Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 https://doi.org/10.1007/s00424-021-02629-9 INTEGRATIVE PHYSIOLOGY Effects of voluntary exercise on the expression of browning markers in visceral and subcutaneous fat tissue of normotensive and spontaneously hypertensive rats Meryem Sevval Karadedeli1 · Rolf Schreckenberg1 · Hanna S. Kutsche1 · Klaus‑Dieter Schlüter1 Received: 5 May 2021 / Revised: 1 September 2021 / Accepted: 27 September 2021 / Published online: 10 December 2021 © The Author(s) 2021 Abstract High physical activity is important to optimize the function of adipose tissue. Dysfunctional adipose tissue contributes to the development of metabolic stress, chronic inflammation, and hypertension. To improve our current understanding of the interaction between physical exercise and adipose tissue, we analyzed the effect of 10 months voluntary running wheel activ- ity of rats on uncoupling protein (UCP) 1 negative white adipose tissue (visceral and subcutaneous adipose tissue, VWAT and SWAT). Analysis was performed via RT-PCR and immunoblot from adipose tissues depicted from adult normotensive and spontaneously hypertensive female rats. UCP1 negative VWAT differed from UCP1 positive WAT and brown adipose tissue (BAT) from interscapular fat depots, by lacking the expression of UCP1 and low expression of Cidea, a transcrip- tional co-activator of UCP1. High physical activity affected the expression of five genes in SWAT (Visfatin (up), RBP5, adiponectin, Cidea, and Nrg4 (all down)) but only one gene (Visfatin, up) in VWAT. Furthermore, the expression of these genes is differentially regulated in VWAT and SWAT of normotensive and spontaneously hypertensive rats (SHR) under sedentary conditions (UCP2) and exercise (Visfatin, Cidea, Nrg4). Keeping the animals after 6 months of voluntary exercise under observation for an additional period of 4 months without running wheels, Visfatin, Cidea, and Nrg4 were stronger expressed in VWAT of SHRs than in sedentary control rats. In summary, our study shows that SWAT is more responsible to exercise than VWAT. Keywords UCP · Visfatin · Cidea · Nrg4 · Hypertension Introduction [1]. This process has been termed browning of WAT. There is evidence that exercise induces browning of SWAT but Obesity is more than ever a raising world health problem. It not VWAT in mice, rats, and humans [1–4]. The release occurs as the consequence of an imbalance between caloric of myokines, that are cytokines or hormones derived from intake and caloric consumption. Energy is stored predomi- the skeletal muscle, may trigger effects of exercise in adi- nantly in white adipose tissues (WAT) in the form of tri- pose tissue [5]. Among the myokines, interleukin (IL)-6 has glycerides; however, there are anatomical and functional addressed most attention. Interestingly, IL-6 induces remod- differences in various types of WAT within the body. In eling of adipose tissue. Therefore, release of IL-6 from example, subcutaneous WAT (SWAT) and visceral WAT skeletal muscle may indeed connect exercise to browning (VWAT) have different localizations and functions. An of WAT [1]. increase in energy expenditure by exercise can induce a phe- WAT is also located in close proximity to brown adipose notypic change of WAT through a transformation from an tissue (BAT), like in the interscapular region (IWAT). IWAT energy-storing tissue to a thermogenic beige adipose tissue located in the vicinity of BAT differs in several aspects from SWAT and VWAT including origin and phenotype. Browning of WAT has initially linked to the expression of * Klaus-Dieter Schlüter uncoupling protein (UCP) 1. UCP1 is a mitochondrial pro- Klaus-Dieter.Schlueter@physiolgie.med.uni-giessen.de tein which uncouples proton transport from ATP produc- 1 Physiologisches Institut, Justus-Liebig-University Giessen, tion and directs the chemical energy of the mitochondrial Aulweg 129, D-35392, Giessen, Germany proton gradient into heat (thermogenesis). Browning in its Vol.:(012 3456789) 206 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 pure