Beneficial effects of running and milk protein supplements on Sirtuins and risk factors of metabolic disorders in rats with low aerobic capacity

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Beneficial effects of running and milk protein supplements on Sirtuins and risk factors of metabolic disorders in rats with low aerobic capacity © 2019 The Author(s) Published version Lensu, S.; Pekkala, S. P.; Mäkinen, A.; Karstunen, N.; Turpeinen, A. T.; Hulmi, J.J.; Silvennoinen, M.M.; Ma, H.; Kujala, U. M.; Karvinen, S.; Koch, L. G.; Britton; S., L.; Kainulainen, H. Lensu, S., Pekkala, S. P., Mäkinen, A., Karstunen, N., Turpeinen, A. T., Hulmi, J.J., Silvennoinen, M.M., Ma, H., Kujala, U. M., Karvinen, S., Koch, L. G., Britton, S., L., Kainulainen, H. (2019). Beneficial effects of running and milk protein supplements on Sirtuins and risk factors of metabolic disorders in rats with low aerobic capacity. Metabolism Open, 4, Article 100019. https://doi.org/10.1016/j.metop.2019.100019 2019 Beneficial effects of running and milk protein supplements on Sirtuins and risk factors of metabolic disorders in rats with low aerobic capacity S. Lensu a , * , S.P. Pekkala a ,A.M € akinen a , N. Karstunen a , 1 , A.T. Turpeinen b , J.J. Hulmi a , M.M. Silvennoinen a ,H.Ma a , c , U.M. Kujala a , S. Karvinen a , L.G. Koch d , S.L. Britton e , f , H. Kainulainen a a Faculty of Sport and Health Sciences, University of Jyv€ askyl€ a, Jyv€ askyl€ a, P.O.B. 35, FI-40014, JYU, Finland b Valio Ltd, R&D, Helsinki, P.O.B. 10, FI-00039, VALIO, Finland c Institute of Dentistry, Faculty of Medicine, University of Helsinki, P.O.B. 63, FI-00014, HY, Finland d Department of Physiology and Pharmacology, Center for Hypertension and Personalized Medicine, The University of Toledo College of Medicine &Life Sciences, Toledo, OH, 2801 W. Bancroft, Toledo, OH, 43606-3390, USA e Department of Anesthesiology, University of Michigan Ann Arbor, 1500 E Medical Center Drive, Ann Arbor, MI, 48109-5048, USA f Department of Molecular &Integrative Physiology, University of Michigan Medical School Ann Arbor, 7744 MS II, 1137 E, Catherine St., Ann Arbor, MI, 48109-5622, USA article info Article history: Received 19 August 2019 Received in revised form 6 October 2019 Accepted 7 October 2019 Available online 10 October 2019 Keywords: Low capacity running rat (LCR) Metabolism Muscle Running Sirtuins Whey abstract Background: Physical activity and dietary intake of dairy products are associated with improved metabolic health. Dairy products are rich with branched chain amino acids that are essential for energy production. To gain insight into the mechanisms underlying the benefit of the sub-chronic effects of running and intake of milk protein supplements, we studied Low Capacity Runner rats (LCR), a rodent exercise model with risk for metabolic disorders. We especially focused on the role of Sirtuins, energy level dependent proteins that affect many cellular metabolic processes. Methods: Forty-seven adult LCR female rats sedentary or running voluntarily in wheels were fed normal chow and given supplements of either whey or milk protein drink (PD)-supplemented water, or water only for 21 weeks. Physiological responses were measured in vivo. Blood lipids were determined from serum. Mitochondrial markers and Sirtuins (Sirt1-7) including downstream targets were measured in plantaris muscle by western blotting. Results: For the first 10 weeks whey-drinking rats ran about 50% less compared to other groups; still, in all runners glucose tolerance improved and triglycerides decreased. Generally, running induced a ~sixfold increase in running capacity and a ~8% decrease in % body fat. Together with running, protein supplements increased the relative lean mass of the total body weight by ~11%. In comparison with sedentary controls, running and whey increased HDL (21%) and whey, with or without running, lowered LDL (34%). Running increased mitochondrial biogenesis and Sirtuins 3 and 4. When combined with exercise, both whey and milk