uptake of glucose from basolateral membrane in the intestine (21) we hypothesized that exercise would increase the

325 expression of Glut2 in enterocytes to enhance the intestinal glucose uptake and that the increase would be higher in

326 MICT compared to HIIT due to higher training volume. We found that both HIIT and MICT increased intestinal Glut 2

327 expression in rats with no differences between the groups. The reason why the increased GU was seen only after MICT

328 in humans, while Glut 2 expression increased in both training groups in rats is unclear. However, it might be that

329 although two weeks of low volume HIIT was enough to induce changes in protein level in rats, longer time is need to be

330 able to detect a change in tissue level non-invasively in humans.

331 The discrepancy in glucose uptake in different parts of the intestine agrees with the findings of Makinen and co-

332 workers, and may be due to the differences in the location of Glut2 receptor in the enterocytes (41). In humans, Glut2

333 has been observed in the apical membrane of an enterocyte in the jejunum but not in the duodenum (3). The

334 discrepancy in substrate uptake in different parts of the intestine is possibly also related to the different digestive tasks

335 between the small and large intestines and how exercise training strains these mechanisms.

336 The results in this study demonstrate a decreased free fatty acid uptake in the duodenum after the training

337 intervention in both training groups. The digestion and delivery of dietary fats throughout the body is mediated by the

338 small intestine. In the small intestine, inside the enterocytes, the dietary fats are resynthesized into triacylglycerols

339 (TAG) and secreted into the circulation or stored in cytoplasmic lipid droplets. Postprandially, the increased secretion of

340 TAG from the small intestine leads to an increment in the circulating TAG levels; however, during a fast the levels

341 decrease as a result of clearance by peripheral tissues (30). Recently, Hung and co-workers showed that in rodent's

342 endurance training leads to enhanced lipid turnover and more efficient fatty acid oxidation for energy utilization within

343 the enterocytes (18). Our data regarding the higher CD36 expression, in both HIIT and MICT trained rats, is in agreement

344 with the results of Hung et al. (18). In spite of the higher CD36 expression the reduced intestinal FFAU after training in

345 the present study could be due to the more efficient fatty acid oxidation. This is because enhanced fatty acid oxidation

346 means that less fatty acids are needed to produce the same amount of energy.

347 Another possible mechanism for the decreased intestinal FFAU could be the reduced free fatty acid flux in the

348 intestine. In fact, we found in the present study an almost significant (p = 0.052, Table 1.) drop in the levels of circulating

349 plasma free fatty acids after the training during the FTHA-PET study (fasting). The lower free fatty acid levels can be

350 explained by decreased visceral fat mass and increased whole body insulin sensitivity post training, as both reduce the

351 adipose tissue lipolysis and thereby circulating FFAs (Table 1) (31; 34; 38).

352 At the moment little is known about the different mechanisms how exercise training could strain the intestinal

353 metabolism, yet some data exists about exercise and splanchnic bed. Splanchnic blood flow reduces during dynamic

354 training and as a function of exercise intensity. However it has been shown that the reduction in splanchnic blood flow

355 during exercise attenuates as a response to long term training. (32; 33) The smaller reduction in splanchnic blood flow

356 during exercise after regular training seems to be related to the enhanced vasodilation and reduced vasoconstriction of

357 splanchnic and renal vasculature which further could indicate improved nutrient supply and utilization during exercise in

358 a trained state. (33) In the present study we did not measure intestinal blood flow in humans. In rodents we found

359 higher VEGFR2 (a marker of angiogenesis) expression level in enterocytes in HIIT compared to MICT and CON group (Fig.

360 4). Thus angiogenesis could be also one factor explaining the attenuated reduction in the intestinal blood flow shown

361 after exercise training (33). The difference in VEGFR2 levels between the groups in the present study might be due to

362 higher transient reduction of flow into the splanchnic area during HIIT compared to MICT. HIIT is extremely intense

363 exercise mode and during the intervals body concentrates to supply blood mainly to the working muscles which may

364 induce hypoxic condition in splanchnic area and further stimulate intestinal angiogenesis. Other possible factors

365 regulating intestinal metabolism could be peristaltic movements and colon transit time (37; 43).

366 We used two different training modes in this study. These both included six training sessions within an

367 intervention period of two weeks. Both the time spent during the training (time HIIT 15 vs. MICT 300 minutes) and the

368 average calculated energy consumption during the training (403 and 2680 kcal, respectively (7)) were much less in HIIT

369 than MICT. Despite this difference, both training modes improved whole body insulin sensitivity (M-value, HIIT 12% and

370 MICT 7%) and aerobic capacity (VO2peak, HIIT 6% and MICT 3%) without significantly different responses between the

371 training modes. In contrast to this, intestinal metabolism seems to be more sensitive to MICT than HIIT. As intestine

372 mediates the delivery of nutrients throughout the body, it may be that the aerobic training mode and longer exercise

373 time per session in MICT compared to HIIT challenges the intestinal metabolism more and thus may be a more rapid and

374 effective way to improve intestinal metabolism.