sense means an induction of UCP1 expression in WAT running time, kidney function, blood pressure, and adapta- located in close proximity of BAT. This type of WAT is tion to exercise in kidney and skeletal muscle were reported then claimed “beige” adipose tissue. Nevertheless, SWAT earlier for these the rats [10–13]. Briefly, running distance and VWAT are also affected by exercise. In this context, it was 74 ± 33 km/week (Wistar) and 94 ± 16 km/week (SHR). must be noted that adaptations of adipose tissue to increased Running time was 21 ± 9 h/week (Wistar) and 30 ± 4 h/week energy demand are not limited to the induction of UCP1 [6]. (SHR). Systolic blood pressure was 127 ± 7 mmHg (Wistar) Furthermore, adipose tissue is not only involved in energy and 190 ± 11 mmHg (SHR). Diastolic blood pressure was storage and energy supply but also linked to regulation of the 80 ± 6 mmHg (Wistar) and 122 ± 10 mmHg (SHR) (see Ref. metabolic homeostasis, appetite, angiogenesis, immunity, 13 for technical details). and cardiovascular function. Therefore, a characterization Male or female UCP2−/− rats and their wild-type litter- of the effects of exercise on adipose tissue must cover them mates were used at an age of 3 months to isolate VWAT in a broad way. and SWAT. Obesity is a risk factor often associated with other risk factors such as hypertension and insulin resistance Tissue material — together referred to as the metabolic syndrome. Spon- taneously hypertensive rats (SHR) have several metabolic The study was intended to investigate the effect of free run- differences to normotensive Wistar rats such as reduced ning wheel activity on cardiovascular adaptations. There- expression of CD36, increased norepinephrine turnover in fore, rats were anesthetized using isoflurane inhalation and adipose tissue, and insulin insensitivity [7–9]. Although subsequently sacrificed by cervical dislocation at an age of SHRs are lighter in weight compared to normotensive Wistar 11.5 months. In addition, we now analyzed molecular adap- rats, they show metabolic dysfunction and cardiovascular tation in fat tissue. Therefore, after removing the heart and abnormalities seen also in patients with metabolic syndrome. lung for isolation of the heart, SWAT and VWAT were dis- In this study, we used normotensive Wistar rats to clarify sected and quickly frozen in fluid nitrogen and subsequently the effect of life-long voluntary exercise on the molecular stored at −80 °C until use (Fig. 1). In the same manner, signature of SWAT and VWAT in rats and compared these interscapular BAT and WAT were dissected from fat depots results to those generated with SHRs. The study is aimed at in the neck region and the UCP1 positive WAT was then improving our current understanding of interaction between separated from the BAT. physical activity, WAT function, and co-morbidities such as hypertension. Material and methods The investigations are in agreement with the “Guide for the Care and Use of Laboratory Animals” purchased by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The study was approved by the local authori- ties (RP Gießen; V 54–19 c 20 15 h 01 GI 20/1 Nr. 76/2014 and Nr. 77/2014). Animal model Female Wistar rats and SHRs were randomized selected and kept either under standard conventional housing conditions (WIS-Sed and SHR-Sed) or received free access to a run- ning wheel during night starting at the age of 6 weeks (pre- hypertensive state of SHR) and maintained for 10 months before rats were sacrificed (WIS-Run and SHR-Run). In some groups, SHRs access to free running wheels was ceased for the last 4 months (SHR-ExR). Another group of rats had free excess to running wheels for 4 weeks with Fig. 1 Anatomic localization of the different sources of adipose tis- cessation for another 4 weeks and again access to running sues used in this study (S, subcutaneous; V, visceral; WAT, white adi- wheels (intermittent exercise; SHR-IR). Running distance, pose tissue; BAT brown adipose tissue) 1 3 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 207 RNA