protein drink induced about a 4-fold increase in Sirt3, compared to runners drinking water only, and about a 2-fold increase compared to the respective sedentary group. Protein supplements, with or without running, enhanced the phosphorylation level of the acetyl-coAcarboxylase, suggesting increased fat oxidation. Both supplemented diets increased Sirt5 and Sirt7 without an additional effect from exercise. Running diminished and PD supplement increased Sirt6. Conclusion: We demonstrate in rats new sub-chronic effects of milk proteins on metabolism that involve Sirtuins and their downstream targets in skeletal muscle. The results show that running and milk proteins act on reducing the risk factors of metabolic disorders and suggest that the underlying mechanisms *Corresponding author. Faculty of Sport and Health Sciences, University of Jyv€ askyl€ a, PO Box 35, FI-40014, JYU, Jyv€ askyl€ a, Finland. E-mail addresses: sanna.t.k.lensu@jyu.fi(S. Lensu), satu.p.pekkala@jyu.fi(S.P. Pekkala), [email protected] (A. M€ akinen), niina.karstunen@firstbeat.com (N. Karstunen), Anu.Turpeinen@valio.fi(A.T. Turpeinen), juha.j.t.hulmi@jyu.fi(J.J. Hulmi), mika.m.silvennoinen@jyu.fi(M.M. Silvennoinen), ma.hongqiang@helsinki.fi (H. Ma), urho.m.kujala@jyu.fi(U.M. Kujala), sira.m.karvinen@jyu.fi(S. Karvinen), [email protected] (L.G. Koch), [email protected] (S.L. Britton), heikki.s.o. kainulainen@jyu.fi(H. Kainulainen). 1 Present address: Firstbeat Technologies Oy, Jyv€ askyl€ a, Yliopistonkatu 28A, FI-40100 Jyv€ askyl€ a, Finland’. Contents lists available at ScienceDirect Metabolism Open journal homepage: www.journals.elsevier.com/metabolism-open https://doi.org/10.1016/j.metop.2019.100019 2589-9368/©2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Metabolism Open 4 (2019) 100019 may involve Sirtuins. Notably, we found that milk protein supplements have some favorable effects on metabolism even without running. ©2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Epidemiological and experimental evidence suggest that physical activity [1], and consumption of dairy products [2,3] associate with decreased risk for metabolic disorders (MD). Metabolic disorder is a constellation of risk factors that commonly occur together, including high blood glucose, triglycerides and blood pressure, and low high-density lipoproteins (HDL). Low-density lipoproteins (LDL) have been associated with traits of metabolic disorder independent of obesity [4]. The effects of dairy products for improved metabolic health seem to be mediated by the protein components and/or minerals of milk [5]. Specifically, casein and whey are accounted to produce these beneficial effects [5]. Compared to casein, whey is especially rich in branched chain amino acids (BCAAs) and thus, the difference in their metabolic effects may arise from the difference in their essential amino acid compositions [6]. Casein and whey also differ in their postprandial kinetics and satiating effect, exemplified by slower stomach emptying of casein than whey [7,8]. BCAAs are essential amino acids used for energy production especially in the skeletal muscle, but also in other tissues, such as brain and adipose tissue. In contrast to other amino acids, BCAAs do not need the first-pass hepatic catabolism for their degradation. BCAAs have numerous physiological functions, one of those being a regulatory role for protein synthesis in skeletal muscle [9]. Although the beneficial effects of BCAAs on metabolic health [9,10] have been linked with the activity of Sirtuin 1 [11,12], the detailed mechanistic pathways are not thoroughly known. The possible links to all seven Sirtuins with exercise and BCAA-supplementation have not been previously studied. Sirtuins are energy level (NAD þ ) -regulated enzymes. They can function as e.g. deacetylases, desuccinylases, demalonylases or ADP-ribosylases, to modify post-translationally a large set of proteins from histones to enzymes of energy metabolism [13,14]. Of the sirtuins, Sirt1 has various nuclear and cytosolic