375 It is also possible that the difference in the daily habitual physical activity levels or in dietary intake affects to the

376 observed findings. In the present study subjects were instructed not to perform any additional physical activity except

377 daily normal living and they reported having done so. However no pedometer or any other device was used to follow

378 the activity. Thus we cannot completely rule out the possible effect of habitual physical activity on our results. Subjects

379 were also instructed to maintain their normal dietary habits and they kept dietary logs for three days before and during

380 the exercise intervention. According to the dietary logs there were no changes in the total caloric intake or in the caloric

381 content before and after the intervention in either study group (data not shown).

382 Most of the beneficial effects of exercise on the whole body are attributed to skeletal muscles and thus it is

383 interesting to compare these intestinal findings to our previous findings concerning skeletal muscles in these same

384 subjects (10). In skeletal muscles, both training modes increased insulin-stimulated GU in the main working muscle, the

385 quadriceps femoris (QF), while no changes were observed in deltoid and other upper body muscles (Fig. 2). In addition,

386 no significant changes were observed in the FFAU in any of the studied muscles. (10) Adding the findings from the

387 present study to the overall picture, it is interesting to note that intestinal metabolism seems to respond more readily to

388 MICT than the metabolism in the non-working upper body muscles (Fig. 2).

389 Previously intestinal insulin-stimulated glucose uptake has been shown to be associated with whole body

390 glucose uptake (M-value), both in healthy and obese subjects (23). Our data is in line with these previous findings

391 showing that whole body glucose uptake associates positively with insulin-stimulated glucose uptake in the colon and

392 inversely with the duodenal free fatty acid uptake. Furthermore, the jejunal glucose uptake correlated positively with

393 the VO2peak and negatively with visceral fat mass and HbA1c, which are both known risk markers for metabolic diseases.

394 Thus, although exercise training induces major health benefits through the body's muscular system, also its effects on

395 the intestine, with an average weight of 3-4 kg and surface of 200-300 m2, warrants further research.

396 There are some limitations in this study. Firstly, the location of the intestine; this is because even though the

397 duodenum has a relatively fixed location in the abdomen, the distal segments of the intestine move within the

398 abdomen. This issue was addressed by confirming the drawn ROIs with a CT scan. Secondly, the results might have been

399 affected by spill-over and partial volume effects due to the trans-axial resolution of the PET scanner and the thinness of

400 the intestinal mucosal wall. However, this effect was demonstrated to be minimal in our previous validation study (17).

401 Thirdly, in this study, we measured the substrate uptake from the circulation into the enterocytes without knowing the

402 release from the enterocytes into the circulation (i.e. from lumen to circulation). Fourth, due to the radiation dose limits

403 we cou ld not perform the [18F]FDG and [18F]FTHA PET scans both at fast and during euglycemic hyperinsulinemic clamp.

404 Thus we studied the FFAU at fasting state and GU during euglycemic hyperinsulinemic clamp, in situations when the

405 FFAU and GU, respectively, are at highest. Finally, the exercise duration in this study was only two weeks. Although this

406 kind of intervention has been shown to be effective (7; 12; 14; 15; 44), it must be emphasized that the findings show

407 only the early training response and, therefore, the long term effects of these training modes on intestinal metabolism

408 should be studied further in future experiments.

409 In conclusion, this study shows that intestinal insulin sensitivity associates positively with aerobic capacity and

410 inversely with the metabolic risk markers visceral adiposity and HbA1C. Two weeks of regular training (HIIT and MICT)

411 was shown to already improve aerobic capacity and whole body insulin sensitivity, and specifically MICT to induce

412 positive changes in intestinal substrate metabolism in middle-aged, healthy men. The changes in intestinal substrate

413 uptake seem to be related to improvements in Glut2 and CD36 protein levels. It is likely that regular long term training

414 has pronounced effects on intestine and whole body metabolism and thus the role of exercise training on intestinal

415 substrate uptake in patient populations warrant further studies.

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548 Figure 1. Study design: Subjects were studied on three separate days before and after the exercise intervention. OGTT,

549 oral glucose tolerance test; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; PET, positron

550 emission tomography; FTHA, 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA); PET-FDG,[18F]fluoro-2-deoxy-D-

551 glucose ([18F]FDG).