isolation and cDNA synthesis performed as described before [6], based on the ΔΔCT method. Total RNA was isolated from adipose tissue using peqGold TriFast™ (peqlab, Biotechnologie GmbH, Germany) accord- Western blot ing to the manufacturer’s protocol. In brief, small pieces of adipose tissue were homogenized in 1 ml TriFAST™ solu- Total protein was extracted from tissues using cell lysis buffer tion for 20 s at 5000 rpm using Precellys® homogenizer. (Cell Signaling, Technology, Frankfurt, Germany), according After adding 200 μl chloroform, samples were centrifuged to the manufacturer’s protocol. Briefly, the homogenates were for 10 min at 12500 rpm and the homogenate was allowed to centrifuged at 14,000 g for 10 min and the supernatants were separate into a clear upper aqueous layer (containing RNA), treated with Laemmli buffer (Sigma-Aldrich, Taufkirchen, an interphase, and a red lower organic layer. The RNA was Germany). The protein concentration was adjusted to either precipitated from the aqueous layer with isopropanol at −20 4 g/l for tissue extracts or 2 g/l for isolated cells. Protein °C. To remove genomic DNA contamination, isolated RNA samples were loaded on NuPAGE Bis-Tris Precast gels (10%; samples were treated with 1 U DNase/μg RNA (Invitrogen, Life Technology, Darmstadt, Germany) and subsequently Karlsruhe, Germany) for 15 min at 37 °C. One microgram of transferred onto nitrocellulose membranes. The expression total RNA was used in a 10 μl reaction to synthesize cDNA of UCP2 was analyzed with an antibody (kindly provided by using Superscript RNaseH Reverse Transcriptase (200 U/ Prof. Dr. E. Pohl), whose specificity was evaluated before [14, μg RNA, Invitrogen, Karlsruhe, Germany) and oligo dTs as 15]. Expression of UCP2 was normalized to the expression primers. RT reactions were performed for 50 min at 37 °C. of actin using an antibody produced in rabbit (Cell Signaling, Technology, Frankfurt, Germany). Secondary antibodies RT‑PCR (horseradish peroxidase-coupled secondary antibody) directed against rabbit IgG or mouse IgG were purchased from Dako Quantitative real-time PCR was performed using the (now Agilent Technologies, Santa Clara, CA, USA). CFX Connect Real-Time PCR Detection System (Bio- Rad, Munich, Germany) in combination with IQ SYBR Statistics green real-time supermix (Bio-Rad, Munich, Germany) as described before [10]. The sequences of the primers used All data are presented as means ± S.D. p values were calcu- in this study are indicated in Table 1. Quantification was lated by unpaired t-tests (Figs. 3 and 4) and p values below Table 1 Primer sequences used in this study Gene Forward Reverse Ampl.-length Adiponectin GGC CGT TCT CTT CAC CTA CG TGT CCC CTT CCC CAT ACA CT 122 bp (NM_144744.3) AR-β3 GGT TGG GCT ATG CCA ACT CT CCT GTT GAG CGG TGA GTT CT 176 bp (NM_013108.2) B2M GCCG TC GTG CTTG CC ATTC CTG AGG TGG GTG GAA CTGA GAC 117 bp (NM_012512.2) Cidea AGA AAT GGA CAC CGG GCA AT TGA AGC TTG TGC AGC GGA TA 177 bp (NM_001170467.1) Leptin ACC AGA CCC TGG CAG TCT AT TTG GAG AAG GCC AGC AGA TG 118 bp (NM_013076.3) Nrg4 CCA GGC ACA GGT CAT TTT GC AGC TGC CGA CAG GTT ACT TT 161 bp (NM_001191109.1) RBP4 CAA GGG ACG AGT CCG TCT TC GTC ATC GTT TCC TCG CTG GA 139 bp (NM_013162.1) TGF-β1 ATT CCT GGC GTT ACC TTG G CCT GTA TTC CGT CTC CTT GG 117 bp (NM_021578) UCP-1 ATC TTC TCA GCC GGC GTT TC AGG GTG GTG ATG GTC CCT AA 146 bp (NM_012682.2) UCP-2 CAC CGT CAT TGC CTC CCC CG CGG AGC ATG GTC AGG GCA CA 102 bp (NM_019354.2) Visfatin CTG TGT CTG TGG TCA GCG AT GTG TCG AGC GGA TTT CCA GA 142 bp (NM_177928.3) 1 3 208 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 p ≤ 0.05 are indicated by asterisks. One-way ANOVA with expressed in BAT but not VWAT (Fig. 2). Collectively, these Student-Newman-Keuls post hoc analysis was performed for results demonstrate differences between WAT in close prox- data analysis of Figs. 2 and 5. Data pairs with p ≥ 0.05 are imity to BAT and VWAT. labeled with the same letter and data pairs with p ≤ 0.05 with different letters. Effect of exercise on the molecular signature of SWAT