targets controlling for instance cell cycle and energy homeostasis [13] and is considered as one of the key regulators of mitochondrial biogenesis [12]. Sirt2 regulates gluconeogenesis [15], and is highly expressed in adipocytes playing a role in metabolic functions [16]. In skeletal muscle, Sirt3 regulates mitochondrial substrate selection and metabolic flexibility [13]. Sirt3 level increases e.g. due to caloric restriction or exercise [17,18] and can be downregulated in MD [19]. The main enzymatic activity of Sirt4 is to control BCAA catabolism: without Sirt4 BCAA metabolism is reduced in hepatic and cardiac mitochondria [20]. Sirt5 desuccinylates, demalonylates and deglutarylates a major portion of proteins related to mitochondrial energy metabolism from Pyruvate Dehydrogenase (PDH) complex, b - oxidation, BCAA catabolism, and Krebs cycle to electron transport chain and ATP synthesis, thus being an important regulator of energy metabolism [21]. Sirt6 is a multifunctional protein [14,22], and acts as one of the key regulators of glucose homeostasis [23], which is disturbed in MD. Sirt7, in addition to its regulatory tasks e.g. in ribosome biogenesis, genome stability, transcription and RNA metabolism, is a regulator of mitochondrial homeostasis and hepatic lipid metabolism [24]. An overview of the roles of Sirtuins in metabolic pathways is shown in Figure S1. Because the protein levels of sirtuins adapt to acute or long-term stimuli like exercise or caloric restriction [14], and skeletal muscles account ~40% of the body mass and almost 30% of the resting energy consumption in normal human [25], we sought to study the interplay between skeletal muscle metabolism, Sirtuins and BCAAs. We previously showed that low capacity runner (LCR) rats score high for developing MD [26], have greater adiposity [27] and develop hepatic steatosis even without a high fat challenge [28], have reduced lifespan [29], and express less BCAA degradation and fatty acid metabolism-related genes than the high running capacity (HCR) rats [30]. Thus, it was of special interest to use LCRs to study the separate and combined effects of long-term BCAA supplementation, and exercise on the variables of metabolic health. We hypothesized that either exercise and/or BCAA-rich milk protein supplements would ameliorate the MD risk factors or concurrent regulatory changes would be seen in skeletal muscle Sirtuin levels. HCR/LCR rat model is a polygenic model [26], and thus, the data derived from them are likely more relevant for translational to humans than that of the commonly used inbred laboratory rodents. 2. Materials and methods 2.1. Animals and intervention measurements LCR and HCR rats are produced by two-way artificial selective breeding [26,31]. Shortly, breeding started with a large founder population of genetically heterogeneous, outbred rats (N:NIH stock). After testing the running capacity (i.e. phenotyping) at 11 weeks of age, the individuals with the lowest and highest running distance were mated to produce the two segregating lines for intrinsic aerobic capacity [31]. For this study full-grown, phenotyped, female LCR rats from generation 28 of selection were transported from the University of Michigan, USA to Finland at the age of ~5 months. Animals were single-housed in standard conditions (temperature 21 ±2  C, humidity 55 ±10%, light period from 8.00 to 20.00). The experiment was approved by the Regional State Administrative Agency, Southern Finland, Finland (ESAVI-201007989/Ym-23), and the Institutional Animal Care &Use Committee (IACUC) at the University of Michigan. Experiment was conducted in accordance with the Guidelines of the European Community Council directives 86/609/EEC, and European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg 1985). The study is reported according to ARRIVE guidelines. Before the running and diet interventions, we conducted a maximal treadmill running capacity test, and measured body composition, spontaneous activity, and glucose tolerance and insulin