552 Figure 2. Insulin stimulated glucose uptake a) and fasting free fatty acid uptake b) in different tissues before and after

553 two weeks of either high intensity interval training (HIIT) black) and moderate intensity continuous training (MICT)

554 ( grey). The muscle (QF + Deltoid) results have been adapted from the Eskelinen et al 2015 (7). All values are

555 expressed as model-based means and bars are confidence intervals [95 % CI]. P-value for time interaction (i.e. the

556 groups behaved similarly for the change in parameter with no differences between the training modes). P-value for

557 time*training interaction (i.e. the groups behaved differently for the change in parameter with significant difference

558 between them). QF, quadriceps femoris; HIIT, high intensity interval training; MICT, moderate intensity continuous

559 training.

560 Figure 3. Correlation between insulin-stimulated jejunal glucose uptake and VO2peak a) and visceral fat mass b) in pooled

561 analysis of MICT ( grey) and HIIT black) subjects'. In figure c) correlation between insulin-stimulated colonic

562 glucose uptake and whole body glucose uptake (M-value) in MICT ( grey) subjects. V02peab aerobic capacity; HIIT, high

563 intensity interval training; MICT, moderate intensity continuous training.

564 Figure 4. a) Relative expression of CD36, Glut2 and VEGFR2 on in duodenum where n is 6-8. All values are expressed as

565 model-based means with error bars representing the confidence intervals [95 % CI]* p-value <0.05. b) Western blots of

566 CD36 (75kDa), Glut2 (55kDa) and VEGFR2 (105 kDa). Animals without detectable band were excluded from the analysis.

567 HIIT, high intensity interval training; MICT, moderate intensity continuous training; CON, control group.

Figure 1

Figure 2

Duodenum

Jejunum

Deltoid

—*—HUT MICT

■E 1-1 a. ^

20 18 16 14 12 -10 8 6 4 2 ■ 0

Training Time

Time^training

(= = 0.27 p = 0.99 p = 0.44 18

Training Time

Time*t raining

p - 0.77 p = 0.86 p=0.08

20 18 16

14 12 10

Training Time

Time'training

p ' 0.44 p = 0.04 p = 0.02

20 18 16 14 12 10 8 6 4 2 0

Training p=0.50

Time p = 0.18

Time+traimng p-0.30

Training p = 0,40

Time p < 0.001

Time*traintng p = 0.27

Baseline

2 weeks

Baseline

2 weeks

Baseline

2 weeks

<s TB a. o = 3

a> •—■

Training p = 0.54

Time p = 0.01

Time'training p = 0.28

Training p=0.14

Time p=0.13

Time'training p=0.76

Training Time

p = 0.70 p = 0.10

Time'training p = 0.08

Training p = 0.99

Time p = 0.65

Time'training p = 0,20

Training p = 0.65

Time p = 0.66

Time*training p = 0.39

Baseline 2 weeks

II0Z '82 M0JB|/\| uo IZZ'OZZ'Ol Äq /ßJ0 Äß0|0!SÄqd def//:dHM luoj] p8pe0|UM0Q

Figure 3

- ---- — i—- -----r.

• - —---

150 — • 1005037-

ß-ACT

VEGFR ß-ACT

Table 1: Subject characteristics at baseline and after the exercise intervention

CT) c n O

CT) c n O

o O </> (D

(D O C

n> cr o

o ■a o 3

(JJ 00

00 (jj

UJ 00 --J NJ

ui Ln "an

(jj NJ

(jj --j

OJ "(JJ OJ 4^ 00

OJ --j

--j OJ

4^ 00 "(JJ

an "an

ID OJ --J

"(JJ NJ

OJ --J

--j an

Tn Ln an Ld

"oj an

NJ CD 4^ NJ

--J ID

ui cn "ai

OJ --J

00 OJ 4^

NJ CD 4^ NJ

II —

o o -c*

o ■c»

--J --J

NJ --J

--J OJ

Time x group interaction

M-value (^mohkg^min-1) 38.2 [30.1, 46.4] 42.8 [34.5, 51.0] 12 31.9 [23.1, 40.7] 34.2 [25.4, 43.1] 7 0.03 0.45 Lipid Profile

FFA fasting (mmd-!1) 0.61 [0.50, 0.71] 0.59 [0.48, 0.70] -3 0.78 [0.67, 0.89] 0.67 [0.54, 0.79] -15 0.052 0.14

FFA damp (mmd-!1) 0.06 [0.05, 0.08] 0.06 [0.05, 0.08] 0 0.08 [0.06, 0.10] 0.07 [0.05, 0.09] -14 0.41 0.43