and VWAT in normotensive rats Results The classical browning marker UCP1 could not be detected in either SWAT or VWAT. Therefore, we addressed the Classical browning parameter in BAT and different question whether the expression of browning markers dis- types of WAT tinct from UCP1 differs between SWAT and VWAT. The expression of several browning markers was analyzed in First, we confirmed a high expression of UCP1 in interscap- these two different types of adipose tissue in normotensive ular BAT and also in WAT located in the immediate vicinity rats that were kept under standard housing conditions (sed- of BAT (Fig. 2). In contrast, UCP2, acting as a metabolic entary conditions; VWAT-Sed and SWAT-Sed). Cidea-4 switch in myocardial cells [16], shows the opposite type of was present in three out of six samples in the VWAT but expression (Fig. 2). As expected, Cidea-1, known to act as in all samples from SWAT. However, the level of expres- an upstream activator of UCP1 expression [17], was strongly sion of Cidea-4 in SWAT was lower than in Cidea-positive expressed BAT (Fig. 2). Neuregulin (Nrg)-4, an activator VWAT (Fig. 3). Similarly, expression of Nrg-4 (Fig. 3), of erb-b2 receptor tyrosine kinase 4 [18], is also strongly adiponectin (Fig. 3), and retinol-binding protein (RBP)-4 was lower expressed in SWAT than in VWAT (Fig. 3). Visfatin, a member of the family of adiponectins, was detected in three out of six samples of VWAT and in all samples from SWAT but again at low level of expression in Visfatin positive samples from SWAT compared to VWAT (Fig. 3). Collectively, these data suggest that browning and metabolic stress markers are stronger expressed in VWAT than in SWAT. Subsequently, we analyzed the effect of exercise on these markers. Voluntary exercise in normotensive Wistar rats affected the mRNA expression of Cidea, Nrg-4, adiponectin, and RBP-4 in SWAT but not VWAT (Fig. 3). All these four genes were downregulated in rats performing long-term exercise. In contrast, Visfatin, the fifth gene that was differentially affected by exer- cise, was commonly upregulated in SWAT and VWAT (Fig. 3). Comparison of the molecular signature of WAT between normotensive and spontaneously hypertensive rats SHRs have occasionally been described as a model of metabolic dysfunction but whether this is accompanied by a different profile of mRNA expression in WAT remains unclear. Major differences between the expression of Fig. 2 Expression of typical browning markers in WAT and BAT browning markers and metabolic molecules were found from interscapular fat depots and in visceral white adipose tis- in WAT between both strains. The mRNA expression of sue (VWAT). Data are means + S.D.; analysis was performed by ANOVA and Student-Newman-Keuls post hoc analysis. Groups are UCP2 was lower in VWAT and SWAT from SHRs com- labeled with identical letters when the difference was >0.05. Different pared to normotensive Wistar rats (Fig. 4). In SWAT from letters indicate group differences with p < 0.05 SHR, UCP2 levels of mRNA were below the detection 1 3 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 209 Fig. 3 Expression of typical browning markers in VWAT and SWAT from normoten- sive Wistar rats and the effect of voluntary exercise (Run) compared to sedentary controls (Sed). Data are means + S.D.; t-tests were performed between two columns and indicated by *, if p < 0.05 level and differences in protein expression in SWAT were detectable in SWAT from SHRs, was stronger expressed stronger than in VWAT. As uncoupling proteins are of in VWAT of SHRs compared to VWAT from normoten- main interest in terms of energy consumption, we con- sive rats (Fig. 4). The expression of leptin was strongly firmed low expression of UCP2 on the protein level in reduced in VWAT and SWAT from SHRs compared to VWAT and SWAT in tissues from SHR versus that of normotensive rats (Fig. 4). Collectively, these data sug- Wistar rats (Fig. 4). In addition, the expression of TGF-β1 gest major differences in the molecular composition of (Fig. 4), Cidea (Fig. 