sensitivity (see Fig. 1 for study setup). Then, the rats (n ¼47, aged 8.7 ±0.6 months) were divided into six ageand weightmatched groups. Three groups were sedentary and three respective groups were provided running wheels (RW) in their home cages (Fig. 1). During diet intervention, protein supplements were given in drinking water. Sedentary control or running control animals had free access to tap water, whey (W) groups received whey protein (5.28 ±0.04 g/kg, Valio) and the protein drink (PD) groups received milk protein drink (5.13 ±0.02 g/kg, Valio) for 21.4 weeks (150 ±3 days; ~5months). All rats had aspen-chips (Tapvei, Kaavi, Finland) as a bedding and nesting material. Sedentary animals were housed in Macrolon IV cages (Techniplast 1354G, Buguggiate, Italy). For the exercise animals, running wheels were mounted onto the S. Lensu et al. / Metabolism Open 4 (2019) 1000192 housing cages (Techniplast 2154F0105, Buguggiate, Italy), and the wheel (Ø 345 mm) revolutions were recorded 24/7 with a selfconstructed computerized system [32]. All rats were given pelleted rodent diet (R36, Labfor/Lantm€ annen, Malm€ o, Sweden) ad libitum. Both diet drinks were given each day (seven days a week) as fresh and were available ad libitum. Food intake and weight gain were measured at least twice a week. Details of diets are given in the Supplement 1, as are the methods for the functional measurements that were done preand post-intervention (Fig. 1). 2.2. Necropsy Animals were euthanized in random order at metestrous/diestrous -phase. The estrous stage was determined by vaginal smear, stained fresh with Giemsa on objective glass [33]. Running wheels were blocked 24 h before necropsy to avoid acute effects of running. The rats were fasted overnight, weighed, quickly anesthetized with mixture of air and CO 2 , and then euthanized by cardiac puncture to collect serum samples. Tissue samples were weighted, immediately snap frozen in liquid nitrogen and stored at 80  C. For the weight of plantaris muscle we used the mean value of the left and right plantaris. 2.3. Western blot We used previously published protocols [34] for plantaris homogenization, and Western blotting of the samples. Antibodies against the following proteins (with the target function) were: cytochrome C (cytC, mitochondrial hemeprotein, describes number of mitochondria), acetyl-CoA carboxylase (ACC) and its phosphorylated form pACC (enzyme involved in fatty acid biosynthesis), hypoxia inducible factor 1 a (HIF1 a , transcriptional regulator of cellular response to hypoxia), Pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4, regulator of glucose/fatty acid metabolism), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 a , master regulator of mitochondrial biogenesis), phosphofructokinase (F6PK, a key regulatory kinase in the glycolysis), and Sirtuins 1e7. Details of the primary antibodies and methods are given in the Supplement 1. All results were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, glycolytic enzyme that catalyzes break down of glucose). In addition, the expression of mitochondrial Sirt3-5 were normalized to the markers of mitochondria: number (cytC) and activity (citrate synthase activity). 2.4. Citrate synthase activity, blood lipids, glucose, insulin and HOMA-index To determine the activity of mitochondria we measured the level of citrate synthase (CS, catalyzes the first reaction of the citric acid cycle) enzyme activity, in plantaris muscle as previously [35]. Blood lipids were assayed from necropsy serum sample by a kinetic photometric method with KoneLab. Fasting blood samples (in vivo) were taken from vena saphena. Blood glucose was measured with glucose analyser (HemoCue Glucose 201 þ , HemoCue AB, € Angelholm, Sweden). Serum insulin was assayed with ELISA (Mercodia AB, Uppsala, Sweden). Calculation of HOmeostatic Model Assessment for insulin resistance (HOMA-index) is a method to assess function of pancreatic b -cells, and insulin resistance, and was calculated with an equation: (fasting blood glucose/fasting insulin *22.4) [36]. 