Cholesterol (mmoH-1) 5.3 [4.8, 5.7] 4.6[4.1, 5.0] -14 4.7 [4.3, 5.2] 4.4 [3.9, 4.9] -7 <0.001 0.06

HDL (mmolT1) T 1.4 [1.2, 1.6] 1.2 [1.1, 1.4] -10 1.4 [1.2, 1.5] 1.3 [1.1, 1.5] -5 <0.001 0.28

LDL (mmolT1) 3.4 [3.0, 3.8] 2.8 [2.4, 3.3] -16 2.9 [2.5, 2.3] 2.7 [2.3, 3.1] -6 <0.001 0.03

HDL Ox 28.7 [26.3, 31.1] 29.4 [27.0, 31.9] 3 27.4 [24.9, 30.0] 27.6 [25.1, 30.1] 1 0.58 0.74

LDL Ox 30.3 [26.0, 34.5] 31.9 [27.6, 36.1] 5 28.0 [23.6, 32.4] 28.4 [24.0, 32.9] 2 0.26 0.50

Triglycerides (mm°H 1)_1.02 [0.85, 1.19] 0.97 [0.79, 1.15] -5 0.96 [0.78, 1.13] 0.80 [0.62, 0.98] -16_0.07_0.37

All values are mean [SE]. BMI, body mass index; AUC, area under the curve; HbAlc, glycosylated hemoglobin; HDL, high density lipoprotein; LDL, low density lipoprotein; HDL Ox , oxidized high density lipoprotein; LDL Ox, oxidized low density lipoprotein; MICT, moderate intensity continuous training; HIIT, high intensity interval training. ^ Log transformation was done to achieve normal distribution. The p-value for time indicates the change in the whole study group. The p-value for time x group interaction indicates if the change in the parameter was different between the HIIT and MICT training modes.

¿1-03 '83 M3JEIAI UO I WOZZ'OI /ßJ0 Äß0|0!SÄi)d dEi//:dni) WOJJ p8pe0|UM0Q

Table 2:Animal characteristics at baseline and the changes induced after the exercise intervention

■a c ro a) c

"¡5 10 ro -Q

ro sz o

«N JU -Q m

Parameter

CON n=8

HIIT n=8

MICT n=8

P value

Anthropometrics

Weight (g)

Fat free mass (%) Fat mass (g)T Fat (%)

VO2max (ml/min/kg) OGTT

Glucose 0 (mmolT1) Glucose 120 (mmolT1) Glucose AUC (mm*mmoM-1)

282 [269, 294] 351 [338, 364]* 239 [229, 248] 282 [271, 294]

11.9 [11.0, 12.9] 12.7 [11.6;13.8]* 6 11.7 [10.8, 12.7] 10.7 [9.7, 11.8]* 69.0 [65.1, 72.9] 68.9 [65.0, 72.8] 0 70.1 [66.2, 74.0] 72.9 [69.0, 76.8]*

5.0 [4.6, 5.4] 5.5 [5.0, 6.1] 840 [779, 900]

4.9 [4.5, 5.3] 5.3 [4.9, 5.8] 813 [767, 859]

-2 5.1 [4.7, 5.5] -3 4.8 [4.3, 5.4] -3 806 [745, 866]

4.9 [4.5, 5.4] 5.2 [4.8, 5.6] 786 [728, 844]

-3 4.9 [4.5, 5.3] 8 5.3 [4.7, 5.8] -2 774 [713,834]

4.7 [4.2, 5.1] 4.9 [4.4, 5.3] 742 [693, 791]

25 297 [285, 309] 346 [331, 360]* 16 281 [269, 293] 350 [337, 364]* 25 18 253 [244, 263] 296 [285, 307] 17 248 [238, 257] 291 [279, 302] 17

36.8 [33.6, 40.4] 47.2 [42.2, 52.7]* 28 38.4 [35.0, 42.1] 40.5 [36.3, 45.2] 6 35.9 [32.7, 39.4] 40.4 [36.2, 45.1]* 13

-8 11.4 [10.4, 12.3] 10.8 [9.7, 11.9]* -5 4 71.2 [67.3, 75.1] 72.8 [68.9, 76.7] 2

-5 -8 -4

<.0001 <.0001

<.0001

0.31 0.73 0.18

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<0.001 <.001 0.05

0.93 0.23 0.97

All values are mean [95 % confidence intervals]. AUC, Area under the curve; CON, control group no exercise; MICT, moderate intensity continuous training; HIIT, high intensity interval training. \og transformation was done to achieve normal distribution. The p-value for time indicates the change in the whole study group. The p-value for time x group interaction indicates if the change in the parameter was different between the CON, HIIT and MICT training modes and * pre vs post p value < 0.05.

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