4), Nrg-4 (Fig. 4), and adiponectin VWAT between SHRs and normotensive Wistar rats and (Fig. 4) were all lower in VWAT from SHRs than in nor- with the exception of leptin these differences were stronger motensive rats. The expression of adrenoceptor 3, barely in VWAT than in SWAT. 1 3 210 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 Fig. 4 Expression of typical browning markers in VWAT and SWAT in normotensive rats (WIS) and spontaneously hypertensive rats (SHR). Data are given for mRNA (all) and protein (UCP2). Data are means + S.D.; t-tests were performed between two columns and indi- cated by *, if p < 0.05 These data suggest that the strong decrease in UCP2 Effect of voluntary exercise on browning parameters expression in SWAT from SHR, a protein located in the in SHRs inner mitochondrial membrane, may trigger the different expression of Cidea and leptin in SWAT from SHRs. We In normotensive rats, voluntary exercise significantly tested this hypothesis in UCP2−/− rats. The mRNA expres- affected the expression of Visfatin, Cidea, and Nrg-5 in sion of U CP2−/− was strongly reduced in VWAT and SWAT SWAT. However, in SHRs, SWAT did not respond to volun- from knockout rats (Fig. 5). However, only in SWAT, but tary running activity in a similar way (Fig. 6). Unexpectedly, not in VWAT, this was accompanied by a similar decrease we observed an upregulation of Cidea and Nrg-4 and a simi- in Cidea and leptin expression as in SWAT from SHRs lar trend for Visfatin in SHRs with cessation of free running (Fig. 5). wheel activity after 6 months (Fig. 6; ExR). No differences 1 3 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 211 functions such as energy storage, energy homeostasis, ther- moregulation, and regulation of appetite. Furthermore, it is involved in fine-tuning of the immune response and the cardiovascular system via the release of adipokines [19]. To cover all these functions, adipose tissues are widely spread throughout the body. However, different locations of adi- pose tissue go along with different phenotypes suggesting that different types of adipose tissue are linked to differ- ent functions. In terms of obesity, a dysregulation between energy intake and energy consumption leads to extension of adipose tissue size and changes in adipose tissue function. High physical activity should affect this dysregulation by improving energy expenditure. Adipose tissue is involved in the adaptation to high physical activity as it improves glucose metabolism and supports the energy requirements of the muscle by lipolysis [20–22]. Investigating the role of adipose tissue in response to physical activity, a lot of attention has been given to the mechanism of browning of WAT. The molecular marker of this process is UCP1 that is responsible for thermogenesis. We confirmed high expres- sion of UCP1 and its upstream regulator Cidea in BAT. However, only WAT in the immediate proximity of BAT from the interscapular region had detectable expression of UCP1 whereas UCP1 could not be detected in either SWAT or VWAT. In some mice strains but not in mice in general, Fig. 5 Effect of genetic depletion of UCP2 on the mRNA expression UCP1 expression as a measure of browning was induced of Cidea and leptin in VWAT and SWAT. Data are means + S.D.; t-tests were performed between two columns and indicated by *, if by exercise. In this case, it was induced in UCP1 positive p < 0.05 WAT [2]. This adipose tissue was termed “brite” adipose tissue indicating that brown adipocytes are present in WAT. High physical activity did not induce the expression of were obtained when rats had access to running wheels only UCP1 in either SWAT or VWAT. In this aspect, our study every second month (interrupted running; Fig. 6; IR). conformed to previous reports that exercise does not induce browning of WAT in humans or rodents [20, 23–26]. Only few reports have reported conflicting results [3, 4]. From a Discussion mechanistic point of view, induction of UCP1 in adipose tissue in response to muscle work does not make sense as it Our study compared