2.5. Statistics Data were analyzed with MS-Excel 2010 and SPSS (version 22, IBM©SPSS Statictics). Results are shown as mean ±SEM. Data were assessed with Univariate Anova (UniANOVA) or two-way analysis of variance using general linear model (GLM) after verifying homogeneity of variances by the Levene’s test. In case of a statistically significant interactive effect with diet and running, a full-factorial model with contrast coefficient was calculated to assign the statistical differences among the groups. If the interaction was not statistically significant, data were analyzed by the analysis of variance followed by post-hoc testing (Sidak’s test or Dunnett’s 2-sided ttest). The repeated observations from the same rat were accounted using linear mixed model. Type III tests of fixed effects with Sidak’s adjustment for multiple comparisons were used. Pearson’s correlation was calculated to estimate correlation between variables. The level of statistical significance was set at p 0.05. 3. Results 3.1. Energy intake, body weight and body composition Energy intake [kcal/body weight] was highest in PD groups (Figure S2a), being significantly higher than in water drinking controls (sedentary or running, p <0.001). Running increased food (p <0.001, Figure S2b) and BCAA intake (p <0.001, Figure S2c) in all protein-supplemented groups. The intake of main nutrients differed between diet-groups (Figure S3), but within groups the levels remained constant throughout the intervention. Intervention did not affect continuous weight gain in any group (Figure S2a), but between the two body composition measurements, both the sedentary PD-group (pre vs. post intervention, p ¼0.023) and RW-group (pre vs. post intervention, p ¼0.001) gained weight (Fig. 2a). When combined with running, both PD and whey boosted the gain in relative lean mass (of the total body weight, p <0.05; ~12 and 10.5%, respectively), while running alone had no effect (Fig. 2b). However, in all runners (Fig. 2c), the amount of fat of the total body mass decreased, the drop being largest in supplemented groups (p <0.05; PDþRW, 45%; WþRW -35%). Further, absolute lean mass (g) increased in all runners (p <0.001, data not shown). 3.2. Running, aerobic capacity, and spontaneous activity The amount of voluntary running increased in all groups during the first 8e11 weeks (Fig. 3a), but to a lesser extent in the WþRW group. In the RW group, the total running distance was 740 ±154 km (range 93e1556 km), in PDþRW 932 ±148 km (range Fig. 1. Schematic drawing of the study setup showing the groups and the timeline for the measurements. S. Lensu et al. / Metabolism Open 4 (2019) 100019 3 380e1515 km, PDþRW vs. RW: p ¼0.553) and in WþRW 495 ±97 km (range 70e1023 km, WþRW vs. RW: p ¼0.365). Although the total running distance in the end of intervention did not differ between the groups, the average daily distances were different (p <0.001, Fig. 3a), the PDþRW having the highest and the WþRW the lowest amount of daily running. Voluntary training improved running capacity about 8-fold in all running groups (p <0.001; Fig. 3b) while diet supplements had no effect. Running in running wheels introduced a 10-fold difference in the spontaneous activity in running rats compared to the sedentary (Fig. 4a, b), the difference being especially clear during the darkness (Sed vs. RW rats, daytime: p ¼0.081, and darkness: p <0.001). Sedentary lifestyle flattened the circadian spontaneous activity pattern (Fig. 4a, b). All rats were handled during the daytime between 14 and 17 h that introduced an insignificant induction in the activity (Fig. 4aec). 3.3. Glucose tolerance, insulin sensitivity, and fasting blood glucose Running improved glucose tolerance in all groups shown by the decreased AUC-values (Table 1;p¼0.001). Whey intervention brought about a ~18% decrease in the glucose sensitivity (Table 1, glucose tolerance test, pre vs. post value: p ¼0.025); whereas running had no effect; there was no difference between pre and post AUC-values (WþRW, pre vs. post: p ¼0.115). All groups became more resistant to exogenous insulin during the intervention (Table 1,Figure S4). At the insulin sensitivity test before intervention, blood glucose plummeted rapidly, within 10 min in all groups, while in the test after intervention, the glucose drop was significant only at 20 min after the insulin injection (Figure S4b). Insulin sensitivity decreased 20e22% in all sedentary and running PDand W-groups (p <0.05, Table 1). Fasting blood glucose values did not differ between the groups before or after the intervention (Figure S4). 3.4. Serum lipids, HOMA-index, and glucose levels in the end of intervention At necropsy, fasting insulin and glucose levels, and thereby HOMA-indices, were equal between the groups (Suppl. Table 2). Diet had a general decreasing effect on serum LDL levels (Table 2; effect of PD, 33%, p ¼0.062; W, 34%, p <0.05) although none of the treatments had a significant effect on serum total cholesterol. Neither the diets nor the running alone affected HDL level, but whey together with running increased HDL by 0.44 mmol/l (p <0.05, contrast to sedentary controls, Table 2). Running decreased triglycerides (Table 2;0.18 mmol/l, 25%, p ¼0.014), while PD or W did not. In comparison with the sedentary groups, running also decreased free fatty acids (0.21 mmol/l, p ¼0.007, Suppl. Table 2). Total running amount (total distance during intervention) did not associate with blood lipids. Serum LDL was inversely associated with protein intake [r ¼0.39, p ¼0.008]. Fig. 2. Changes in body composition during a 5-month intervention. (a) Weight (g) was measured with a scale, while for the (b) lean body mass (g) [relative to body weight, BW (g)], and (c) fat mass (g) [relative to BW (g)] rats were anaesthetized and measured with dual-energy X-ray absorptiometry. In the panels, * indicates significant change within group during intervention, pre vs. post value, linear mixed model,* p <0.05; ** 0.01 >p0.001; *** p <0.001. # indicates significant difference as compared with ctrl-group, # p <0.05, ## 0.001 <p<0.01, ### p <0.001, GLM. Fig. 3. (a) Daily voluntary running distances [meters (m), group average ±SEM] were followed using the computerized recording system, the functioning of which was confirmed using uninterruptible power source. * depicts significant difference of the PDþRW to the RW-group, p <0.05, # depicts significant difference of the WþRW to the RW-group, p<0.05, linear mixed model. For the aerobic fitness i.e. running capacity we conducted treadmill exercise tests, and panel (b) shows the difference between the pre and postvalues as a change in running distance (m). Effects are shown as *** p <0.001, linear mixed model. S. Lensu et al. / Metabolism Open 4 (2019) 1000194 3.5. Mitochondrial markers cytC, CS activity, and PGC-1 a Running increased the expression of cytC by ~2-fold (p <0.001) while the diets had no effect on it (Fig. 5a). Generally, both running and diet supplement increased CS activity in plantaris muscle (Suppl. Table 2, running by 1.7-fold, p <0.001, diets by 1.2-fold, p¼0.043). Especially whey together with running increased CS activity by 1.8 fold (p <0.001). Expression of PGC-1 a increased by Fig. 4. Continuous diurnal spontaneous activity index, shown as a mean value (±SEM, please note that error bars are slightly dissociated to diminish their overlapping) in 3 h bins, was measured during the intervention in sedentary groups (a) and in running groups (with availability to run during the activity measurement, panel b). Measurement was done for 6 animals in each group, the exception being the water drinking ctrl-group from which 4 animals were measured. In panel (c), activity of sedentary animals is shown in a larger scale. Black-and-white bars near the x-axes indicate the lighting periods in the room (lights were on between 8 a.m. and 8 p.m.). Daily handling of the rats occurred between 2e5 p.m. (rounded rectangle in line graphs a, b, c). In the panel (d), diurnal