SWAT and VWAT of normotensive further increases heat production. On the other hand, it has and hypertensive rats and investigated the effect of exercise been argued that increased energy consumption is required on these tissues in both strains. There are two main find- to maintain proper fatty acid control under conditions of ings in our study. At first, recently discovered adaptations lipolysis. Some authors describe that re-browning of WAT of SWAT in normotensive rodents to high physical activity is associated with less UCP1 expression in BAT [3, 4]. Fur- do not allow to translate these findings to hypertensive rats. thermore, some results may be explained by heterogeneous This result is important as many obese patients have hyper- behavior of different types of adipose tissues and does not tension as an important co-morbidity and our data suggest exclude that other parts of the adipose tissue respond by that metabolic improvements by high physical activity are re-browning. Whatever the explanation for these divergent more complex than expected from such studies. Second, our results is, it is clear that a general re-browning of WAT by results show that a different responsiveness between SWAT induction of UCP1 does not occur. In this aspect, our study and VWAT may be associated with reduced tissue expres- is in line with other reports [20, 25, 26]. sion of UCP2. SWAT and VWAT differ not only in anatomic localiza- Regulation of energy metabolism of the whole body tion but more importantly in their physiological function. requires a fine-tuning between the main tissues involved Specifically, the VWAT located in vicinity to organs like in this process, namely, liver, skeletal muscle, and adipose kidney, liver, and heart are of interest because they release tissue. Adipose tissue has several important physiological adipokines that directly affect non-adipose tissues [1]. This 1 3 212 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 Fig. 6 Expression of typical browning markers in VWAT and SWAT in spontaneously hypertensive rats (SHR) with different running protocols (Run, 10 months access to running wheels; ExR, 6 months access to running wheels and 4 months cessation; IR; free access to running wheels every second month). Data are means + S.D.; analysis was performed by ANOVA and Student-New- man-Keuls post hoc analysis. Groups are labeled with identi- cal letters when the difference was >0.05. Different letters indicate group differences with p < 0.05 was formerly studied for vascular adipose tissue and the VWAT. Nrg-4 is known to be stronger expressed in VWAT impact of vascular adipose tissue on endothelial function. It than SWAT [27] as confirmed in this study. One of its physi- is clear that different types of WAT require the expression of ological functions is to fine-tune the activity of adipose tis- a different type of proteins to fulfill the different functions. sue and the liver [28, 29]. Adiponectin is an organ-protective In this study, we observed such differences for the expres- protein that is exclusively expressed by adipocytes; however, sion of Nrg-4, adiponectin, and RBP-4 between SVAT and its expression is markedly reduced in obese patients [19, 1 3 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 213 30]. RBP-4 is upregulated in obese rodents and contributes lower than that of normotensive rats [34]. This may indicate to insulin resistance. Collectively, the molecular expression that the observed differences are compensatory mechanisms of factors coupling adipose tissue with metabolism is lower to normalize lipolytic response to catecholamines. Due to in SWAT than VWAT suggesting that SWAT is acting more the inability of catecholamines to increase lipolysis in adipo- like an energy storage tissue and VWAT involved in adipose cytes from SHR, exercise does not increase lipolysis in such tissue-dependent control of metabolism. Exercise, as per- cells [35] and we did not find an exercise-dependent increase formed in this study by voluntary running wheel activity, in adrenoceptor-β3 