spontaneous activity (during one representative day, 24 h) is shown as divided into daytime and darkness (12 h). Number within each pie represents the group mean value of the sum of the total activity during daytime (12 h) or darkness, and the percentages show the distribution of the activity across circadian phases in each group. Differences between the groups were statistically assessed by the linear mixed model, * p <0.05, difference between PD and crtl; # p<0.05, difference between W and ctrl. S. Lensu et al. / Metabolism Open 4 (2019) 100019 5 1.8 fold in response to running (Fig. 5b; p <0.001). Markers of mitochondrial biogenesis and activity were associated with increase in running capacity: PGC-1 a (r ¼0.56, p<0.001), cytC (i.e. mitochondrial quantity, r ¼0.44, p ¼0.002), and CS (i.e. mitochondrial activity, r ¼0.74, p <0.001). 3.6. Intermediary metabolic proteins Neither diets nor exercise had an effect on PDK4 (Fig. 5c) that plays a key role in regulation of glucose and fatty acid metabolism. In sedentary animals PD and W diminished ACC while in running groups both PD and W enhanced ACC [Fig. 5d, interactive effect of running and diets (p <0.001)]. Phosphorylation of ACC, that enhances fat oxidation, increased (Fig. 5e) in response to whey and to running. Thus, both diets (PD þ51% and W þ89%) and running without supplements (þ11%) enhanced the amount of phosphorylated ACC relative to the total amount of ACC (Fig. 5f). PD increased the expression of glucose metabolism-regulating Hif1 a by 4.5-fold and 3.2-fold in comparison to controls and whey (respectively) (Fig. 6a). Similarly F6PK, the rate limiting enzyme of glycolysis, was induced by PD by 3-fold and by 1.8-fold (Fig. 6b) in comparison to controls and W, respectively. Running had no effect on either Hif1 a or F6PK. 3.7. Sirtuins Both diets had a similar general tendency to induce Sirt 1 (about þ31%, p ¼0.092, Fig. 7a), that is one of the key regulators of mitochondrial biogenesis [12]. PD combined with running had most pronounced effect by enhancing Sirt1 level by 1.8-fold in comparison to controls (p ¼0.034, Fig. 7a). Neither running nor protein supplements had an effect on Sirt2 (Fig. 7b). Of the effects of intervention on the other non-mitochondrial sirtuins, running decreased Sirt6, which acts as one of the key regulators of glucose homeostasis [23], by 25% (p <0.001, Fig. 7c), and the difference was significant between all diet-matched, sedentary and RW groups (p <0.01 for all). However, protein drink increased Sirt6 expression in comparison to both control diet (1.5-fold, p <0.001) and whey (1.4-fold, p <0.001). Sirt6 associated with fat intake (r ¼0.61, p<0.001), increased adiposity (r ¼0.33, p ¼0.023), and with the downstream targets of it: Hif1 a (r ¼0.60, p <0.001) and F6PK (r ¼0.56, p <0.001). An inverse association was found with Sirt6 and improved running capacity (r ¼0.33, p ¼0.023), CS activity (r ¼0.44, p ¼0.002), and expression of PGC1 a (r ¼0.33, p¼0.022) and total ACC (r ¼0.5, p <0.001). Running and diets had an interactive effect on Sirt7 (Fig. 7d; p¼0.002). Running alone had no effect but both diets increased Sirt7 in comparison with normal diet (PD, þ53%, p <0.001 and W, þ82%, p <0.001, Fig. 7d). Thus, both BCAA (r ¼0.68, p <0.001) and protein (r ¼0.77, p <0.001) intakes were associated with Sirt7, the regulator of mitochondrial homeostasis and hepatic lipid metabolism [24]. Because running induced expression of mitochondrial markers, the data of all mitochondrial sirtuins (Sirt3, 4 and 5) are shown as expression data, and as normalized to mitochondrial markers CS and cytC (Fig. 8). Sirt3 was associated with an increase in running capacity (r ¼0.5, p <0.001), CS activity (r ¼0.75, p <0.001), PGC1 a (r ¼0.71, p <0.001), and cytC expression (r ¼0.51, p <0.001), Both diets (PD, þ65% p ¼0.001; W, þ80% p <0001) as well as running (þ114%, p <0.001) increased significantly Sirt3 (Fig. 8a). Despite normalization to CS the inducing effect of running and both protein supplements on Sirt3 remained significant (RW, p ¼0.011; PD, p¼0.011, and W, p <0.001), but after normalization to cytC Sirt3 induction by running was not significant. Sirt3 was associated with increased lean mass (r ¼0.57, p <0.001) and an inverse association was found with Sirt3 and D AUC in glucose tolerance tests (r ¼0.34, p ¼0.022). Sirt3 was also inversely associated with increased adiposity ( D fat%, r ¼0.51, p <0.001), and serum triglyceride level (r ¼0.35, p ¼0.018). Compared to sedentary controls, Sirt4 (whose the enzymatic Table 1 AUC-indices: Area under the blood glucose curve was calculated from blood glucose values in glucose tolerance and insulin sensitivity tests (mean ±SEM), before and in the end of the intervention. Group AUC, glucose tolerance test (a AUC, insulin sensitivity test Before intervention End of intervention Before intervention End of intervention ctrl 1187 ±61 1267 ±107 183 ±6 186 ±5 PD 1164 ±64 1252 ±78 179 ±5 216 ±5* W1182 ±90 1397 ±105* 174 ±4 213 ±15* RW 1107 ±68 1003 ±51 181 ±7 200 ±6 PDþRW 1115 ±76 989 ±42 176 ±8 214 ±13* WþRW 1169 ±58 1020 ±53 172 ±4 206 ±13* Number of animals in each group is 8, except in PDþRW n ¼7. a Significant effect of running, p ¼0.001 (GLM). * Significant effect of intervention, difference between pre and post -value, p <0.05 (GLM). Table 2 Blood lipids (mean ±SEM) in the end of intervention, measured after overnight fasting from serum sample. Group Triglycerides (mmol/l) a Total cholesterol (mmol/l) HDL (mmol/l) b LDL (mmol/l) c,d ctrl 0.79 ±0.12 3.3 ±0.2 2.06 ±0.08 0.93 ±0.15 PD 0.70 ±0.08 3.4 ±0.2 (n ¼7) 2.41 ±0.12 (n ¼7) 0.59 ±0.13 (n ¼7) W 0.71 ±0.10 2.8 ±0.1 1.96 ±0.14 0.55 ±0.10 RW 0.64 ±0.10 3.4 ±0.2 2.16 ±0.09 0.96 ±0.11 PDþRW 0.43 ±0.07* 3.1 ±0.3 2.19 ±0.15 0.69 ±0.15 WþRW 0.57 ±0.05 3.5 ±0.2 2.50 ±0.23 b 0.66 ±0.12 a Significant effect of running, p <0.05. b Interactive effect of running and protein supplements, p <0.05; contrast of WþRW to ctrl p <0.05). c Significant effect of whey, p <0.05. d Main effect of diet, p <0.05. S. Lensu et al. / Metabolism Open 4 (2019) 1000196 Fig. 5. Protein content of (a) cytochrome C, (b) PGC-1 a , (c) PDK4, (d) Acetyl-CoA-carboxylase (ACC), and (e) phosphorylated Acetyl-CoA-carboxylase (pACC) in plantaris muscle. Panel (f) shows the expression of phosphorylated Acetyl-CoA-carboxylase (pACC) in relation with total ACC. Representative examples of western blots are shown above each graph, gel blots are divided because the order of samples in the gel was not equal for the order of groups in results-section. Nonetheless, all gel figures originate from the samples run in the same gel. Each gel contained protein samples of all groups, and the samples run on different gels were normalized to a control sample, that was repeated in each gel. In the panels, * indicates significant difference to sedentary control (ctrl), * p <0.05; ** 0.01 >p0.001; *** p <0.001. S. Lensu et al. / Metabolism Open 4 (2019) 100019 7 Fig. 6. Protein content of (a) Hif1 a and (b) phosphofructokinase (F6PK) in plantaris muscle. Representative examples of western blots are shown above each graph. Gel blots are divided because the order of samples in the gel was not equal for the order of groups in results-section. Nonetheless, all gel figures originate from the samples run in the same gel. Western blot gels were run equally as in Fig. 5. In the panels, * indicates significant difference to sedentary control (ctrl), * p <0.05; ** 0.01 >p0.001; *** p <0.001. Fig. 7. Protein content of (a) Sirt1, (b) Sirt2, (c) Sirt6, and (d) Sirt7 in plantaris muscle. Representative examples of western blots are shown above each graph. Gel blots are divided because the order of samples in the gel was not equal for the order of groups in results-section. Nonetheless, all gel figures originate from the samples run in the same gel. Western blot gels were run equally as in Fig. 5. In the panels, * indicates significant difference to sedentary control (ctrl), * p <0.05; ** 0.01 >p0.001; *** p <0.001. S. Lensu et al. / Metabolism Open 4 (2019) 1000198