expression. Most of the transcriptional strongly reduced the expression of such molecules in SWAT differences between WAT from normotensive and hyperten- but not VWAT. In general, this conformed to earlier reports sive rats, except that of leptin, were specific for VWAT and that SWAT is more sensitive to exercise than VWAT [1]. barley seen in SWAT. An exception to this rule is the upregulation of Visfatin in The aforementioned findings show significant differences both types of adipose tissue. Mechanistically, induction of in the molecular signature of adipose tissues in SHRs ver- Visfatin in rats with high physical activity makes sense as it sus normotensive rats. Similar conclusions hold also for the increases the glucose uptake in muscles [31]. The transcrip- effect of exercise on the molecular signature of SWAT and tional regulation of Visfatin seems to be strain specific, as VWAT. More specifically, there was a lack of responsiveness voluntary exercise increased Visfatin expression in SWAT with respect to Visfatin. While exercise did not affect the and VWAT of normotensive rats but not in SHRs. This is a molecular signature in SWAT and VWAT in SHRs, cessa- new finding from our study. tion of exercise increased the expression of some browning SHRs have high blood pressure and other cardiovascu- makers in VWAT (Cidea, Nrg-4). One may assume that ces- lar risk factors such as insulin resistance [9]. In the cur- sation of increased energy expenditure by exercise requires a rent study, we first addressed the question whether SHR and proper compensatory upregulation of pathways that increase normotensive Wistar rats differ in the expression on metab- energy expenditure, but this requires further analysis to be olism-related genes in WAT. In total, seven differentially properly confirmed. If these data can be translated into regulated genes were identified, of which UCP2, adiponec- human behavior, it would suggest that high physical activ- tin, TGF-β1, Cidea, Nrg-4, and leptin are lower expressed in ity is more important for the regulation of adipose tissues VWAT from SHRs versus VWAT from normotensive rats. than continuation of high physical activity. UCP2 expression in adipose tissue is depressed by vitamin Transcriptional adaptations of tissues to high physical D3 [32]. However, SHRs have lower vitamin D levels than activity are potentially triggered by the release of myokines normotensive rats but still lower UCP2 expression indicating from the skeletal muscle. However, such a release is typi- other mechanisms leading to the low levels of UCP2 expres- cally seen after the onset of muscle work. In this case, we sion [33]. UCP2 is of specific interest as it may replace expected that alternation in physical activity as investigated the classical browning marker UCP1 due to its structural by access to running wheels every second month (inter- similarity. Therefore, it is important that the lower mRNA rupted running, IR) would have a stronger impact on adapta- expression was accompanied by lower protein expression as tion in fat tissue than permanent activity. That was, however, well. Importantly, selective knockdown of UCP2 in VWAT not the case. It might be that 4 weeks of running perfor- and SWAT caused similar changes in Cidea and leptin mance interrupted by 4 weeks of resting periods was too expression in SWAT but not VWAT. The data underline the long to indicate such an effect as we have previously shown importance of the protein of the inner mitochondrial mem- that an increased expression of IL-6 in skeletal muscles, an brane for the physiology of SWAT. The reduced expression indicator of increased myokine release, was significant after of adiponectin and leptin in SHRs suggests that the impact 2 days but not in the IR group [13]. of VWAT-derived hormones on energy homeostasis is less In conclusion, the current study conformed to two major pronounced in these rodents. The lower expression of TGF- findings from former research, namely, that exercise does β1 in VWAT from SHRs, an anti-inflammatory cytokine of not upregulate UCP1 in VWAT and SWAT and that exer- adipose tissues, suggests increased inflammatory stress. The cise mainly affects the molecular signature of SWAT. A new lower expression of Cidea and Nrg-4 suggests an impaired finding of this study is that we identified an upregulation of regulation of glucose metabolism within adipocytes. How- Visfatin as a potential link between muscle load and adipose ever, the expression of adrenoceptor β3 was significantly tissue function and show for the first time that this effect was increased. It is known that tyrosine hydroxylase activity in abrogated in SHRs. Furthermore, the data analyzed in our adipocytes from SHRs is more activate than in normotensive two rat models show that SHRs have a phenotypic different rats [8]. The upregulation of the corresponding receptors VWAT and that exercise does not exert a similar adaptation may simply reflect an over-activation of adrenergic stimu- in this strain compared to normotensive Wistar rats. The data lation in the adipose tissue. However, the norepinephrine- may also explain the large discrepancy in the literature con- dependent lipolytic response of adipocytes from SHRs is cerning the interaction between exercise and adipose tissue 1 3 214 Pflügers Archiv - European Journal of Physiology (2022) 474:205–215 function. Genetic variability as between these two rat strains Genetic, physiological and comparative genomic studies of hyper- used in this study is remarkable and may affect the outcome. tension and insulin resistance in the spontaneously hypertensive rat. Disease Models Mechanisms 10:297–306. https://d oi.o rg/1 0. 1242/ dmm. 026716 10. Braun K, Atmanspacher F, Schreckenberg R, Grgic I, Schlüter Funding Open Access funding enabled and organized by Projekt K-D (2018) Effect of free running wheel exercise on renal expres- DEAL. This study was supported in part by the Deutsche Forschun- sion of parathyroid hormone receptor type 1 in spontaneously gsgemeinschaft (DFG) within the CRC 1213, project B05. hypertensive rats. Physiol Rep 6:e13482. https://d oi.o rg/1 0.1 4814/ phy2. 13842 Declarations 11. da Costa Rebelo RM, Schreckenberg R, Schlüter K-D (2012) Adverse cardiac remodelling in spontaneously hypertensive rats: acceleration by high aerobic exercise intensity. J Physiol Conflict of interest The authors declare no competing interests. 590:5389–5400. https:// doi.o rg/ 10.1 113/ jphys iol. 2012. 241141 12. Schreckenberg R, Horn A-M, da Costa Rebelo RM, Simseky- Open Access This article is licensed under a Creative Commons Attri- ilmaz S, Niemann B, Ling L, Rohrbach S, Schlüter K-D (2017) bution 4.0 International License, which permits use, sharing, adapta- Effects of 6 months’ exercise on cardiac function, structure, and tion, distribution and reproduction in any medium or format, as long metabolism in female hypertensive rats – the decisive role of lysyl as you give appropriate credit to the original author(s) and the source, oxidase and collagen III. Front Physiol 8:556. https:// doi. org/ 10. provide a link to the Creative Commons licence, and indicate if changes 3389/ fphys. 2017.0 0556 were made. The images or other third party material in this article are 13. Wolf A, Kutsche HA, Atmanspacher F, Karadedeli MS, Schreck- included in the article's Creative Commons licence, unless indicated enberg R, Schlüter K-D (2021) Untypical metabolic adaptation otherwise in a credit line to the material. If material is not included in in spontaneously hypertensive rats to free running wheel activ- the article's Creative Commons licence and your intended use is not ity includes uncoupling protein-3 (UCP-3) and proprotein con- permitted by statutory regulation or exceeds the permitted use, you will vertase subtilisin/kexin type 9 (PCSK9) expression. Front Physiol need to obtain permission directly from the copyright holder. To view a 12:598723. https:// doi.o rg/ 10. 3389/ fphys.2 021.5 98723 copy of this licence, visit http://c reati vecom mons.o rg/l icens es/b y/4.0 /. 14. 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